mwwXaiv lus
Tsev / Kev paub / Ntsiab lus

Kev paub

Immune Responses To Sequential Binocular Transplantation Of Allogeneic Retinal Progenitor Cells Rau Vitreous Cavity hauv nas

Dec 11, 2023

Abstract:

Intravitreal transplantation ntawm allogeneic human retinal progenitor cells (RPC) tuav cov lus cog tseg raws li kev kho mob rau qhov muag tsis pom kev ntawm lub qhov muag. Ua ntej ua haujlwm tau pom tias cov neural progenitors tau txais txiaj ntsig zoo li allografts tom qab txhaj tshuaj ib zaug; Txawm li cas los xij, kev xa tawm ntawm cov hlwb allogeneic nce qhov kev pheej hmoo ntawm tus tswv tsev rhiab heev nrog kev tiv thaiv kab mob tom ntej ntawm grafts. Txoj kev tshawb fawb tam sim no tau tsim los ntsuas seb qhov kev tiv thaiv kab mob puas yuav raug cuam tshuam los ntawm kev hloov pauv intravitreal ntawm allogeneic RPCs siv tus qauv nas. Peb txhaj tshuaj murine retinal progenitor cells (gmRPCs), Ameslikas muab los ntawm cov neeg pub dawb nrog C57BL / 6 keeb kwm caj ces, rau hauv BALB / c cov nas tau txais txiaj ntsig txhawm rau muab cov ntaub ntawv kev nyab xeeb raws li qhov yuav xav tau tom qab rov kho cov neeg mob nrog allogeneic tib neeg cov khoom ntawm tes. . Cov tshuaj tiv thaiv kab mob rau gmRPCs yog me me, suav nrog T hlwb, B hlwb, neutrophils, thiab cov cell killer ntuj, nrog macrophages kom meej meej predominating. Tsiaj txhu tau kho nrog cov tshuaj rov ua dua ntawm gmRPCs tsis tau pom cov pov thawj ntawm kev nkag siab, thiab tsis muaj kev tiv thaiv kab mob kev puas tsuaj ntawm cov grafts. Txawm hais tias tsis muaj kev kho mob tiv thaiv kab mob, allogeneic gmRPC grafts muaj sia nyob tom qab noj tshuaj dua, yog li muab kev txhawb nqa rau kev soj ntsuam ua ntej uas rov txhaj tshuaj ntawm allogeneic RPCs rau hauv cov kab noj hniav yog zam rau cov neeg mob retinitis pigmentosa.

Desert ginseng-Improve immunity (15)

cistanche cog-nce kev tiv thaiv kab mob

Nyem qhov no mus saib Cistanche Enhance Immunity khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Ntsiab lus: qia hlwb; tiv thaiv kab mob; kev tiv thaiv kab mob; kev tsis lees txais nyiaj txiag; Kev txhaj tshuaj intravitreal

1. Taw qhia

Retinitis pigmentosa (RP) encompasses ib chav kawm ntawm genetic rod-cone degeneration thiab ua rau muaj kev puas tsuaj rau qhov muag thiab qhov muag tsis pom. Rau feem coob ntawm cov neeg mob, tsis muaj cov pov thawj kho mob muaj los khaws lossis kho qhov muag; Txawm li cas los xij, ib lub tswv yim los daws qhov kev xav tau kev kho mob uas tsis tau ntsib yog kev hloov pauv ntawm cov qia. Peb pawg neeg tshawb fawb tau tshawb fawb txog kev txhaj tshuaj intravitreal ntawm kab lis kev cai tib neeg retinal progenitor hlwb (RPC) ua ib txoj hauv kev los cuam tshuam hauv RP nrog lub hom phiaj kho mob kom ruaj khov lossis tej zaum thim rov qab cov kab mob. RPGs tuaj yeem muab tau los ntawm cov retinas tsis paub qab hau los ntawm cov ntaub so ntswg pub dawb lossis, tsis ntev los no, los ntawm pluripotent cell kab. Kev tshawb fawb hauv cov qauv tsiaj tau pom tias cov hlwb no muaj peev xwm cawm tau cov photoreceptors los ntawm degeneration tom qab txhaj tshuaj rau hauv lub qhov muag thiab tseem muaj peev xwm sib txawv rau hauv pas nrig photoreceptors hauv lub qhov muag. Muaj qee cov pov thawj los ntawm kev tshawb fawb tsiaj uas txhaj hRPCs tuaj yeem ua haujlwm tau zoo, kev sib koom ua ke ntawm photoreceptors thiab yog li muaj peev xwm ua kom lub retina ruaj khov los ntawm kev hloov cov hlwb ncaj qha. Yog li, txhaj hRPCs tuaj yeem kho RP hauv ob qho tib si neurotrophic zam nrog rau ntawm kev hloov pauv ntawm tes. Txawm li cas los xij, txoj hauv kev ntawm tes no muaj lub tswv yim tsim nyog rau kev kho mob ntawm cov neeg mob RP thiab, muaj peev xwm, lwm yam kab mob ntawm lub qhov muag.

Lwm qhov txiaj ntsig zoo ntawm RPCs, thiab tej zaum neural progenitors feem ntau, yog tus txheeb ze kam rau cov hlwb no tom qab hloov pauv raws li allografts, tshwj xeeb tshaj yog thaum xa mus rau qhov chaw uas muaj kev tiv thaiv kab mob xws li retina [1,2]. Qhov ntawd tau hais tias, kev tshuaj xyuas tsis ntev los no ntawm kev sim tshuaj kho mob tau ua nyob rau hauv thaj chaw ntawm cov tshuaj regenerative txuas ntxiv mus tshaj tawm cov kev sib tw loj thiab tseem ceeb ntsig txog kev mob thiab kev tiv thaiv kab mob [3,4], suav nrog rau kev cuam tshuam rau lub qhov muag xws li kev kho cov noob caj noob caj noob ces [5], xws li zoo li qee qhov tab sis tsis yog txhua txoj kev kho cell [6]. Txawm hais tias ib hom cell hauv zos, cov xim retinal pigment epithelial (RPE) cell tsis raug zam los ntawm cov teeb meem tsis lees paub [7–9]; Yog li ntawd, kev tiv thaiv kev tiv thaiv ntawm cov neeg tau txais kev pab yog tam sim no tus qauv coj [10,11]. Tib neeg RPCs, ntawm qhov tod tes, zoo nkaus li tau txais txiaj ntsig zoo li allografts [6,12–16], txawm hais tias qhov no tsis txuas ntxiv mus rau lawv siv li xenografts, qhov twg yuav tsum muaj kev tiv thaiv kab mob [17]. Lub hauv paus rau kev muaj sia nyob ntawm allogeneic RPCs hauv lub qhov muag yuav muaj ntau yam cuam tshuam nrog cov hlwb siv thiab qhov chaw txais [1,8,18,19]. Hauv kev ua haujlwm dhau los uas siv cov cytometry ntws, peb tau pom tias tib neeg neural progenitors, ob lub hlwb-derived thiab retina-derived, qhia chav kawm I, tab sis tsis yog chav kawm II, loj histocompatibility (MHC) antigens [20,21]. Cov txheej txheem classical ntawm graft rejection muaj xws li cov nonspecific lees paub ntawm txawv teb chaws MHC class II molecules los ntawm CD{23}} party lymphocytes [20–25]. Nyob rau hauv txoj kev ntawd, qhov tsis muaj chav kawm II molecules tuaj yeem tso cai rau cov hlwb grafted progenitor kom tsis txhob tiv thaiv lub cev tiv thaiv kab mob los ntawm cov txheej txheem no. Txawm li cas los xij, qhov pom tseeb "kev tiv thaiv tsis muaj cai" xwm txheej ntawm intravitreally txhaj hRPCs tsis tas yuav tsum muaj tseeb. Thaum murine central nervous system (CNS) progenitors tsis qhia cov chav kawm I lossis chav kawm II MHC cov molecules ntawm lub hauv paus thiab nthuav tawm lub cev tiv thaiv kab mob zoo li allografts [1,2,20], cov hlwb no tuaj yeem tiv thaiv kev tiv thaiv kab mob hauv qee qhov xwm txheej. Cov kev tshawb fawb tau pom tias MHC antigens tuaj yeem raug ntxias los ntawm kev txhawb nqa ntawm CNS progenitors nrog interferon-gamma (IFN) [1,26]. CNS progenitors tuaj yeem raug tsis lees paub tom qab kev nkag siab ntawm tus tswv tsev yav dhau los grafted. Yog li ntawd, CNS progenitor hlwb (xws li RPCs) qhia alloantigens uas tau kuaj pom los ntawm tus tswv tsev tiv thaiv kab mob. Ua ke, qhov no txhais tau hais tias lub cev tiv thaiv kab mob muaj cai ntawm grafted progenitor cells yog ib ntus thiab raug hloov kho, piv txwv li, los ntawm cytokines tam sim no hauv cheeb tsam microenvironment, thiab uas tuaj yeem hloov pauv thaum lub sijhawm degenerations xws li RP. Rau tag nrho cov laj thawj no, lub cev tiv thaiv kab mob rau ntau qhov kev txhaj tshuaj intravitreal RPC yog qhov nyuaj rau kwv yees thiab yuav tsum tau kuaj tshwj xeeb.

Desert ginseng-Improve immunity (9)

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Yog tias kev kho mob RPC ua pov thawj tias kev kho mob tau zoo hauv RP, yuav muaj kev txhawb zog rau kev kho ob lub qhov muag hauv ob tog qhov muag tsis pom kev. Rau qhov laj thawj ntawm kev nyab xeeb yam tsawg kawg nkaus, kev kho mob binocular feem ntau yuav ua raws li, nrog rau lub sijhawm tseem ceeb ntawm ob qhov kev txhaj tshuaj. Nco ntsoov, qhov kev xa tawm ntawm cov hlwb allogeneic no tuaj yeem ua rau tus tswv tsev rhiab heev hauv cov lus teb rau thawj qhov kev txhaj tshuaj, uas tuaj yeem ua rau lub cev tsis muaj zog hauv kev teb rau kev txhaj tshuaj thib ob. Qhov no tuaj yeem ua rau poob ntawm grafts ntawm ob lub qhov muag. Cov ntaub ntawv ua ntej tshuaj xyuas ib qho kev txhaj tshuaj subretinal nrog hRPCs hauv RP [27] lossis rov ua dua nrog tib neeg neural progenitors hauv cov tsiaj [28,29] suav nrog kev tiv thaiv kab mob. Yog li, txoj kev tshawb fawb hauv qab no tau tsim los ua kev tshawb nrhiav kev txhais lus ntawm murine RPCs raws li cov kab ke sib txuas hauv cov kab mob tiv thaiv kab mob uas muaj peev xwm tiv thaiv kab mob nrog cov keeb kwm caj ces sib txawv. Txawm hais tias tsis suav nrog tib neeg cov khoom RPC, txoj haujlwm no tau ua ib feem ntawm IND-enabling cov kev tshawb fawb txhawm rau muab cov ntaub ntawv tsiaj hais txog kev nyab xeeb, piv txwv li, seb kev tiv thaiv kab mob puas tsim nyog rau cov neeg mob tau txais kev kho RPC rov ua dua hauv kev sim tshuaj FDA.

2. Cov txiaj ntsig

Txhawm rau kawm txog kev tiv thaiv kab mob ntawm ob sab RPC txhaj rau hauv cov nas allogeneic, gmRPCs (C57BL / 6 keeb kwm caj ces) tau txhaj tshuaj intravitreally rau hauv ib lub qhov muag ntawm BALB / c tau txais thiab koob thib ob ntawm RPCs tau txhaj rau lwm qhov muag 2 lub lis piam tom qab. Lub sijhawm 2- lub limtiam lub sijhawm raug xaiv kom txaus los tso cai rau thawj zaug graft ua rau ob qho tib si hauv lub cev thiab lub cev tiv thaiv kab mob. Tsis muaj ib tus tsiaj sim tau txais kev tiv thaiv kab mob tiv thaiv kab mob kom cov tshuaj tiv thaiv kab mob hauv lub cev tuaj yeem pom.

2.1.Kev soj ntsuam kev soj ntsuam thiab kuaj qhov muag pom pom tsis muaj qhov txawv txav

Qhov hnyav ntawm cov tsiaj sim tau ntsuas thaum lub sijhawm ntawm txhua qhov kev txhaj tshuaj gmRPC. Txhua tus tsiaj sim tau pom qhov hnyav nce ntawm thawj thiab thib ob txhaj tshuaj, ua raws li kev noj qab haus huv tag nrho. Tsiaj txhu uas raug txiav tawm ntawm hnub 14 thiab hnub 28 ± 1 tom qab txhaj tshuaj gmRPC thib ob raug kuaj xyuas los ntawm kev phais mob. Ob qho tib si sab pem hauv ntej thiab tom qab ntawm feem ntau ntawm lub qhov muag kuaj tau raug txiav txim siab "nyob rau hauv ib txwm txwv" (WNL). Tsis muaj cov tsos mob ntawm tus mob los yog lwm yam pom txawv txav nyob rau hauv kev kho tsis tau, sham-kho, thiab gmRPC-kho pab pawg, qhia tias gmRPC intravitreal txhaj tshuaj tsis txhaum cai inflammatory teb los yog lub cev nqaij daim tawv puas tsuaj los ntawm txoj kev no. Cell pawg uas muaj cov graft tau pom nyob rau hauv vitreous ntawm ob hnub 14 thiab hnub 28 ± 1 tom qab txhaj tshuaj gmRPC thib ob (Daim duab 1). Kev txhaj tshuaj rov ua dua ntawm gmRPCs tsis ua rau pom qhov hloov pauv ntawm qhov loj ntawm cov pawg cell, zoo ib yam nrog cov neeg pub dawb cell ciaj sia taus. Tag nrho cov pathology kuj tau soj ntsuam ntawm qhov kawg ntawm qhov kawg thiab tsis muaj qhov muag loj (qhia txog kev tsim qog) lossis lwm yam txawv txav tau raug sau tseg.

Figure 1. Transplanted gmRPCs visualized in vivo via fundus photography through a surgical microscope (all = left eye, OS). (A, B), sham-treated animals (sham/sham, S/S); (C, D), control animals with sham-treated right eye followed by gmRPCs-treated left eye (sham/cell, S/C), and animals with both eyes treated sequentially with gmRPCs (cell/cell, C/C) were observed and imaged through the surgical microscope. The upper panel (A, C, E) shows images of OS taken on Day 14 post-second injection; the lower panel (B, D, F) shows images of OS taken on Day 28 post-second injection. Allografts were seen as pale-colored cell clumps (arrows) visible in the vitreous of cell-injected left eyes (S/C, C/C) at both time points (Day 14: (C, D); and Day 28: (E, F)). Apart from grafts, there was no obvious inflammation or additional abnormality seen in the anterior and posterior segments, as viewed axially in this manner.


Daim duab 1. Transplanted gmRPCs pom nyob rau hauv vivo ntawm fundus photography los ntawm ib tug phais microscope (tag nrho=sab laug qhov muag, OS). (A, B), sham-kho tsiaj (sham/sham, S/S); (C, D), tswj tsiaj nrog sham-kho sab xis qhov muag ua raws li gmRPCs-kho sab laug qhov muag (sham/cell, S/C), thiab cov tsiaj nrog ob lub qhov muag kho raws li gmRPCs (cell / cell, C / C) tau pom thiab duab los ntawm kev phais microscope. Lub vaj huam sib luag sab saud (A, C, E) qhia cov duab ntawm OS coj hnub 14 tom qab txhaj tshuaj thib ob; lub vaj huam sib luag qis (B, D, F) qhia cov duab ntawm OS coj nyob rau hnub 28 tom qab txhaj tshuaj thib ob. Allografts tau pom raws li xim daj-xim cell clumps (xub xub) pom nyob rau hauv vitreous ntawm cell-injected ob lub qhov muag (S/C, C/C) ntawm ob lub sij hawm ntsiab lus (Hnub 14: (C, D); thiab Hnub 28: ( E, F)). Sib nrug los ntawm grafts, tsis muaj qhov pom tseeb qhov mob lossis qhov txawv txav ntxiv pom nyob rau sab hauv thiab tom qab ntu, raws li pom axially hauv qhov no.

2.2. Immunofluorescent Labeling pom cov kab mob tiv thaiv kab mob hauv Vitreous

Immunofluorescent labeling rau tsib lub cev tiv thaiv kab mob loj (macrophages, neutrophils, ntuj killer cells, T hlwb, thiab B hlwb) tau ua rau ntawm lub qhov muag ntawm lub qhov muag thiab qhia pom qhov zoo ntawm lub cev tiv thaiv kab mob hauv lub qhov muag tom qab txhaj tshuaj gmRPC (Daim duab 2A-F). T cell (CD3), B cell (CD45R), neutrophil (Ly-6G), thiab natural killer cells (CD49b) pom muaj qhov txwv tsis pub muaj nyob rau hauv ob lub qhov muag txhaj nrog gmRPCs, whereas activated macrophages (Iba-1) ) yog lub cev tiv thaiv kab mob tseem ceeb teb rau cov kab mob intravitreal grafts. Sib piv cov ntaub so ntswg los ntawm cov tsiaj tsis kho thiab sham-kho tsiaj tsis pom cov kab mob tiv thaiv kab mob hauv ocular cryosections, qhia tias lub cev tiv thaiv kab mob hauv lub cev pom yog cov lus teb rau gmRPC grafts. Raws li qhov kev soj ntsuam no, lub cev tiv thaiv kab mob tau nyob hauv thaj chaw ib puag ncig gmRPC grafts lossis hauv gmRPC pawg lawv tus kheej.

Figure 2. Immuno-labeling of infiltrated immune cells by treatment group. Immunofluorescent images of (A) control sections (secondary antibody alone), (B) anti-Iba-1 (activated macrophage and microglia marker), (C) anti-Ly-6G (neutrophil marker), (D) anti-CD49b (natural killer cell marker), (E) antibodies anti-CD3 (T cell marker), and (F) anti-CD45R (B cell marker) are displayed in the figure. The limited red signals present indicate positive antibody labeling for leucocyte markers, green signals indicate the gmRPC grafts and blue signals indicate DAPI nuclear labeling. Yellow is a nonspecific overlap of signals, most obvious in the photoreceptor outer segments which exhibit autofluorescence. Treatment groups: untreated (UT), both eyes untreated; S/S, both eyes sham-treated (sequentially) with vehicle; S/C, right eye injected with vehicle followed by left eye with 50,000 gmRPCs and; C/C, both eyes injected with 50,000 gmRPCs (sequentially) per schedule in Section 4


Daim duab 2. Immuno-labeling ntawm infiltrated immune cells los ntawm pab pawg kho mob. Immunofluorescent dluab ntawm (A) tswj seem (thib ob antibody ib leeg), (B) anti-Iba-1 (activated macrophage thiab microglia marker), (C) anti-Ly-6G (neutrophil marker), (D) anti-CD49b (natural killer cell marker), (E) antibodies anti-CD3 (T cell marker), thiab (F) anti-CD45R (B cell marker) muaj nyob rau hauv daim duab. Cov cim liab txwv tam sim no qhia tias muaj cov tshuaj tiv thaiv kab mob zoo rau cov cim leucocyte, cov cim ntsuab qhia txog gmRPC grafts thiab xiav teeb liab qhia DAPI nuclear labeling. Daj yog ib qho kev sib tshooj ntawm cov teeb liab, pom tseeb tshaj plaws nyob rau hauv cov duab sab nrauv uas nthuav tawm autofluorescence. Cov pab pawg kho mob: tsis kho (UT), ob lub qhov muag tsis kho; S/S, ob lub qhov muag sham-kho (sequentially) nrog lub tsheb; S/C, sab xis qhov muag txhaj nrog lub tsheb ua raws li sab laug qhov muag nrog 50, 000 gmRPCs thiab; C/C, ob lub qhov muag txhaj tshuaj nrog 50,{16}} gmRPCs (ib ntus) ib lub sijhawm hauv Tshooj 4

Cov naj npawb ncov ntawm txhua lub cev tiv thaiv kab mob hauv lub qhov muag sib txawv los ntawm hom cell (Daim duab 3A–E). Hauv qhov sib piv, ob txoj kev ANOVA qhia tsis muaj qhov sib txawv ntawm kev tiv thaiv kab mob hauv lub cev thaum sib piv cov tsiaj uas tau txais ib qho kev txhaj tshuaj gmRPC (sham / cell) rau cov uas tau txais kev txhaj tshuaj ob sab (hlwb / cell). Qhov no yog cov ntaub ntawv rau infiltration ntawm macrophages (anti-Iba-1 staining) (Daim duab 3A), neutrophils (anti-Ly-6G staining) (Daim duab 3B), natural killer cells (CD49b staining) ( Daim duab 3C), T cells (CD3 staining) (Daim duab 3D), thiab B hlwb (CD45R staining) (Daim duab 3E).

2.3. ELISPOT Tsis Qhia Antigen Recall Responses

ELISPOT yog ib txoj hauv kev los soj ntsuam cov tshuaj tiv thaiv tshwj xeeb IFN ua rau T cell ua haujlwm. Phorbol 12-myristate 13-acetate (PMA) kev kho mob yog ib qho kev tswj xyuas zoo ua raws li tus neeg xa xov liaison thib ob, DAG, txhawm rau qhib T cell receptor txoj hauv kev thiab, dhau los, ua rau T cell ua haujlwm. Nyob rau hauv daim duab 4A-D, PMA-kho pab pawg tau pom ntau dua IFN qhov chaw suav piv rau cov pab pawg tswj nrog cov hlwb teb ib leeg (piv txwv li, lymphocytes los ntawm CLNs, splenocytes los ntawm spleens). Thaum cov hlwb teb tau sib koom ua ke nrog C57BL / 6 splenocytes (B6 SPL), hnub 14 cov qauv piv rau cov hlwb teb ib leeg, tsuas yog me ntsis siab dua IFN qhov chaw suav, qhia tias tsis muaj T cell ua haujlwm (Daim duab 4A, B).

Figure 3. Quantification of immune cell infiltration following gmRPC transplantation. Infiltrated immune cells were visualized as red fluorescent signals within the imaging field following gmRPC injection for each experimental condition, counted using ImageJ, and plotted in bar graphs. Data from Iba-1 (activated macrophage and microglia marker) (A), anti-Ly-6G (neutrophils marker) (B), anti-CD49b (natural killer cells marker) (C), antibodies anti-CD3 (T cells marker) (D), and anti CD45R (B cells marker) (E) were displayed in the figure. Two-way ANOVA tests were used to test significance between the sham/cell and cell/cell groups; p values were: (A) p < 0.4492, (B) p < 0.9376, (C) p < 0.1190, (D) p < 0.6411, and (E) p < 0.7201 (none were significant).


Daim duab 3. Quantification ntawm lub cev tiv thaiv kab mob infiltration tom qab hloov gmRPC. Infiltrated lub cev tiv thaiv kab mob tau pom tau tias yog liab fluorescent teeb liab nyob rau hauv daim duab teb tom qab gmRPC txhaj rau txhua qhov kev sim, suav siv ImageJ, thiab npaj rau hauv bar graphs. Cov ntaub ntawv los ntawm Iba-1 (activated macrophage thiab microglia marker) (A), anti-Ly-6G (neutrophils marker) (B), anti-CD49b (natural killer cells marker) (C), antibodies anti-CD3 (T cells marker) (D), thiab anti CD45R (B hlwb marker) (E) tau tshwm sim hauv daim duab. Ob txoj kev ANOVA xeem tau siv los ntsuas qhov tseem ceeb ntawm cov sham / cell thiab cell / cell pawg; p tus nqi yog: (A) p < {{10}}.4492, (B) p < 0.9376, (C) p < 0.119{{18 } }, (D) p < 0.6411, thiab (E) p < 0.7201 (tsis muaj qhov tseem ceeb).

Thaum kuaj allogenic pub hlwb siv qhov kev ntsuam xyuas no, cov hlwb teb los ntawm cov tsiaj intraperitoneally txhaj nrog gmRPCs (IP pawg) tsis muaj cov lus teb rov qab los ntawm antigen vim cov qauv tsis qhia ntau T cell ua kom los ntawm IFN qhov chaw suav piv rau cov tsiaj sham yeej tsis raug. rau gmRPCs (sham/sham). Tsis yog cov tsiaj uas tau txhaj tshuaj intravitreally nrog gmRPCs, ib zaug (sham / cell) lossis rov ua dua (cell / cell) qhia cov lus teb rov qab los ntawm antigen. Nyob rau hauv ib rooj plaub, cov lus teb rov ua dua gmRPC koob tshuaj tau tshwm sim ntau dua (Daim duab 4B). Ua ke, cov txiaj ntsig no tau qhia tias gmRPCs pom muaj antigenicity tsawg heev. Nco ntsoov tias C57BL / 6 splenocytes tau txuam nrog gmRPCs 'genetic keeb kwm yav dhau. Tsis xav txog, lwm cov stimulator hlwb, gmRPCs, nce IFN qhov chaw suav nyob rau hauv tag nrho cov kev sim pab pawg piv rau pawg tswj tsis zoo (Daim duab 4A–D). Qhov kev piav qhia muaj peev xwm yuav yog gmRPCs ua rau muaj keeb kwm yav dhau los hauv qhov kev ntsuam xyuas no. Qhov tseem ceeb, tsis muaj qhov sib txawv ntawm qhov tsis kho, sham-kho, ib qho kev txhaj tshuaj gmRPC, thiab rov ua cov pab pawg txhaj tshuaj gmRPC, uas rov qhia tias tsis muaj cov tshuaj tiv thaiv rov qab.

Figure 4. ELISPOT quantification following gmRPC transplantation. ELISPOT assays were set as the following groups: control, responder cells alone (lymphocytes or splenocytes containing T cells isolated from cervical lymph nodes (CLNs) or spleens (SPL) of the experimental animals; PMA, responder cells treated with phorbol 12-myristate 13-acetate (PMA) and Ca ionophore; B6 SPL, responder cells mixed with splenocytes isolated from C57BL/6 animals; gmRPCs, the same responder cells mixed with gmRPCs. (A), The responder cells were the splenocytes from the experimental animals that were sacrificed on day 14 after the second gmRPCs injection. (B) The responder cells were the lymphocytes from the CLNs of the experimental animals that were sacrificed on day 14 after the second gmRPCs injection. (C) The responder cells were the splenocytes from the experimental animals that were sacrificed on day 29 after the second gmRPCs injection. (D) The responder cells were the lymphocytes from the CLNs of the experimental animals that were sacrificed on day 29 after the second gmRPCs injection. Two-way ANOVA tests were used to test significance (*) between the sham/cell and cell/cell groups; p values were: (A) p < 0.5332, (B) p < 0.0001 (significant), (C) p < 0.0207 (significant), and (D) p < 0.3355.


Daim duab 4. ELISPOT kom muaj nuj nqis tom qab hloov gmRPC. ELISPOT cov kev ntsuam xyuas tau teeb tsa raws li pawg hauv qab no: kev tswj hwm, kev teb cov hlwb ib leeg (lymphocytes lossis splenocytes uas muaj T hlwb cais tawm ntawm lub ncauj tsev menyuam cov qog ntshav (CLNs) lossis spleens (SPL) ntawm cov tsiaj sim; PMA, cov hlwb teb kho nrog phorbol {{1} }myristate 13-acetate (PMA) thiab Ca ionophore; B6 SPL, cov hlwb teb sib xyaw nrog splenocytes cais los ntawm C57BL/6 tsiaj; gmRPCs, tib lub hlwb sib xyaw nrog gmRPCs. (A), Cov hlwb teb yog cov splenocytes los ntawm cov tsiaj sim uas tau txi rau hnub 14 tom qab kev txhaj tshuaj gmRPCs thib ob. (b) Cov hlwb teb yog cov lymphocytes los ntawm CLNs ntawm cov tsiaj sim uas tau txi rau hnub 14 tom qab txhaj tshuaj gmRPCs thib ob. (C) Cov hlwb teb. yog cov splenocytes los ntawm cov tsiaj sim uas tau txi rau hnub 29 tom qab txhaj koob thib ob gmRPCs. Ob txoj kev xeem ANOVA tau siv los ntsuas qhov tseem ceeb (*) ntawm pawg sham / cell thiab cell / cell; p tus nqi yog: (A) p < 0.5332, (B) p < 0.0001 (tseem ceeb), (C) p < 0.0207 ( tseem ceeb), thiab (D) p <0.3355.

3. Kev sib tham 

Muaj kev txaus siab rau cov qia cell thev naus laus zis raws li kev kho cov kab mob retinal degenerative thiab peb pab pawg tau tshawb nrhiav kev siv allogeneic hRPCs hauv RP. Txhawm rau sau cov ntaub ntawv hais txog qhov muaj peev xwm tiv thaiv kab mob tshwm sim thaum ntau tshaj ib koob tshuaj ntawm tib neeg RPC cov khoom siv los ntawm kev txhaj tshuaj intravitreal, peb tau ua cov kev tshawb fawb tsiaj tam sim no siv gmRPCs los ntawm C57BL / 6 keeb kwm yav dhau los ua grafts thiab BALB / c nas ua tswv. Cov keeb kwm ntawm caj ces tsis sib xws no tau siv los ua qauv kev kho mob ua ntu zus nrog allogeneic hRPCs thiab muab cov ntaub ntawv kev nyab xeeb ua ntej ua ntej kuaj hauv tib neeg.

Txhawm rau ntsuas cov lus teb tag nrho, cov tsiaj yug tsiaj tsis tau txais kev kho mob tiv thaiv kab mob. Peb piv cov tshuaj tiv thaiv kab mob uas tsim los ntawm ib qho kev txhaj tshuaj gmRPC rau kev txhaj tshuaj ob sab raws li lub sijhawm txhaj tshuaj tau piav qhia. Kev kuaj pom qhov muag tsis pom qhov mob lossis lwm qhov sib txawv tseem ceeb ntawm pawg tsiaj uas tau txais 1 koob tshuaj gmRPCs lossis 2 koob tshuaj gmRPCs, qhia tias cov neeg tau txais kev pab tsis hnov ​​​​tsw los ntawm kev kho gmRPC thawj zaug, lossis qhov kev xav tau hloov maj mam. Immunolabeling cov txiaj ntsig tau pom tias tag nrho tsib lub cev tiv thaiv kab mob tau tshuaj xyuas, xws li T cells (CD3), B hlwb (CD45R), macrophages (Iba-1), neutrophils (Ly-6G), thiab cov cell killer ntuj ( CD49b), tau nkag mus rau hauv lub qhov muag thiab tsom rau RPC grafts tom qab hloov pauv. Txawm li cas los xij, qhov infiltration no kuj yog nruab nrab thiab, txawm hais tias lwm hom ntawm tes tau kuaj pom, nws tau muaj feem ntau ntawm Iba-1 zoo macrophages. Muaj tsawg tsawg ntawm T hlwb, B hlwb, neutrophils, thiab cov cell killer ntuj. Interestingly, kev ciaj sia tau pom los ntawm ib leeg thiab rov ua RPC grafts yog sib npaug, tsis muaj kev poob ntawm grafts nyob rau hauv ob qho tib si, txawm tias muaj cov macrophages. Zuag qhia tag nrho, qhov no qhia tias ob qho tib si hauv lub cev thiab hloov lub cev tiv thaiv kab mob rau cov kab mob sib txawv ntawm RPCs yog nruab nrab, txawm tias kev tiv thaiv kab mob tsis ua haujlwm. Tsis tas li ntawd, ELISPOT qhov kev soj ntsuam tau pom tias tsis muaj cov lus teb rov qab los ntawm cov tshuaj tiv thaiv kab mob hauv gmRPC-kho, raws li kev sib cav hais tias T hlwb tsis ua lub luag haujlwm tseem ceeb hauv kev tsis lees paub ntawm gmRPC grafts los ntawm C57BL / 6 nas. Ib zaug ntxiv, macrophage infiltration ntawm gmRPC grafts tau pom, txawm hais tias qhov no tsis cuam tshuam nrog kev puas tsuaj loj ntawm hom cuam tshuam nrog kev tiv thaiv kev tiv thaiv classical [22-24].

Desert ginseng-Improve immunity (21)

cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob

Ua ke, cov kev soj ntsuam no qhia txog qhov xwm txheej uas txawv ntawm qhov pom dav dav T thiab B cell-mediated allogeneic hloov pauv cov txheej txheem pom hauv lwm cov ntaub so ntswg. Ntau yam tuaj yeem ua rau muaj qhov xwm txheej no. Ntawm qhov tod tes, muaj qhov kev xav ntawm lub qhov muag raws li "kev tiv thaiv tsis muaj cai" qhov chaw, txawm tias qhov twg cov vitreous qhia cov yam ntxwv zoo li no nrog lub cornea thiab retina tsis tau kawm thiab tej zaum muaj kev sib cav ntau. Ntawm qhov tod tes, RPCs lawv tus kheej tshwm sim kom txo qis kev tiv thaiv kab mob raws li allografts, piv rau ntau zaus kawm hom cell. Raws li peb tau pom yav dhau los, murine RPCs tsis muaj cov ntsiab lus qhia ntawm MHC chav kawm I thiab chav kawm II antigens, txawm hais tias cov no yog inducible ntawm cytokine stimulation. Qhov tsis muaj MHC qhia no tuaj yeem ua rau muaj sia nyob ntawm cov hlwb tom qab hloov pauv. Txawm li cas los xij, lub xub ntiag ntawm lub cev tiv thaiv kab mob hauv cov kab mob grafts txhawb lub tswv yim tias kev ua siab ntev rau hauv qhov chaw no tsis yog passive thiab tshwm sim los ntawm ib qho active immunoregulatory phenomenon. Ntawm no, nws yuav tsum tau hais qhia tias tib yam kev tiv thaiv kab mob hauv lub cev tau tshwm sim tom qab kev txhaj tshuaj ib zaug thiab tsis ua kom hnyav dua los ntawm kev rov ua dua, yog li qhia peb qhov kev txiav txim siab tias kev tiv thaiv kab mob hauv lub cev ntawm cov grafts, tshwj xeeb yog los ntawm macrophages, tsis yog qhov tshwm sim ua ntej. host rhiab heev. Nyob rau hauv qhov tsis sib xws ntawm qhov kev sim no, kev siv tshuaj rov ua dua tsis tshwm sim kom txo qis tag nrho cov graft viability, uas yog raws li qhov ua tau kom muaj txiaj ntsig zoo hauv cov ntsiab lus kho. Lwm qhov tsis sib xws, xws li qhov sib txawv ntawm kev txhaj tshuaj, tuaj yeem nce lossis txo qis kev tiv thaiv kab mob hauv lub cev mus rau qhov uas yuav cuam tshuam rau kev siv tshuaj kho mob. Txawm hais tias cov cellular constituents ntawm infiltrates, suav nrog macrophage predominance, kuj tseem siv rau tib neeg tsis paub meej. Tsis tas li ntawd, kev ciaj sia ntawm cov allografts no yuav tsis yog nyob ntawm lub retina ib txwm muaj kev noj qab haus huv ntawm cov ntshav-retinal barrier. Cov neeg tau txais BALB/c siv ntawm no yog albinos thiab raug rau lub teeb puas [30]. Tsis tas li ntawd, kev muaj sia nyob tau raug pom ntau zaus hauv kev ua haujlwm dhau los nrog ntau yam qauv ntawm retinal degeneration (piv txwv li, [31]).

Piv nrog rau graft ciaj sia taus, txoj kev vam meej ntawm cov hlwb hauv grafts nthuav tawm qhov teeb meem nyuaj dua. Muaj qhov paub tias poob ntawm CNS progenitor hlwb uas tshwm sim sai sai tom qab hloov pauv, yuav yog vim muaj ntau yam uas muaj feem cuam tshuam nrog kev loj hlob sai sai. Thaum nyob rau hauv lub qhov muag, cov hlwb dissociated coalesce mus rau hauv sphere-zoo li aggregates, cov chaw uas zoo nkaus li muab ib tug suboptimal microenvironment rau kev loj hlob, tej zaum vim tsis muaj vascularization thiab cov teeb meem rau nutrient diffusion. Qhov pom tseeb tropism ntawm macrophages rau RPC grafts tuaj yeem yog cov lus teb tsis tseem ceeb rau qhov muaj cov hlwb tsis zoo nyob rau sab hauv ntawm cov grafts, nrog rau cov macrophages ua haujlwm hauv lawv lub luag haujlwm ua cov khib nyiab ntawm cov khib nyiab ntawm tes. Xwb, RPCs uas muaj peev xwm ua tau zoo tuaj yeem nyiam cov macrophages, piv txwv li, los ntawm kev qhia ntawm chemo-zoo nkauj cytokines, whereupon lub tswv tsev cell yuav ob peb ntsib cov khib nyiab los ntawm cov tsis muaj peev xwm pub hlwb. Qhov tseem ceeb, qib ntawm macrophage infiltration pom nyob rau hauv cov chaw tshwj xeeb no tsis tshwm sim kom muaj qhov tsis zoo rau kev cog qoob loo lossis tus tswv tsev retina, tsis zoo li cov lus teb rau cov cellular allografts hauv cov chaw tsis muaj zog tiv thaiv kab mob [4]. Thaum kawg, nws tsim nyog hais tias cov txiaj ntsig tau tshaj tawm ntawm no, txawm tias txwv rau tus qauv murine, yuav muaj feem cuam tshuam rau kev ua haujlwm ntawm tib neeg. Nyob rau hauv sib piv rau tus nas, peb paub hais tias kab lis kev cai ntawm tib neeg RPCs qhia txog qib robust ntawm MHC chav kawm I antigens ntawm lub hauv paus; yog li ntawd, qhov kev sib piv yog lees paub tentative. Txawm li cas los xij, nws yog qhov nthuav kom nco ntsoov tias kev kuaj mob ntawm subretinal hRPC grafts los ntawm ib pab pawg hauv Suav teb [13], nrog rau kev hloov pauv ntawm intravitreal hRPC los ntawm peb pab pawg, tau qhia tom qab pom tias muaj sia nyob ntawm cov allografts hauv cov neeg mob uas tsis muaj kev tiv thaiv kab mob nrog RP (JC. -01, [32], cov ntaub ntawv tsis tau tshaj tawm). Qhov no kuj yog qhov tshwm sim tom qab txhaj tshuaj ntawm ob lub qhov muag (JC-01 Extension, cov ntaub ntawv tsis tau tshaj tawm), piv rau cov haujlwm uas tau nthuav tawm ntawm no. Tsis tas li ntawd, kev noj qab haus huv muaj sia nyob tau pom nyob rau hauv kev tshawb nrhiav tom qab ntawm JC-02 [33], nyob rau hauv uas cov koob tshuaj ua ntu zus tau muab rau tib lub qhov muag [34]. Ua ke, cov kev tshawb pom no qhia tau hais tias kev tiv thaiv kev tiv thaiv tsis yog ib txwm tsim nyog nyob rau hauv kev teeb tsa ntawm neural progenitor transplantation, tshwj xeeb tshaj yog rau lub qhov muag, txawm hais tias qhov kev txwv ntawm qhov tshwm sim thiab cov txheej txheem tswj hwm hauv qab tseem yuav tsum tau piav qhia.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Cell Culture

Yav dhau los tus cwj pwm gmRPCs [31] tau raug xaiv los ua cov hlwb pub rau qhov kev tshawb fawb allogeneic. Keeb kwm, gmRPCs raug cais los ntawm GFP-transgenic C57BL / 6 nas hloov caj ces los qhia txog kev txhim kho ntsuab fluorescent protein (GFP). Qhov kev qhia ntawm GFP protein nyob rau hauv gmRPCs yog qhov txaus siab nyob rau hauv uas nws tso cai rau pom kev ntawm grafts tom qab txhaj tshuaj yam tsis tas yuav tsum tau ntxiv daim ntawv lo. Rau txoj kev tshawb fawb tam sim no, gmRPCs tau coj mus kuaj hauv Advanced DMEM/F12 ntxiv nrog N-2 Supplement (1:100, Life Technology, Carlsbad, CA, USA), Glutamax-1 (1:100, Life Technology , Carlsbad, CA, USA), thiab EGF (20 ng/mL, recombinant, Human, Life Technologies, Carlsbad, CA, USA) thiab incubated ntawm 37 ◦C thiab 5% CO2. Cov hlwb tau khaws cia rau hauv cov khoom sib xyaw ua ke / xoob txuas nrog cov kab mob hauv cov ntaub so ntswg uas tsis muaj ntaub so ntswg. Cov hlwb tau dhau los ntawm trypsinization nrog TrypLE Xaiv CTS (Lub Neej Technologies, Carlsbad, CA, USA) diluted 1: 5 hauv PBS rau ib feeb thiab nruab nrab los ntawm 10 npaug ntawm qhov ntim ntawm trypsin ntxiv. Lub cell-trypsin mix yog centrifuged ntawm 140 g rau 2 min nyob rau hauv chav tsev kub, lub supernatant tshem tawm, thiab lub cell pellet resuspended nyob rau hauv tshiab xov xwm ua ntej noob mus rau cov ntaub so ntswg kab lis kev cai flasks ntawm qhov xav tau concentration. Cell concentration thiab viability raug txiav txim los ntawm trypan xiav staining; Kev suav tau ua los ntawm Countess (Life Technologies, Carlsbad, CA, USA) thiab hemocytometer.

Rau kev txhaj tshuaj intravitreal, lub cell pellet tau rov ua dua ntawm 50K hlwb / µL hauv BSS PLUS®. Cell concentration thiab viability raug soj ntsuam ua ntej thiab tom qab txhaj tshuaj. Cov koob txhaj tshuaj yog 50,000 lub hlwb ib qhov muag rau ib qho tshuaj. Cov koob tshuaj thiab lub tsheb raug xaiv raws li peb cov kev tshawb fawb yav dhau los.

4.2. Kev sim tsiaj

Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog txhawm rau tshawb xyuas qhov muaj peev xwm tiv thaiv kab mob tiv thaiv kab mob uas tshwm sim los ntawm kev txhaj tshuaj intravitreal ntawm allogeneic RPCs. Cov tsiaj uas tau txais yog BALB/c nas, uas txawv ntawm caj ces los ntawm C57BL/6 nas uas gmRPCs raug cais los ntawm. Kev siv yam tsawg kawg peb tus tsiaj rau ib pab neeg sim kho rau txhua lub sij hawm taw tes tau pom tias yuav tsum tau tso cai rau kev soj ntsuam ntawm qhov tseem ceeb ntawm cov pab pawg nrog rau cov txiaj ntsig ntawm cov txiaj ntsig thiab cov tsiaj poob thaum lub sij hawm sim. Tsis tas li ntawd, muab ntau yam ntxiv uas muaj feem cuam tshuam rau qhov kev sim tau tshwm sim, xws li cov txheej txheem kev txhaj tshuaj ntawm tes tsis ua tiav lossis muaj peev xwm attrition los ntawm kev sib ntaus sib tua tsiaj, cov txheej txheem txhaj tshuaj tau ua rau tsib tus tsiaj hauv ib pab pawg ntxiv kom ua tau raws li qhov tsawg kawg nkaus uas yuav tsum muaj rau kev txheeb xyuas kev txheeb xyuas.

Cistanche deserticola-improve immunity

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

Table 1 qhia txog plaub pawg tsiaj uas npaj rau txhua qhov ntawm plaub lub sijhawm (4, 7, 14, thiab 28 hnub) ntawm qhov kev ntsuam xyuas ntawm histopathology, suav nrog immunofluorescence, yuav ua. Ob pawg neeg ntxiv ntawm cov tsiaj txhaj tshuaj nrog 10ˆ6 gmRPCs intraperitoneally tau npaj raws li kev tswj tau zoo rau txhua qhov ntawm ob lub sijhawm ntsuas ELISPOT (14 thiab 28 hnub). Table 2 qhia txog cov kev ntsuam xyuas tau teem tseg rau txhua pab pawg.

Table 1. Cov pab pawg sim.

Table 1. Experimental groups.

Table 2. Lub sij hawm ntsuam xyuas cov ntsiab lus.

Table 2. Evaluation time points.


4.3. Kev txhaj tshuaj Intravitreal

Cov gmRPCs tau siv los ntawm kev txhaj tshuaj intravitreal. Ib zaug tshuaj loog, Mydriacyl (1% Tropicamide ophthalmic tov) thiab Phenylephrine (2.5% tshuaj ophthalmic) tau siv rau ob lub qhov muag ntawm tus tsiaj kom txhaj. Thaum ua tiav cov mydriasis txaus lawm, tus tsiaj tau tswj xyuas manually thiab tus tsiaj lub taub hau tig los ua kom lub qhov muag axis ntawm lub qhov muag yuav tsum tau txhaj nrog optics ntawm qhov phais microscope thiaj li pom cov ocular posterior ntu (ie, vitreous thiab retina) . Lub qhov muag tau maj mam muab tso rau ntawm lub siab ntawm daim tawv muag thiab ib rab koob 31G ntawm lub koob txhaj tshuaj insulin tau siv los maj mam tho lub sclera nyob ib sab ntawm lub limbus hauv qhov ntswg qhov ntswg. Lub iav polished micropipette taub uas muaj cov hlwb pub dawb (lossis kev tswj lub tsheb) tau dhau los ntawm kev txiav mus rau hauv cov kab noj hniav nyob rau hauv kev pom ncaj qha. Kev saib xyuas tsis txhob cuam tshuam qhov kev ncaj ncees ntawm lub lens lossis cov qauv ntu tom qab. Cov hlwb lossis lub tsheb ib leeg tau txhaj rau hauv qhov ntim ntawm 1 microliter. Tom qab ob peb lub sijhawm nres kom tso cai rau qhov sib npaug ntawm qhov siab ntawm lub cev, lub taub hau pipette tau maj mam tshem tawm ntawm lub qhov muag, tag nrho hauv kev pom ncaj qha. Txhua yam ntshav intraocular tau sau tseg, nrog rau qhov chaw. Tom qab txhaj tshuaj, tus tsiaj tau muab tso rau hauv ib lub tawb huv si lined nrog cov ntaub ntawv pov tseg tshiab taw qhia nrog absorbent-sab-up / yas-sab-down, kom rov zoo. Kev rov qab tau yooj yim siv lub tshuab cua sov thiab tau txheeb xyuas los ntawm tus tsiaj muaj peev xwm ua kom muaj zog. Tom qab waking, tus tsiaj raug xa mus rau tom qab kev ua haujlwm tawb nrog ad libitum nkag mus rau hauv dej. Tsiaj txhu tau pom kev saib xyuas txhua hnub tom qab ua haujlwm. Tsawg lossis tsis muaj cov cim qhia txog kev txhaj tshuaj intraocular tau pom.

Tom qab cov txheej txheem, cov tsiaj raug soj ntsuam rau rubbing ntawm lub qhov muag ua haujlwm, ruffled pluab, los yog muaj zog lethargy, tag nrho cov no yog lub hauv paus rau euthanasia tam sim ntawd. Muaj ob tug tsiaj raug tshem tawm raws li qhov tshwm sim ntawm kev raug mob ntawm kev sib ntaus sib tua. Tag nrho lwm cov tsiaj tau raug txiav tawm ntawm qhov kawg ntawm qhov kawg. Euthanasia tau ua los ntawm CO2 inhalation.

4.4. Ophthalmic Examination

A Leica Ultimate Red Reflex Surgical Microscope was used for ophthalmic examination and photography of experimental mice. Sedation was performed with a Ketamine Hydrochloride/Xylazine Hydrochloride mixture (50–100 mg/mL Ketamine, 5–10 mg/mL Xylazine) administered by intraperitoneal injection. After sedation, topical mydriatics (Tropicamide, Phenylephrine) were applied to the eye(s) to be imaged in 5 min intervals until adequate mydriasis was achieved, as determined via pupil diameter (>2.5 mm) thiab teb rau lub teeb stimulation. Raws li kev them nyiaj rau qhov poob ntawm blinking, ib qho tshuaj hypromellose (Gonak) tau siv tshuaj pleev ib ce raws li xav tau rau ob lub qhov muag los tiv thaiv pob txha desiccation. Rau cov txheej txheem imaging, anesthetized tsiaj tau muab tso rau ntawm lub tshav kub pad thiab positioned nyob rau hauv sternal recumbency. Thaum xaus ntawm cov txheej txheem, ib nrab thim rov qab ntawm cov tshuaj loog tau ua tiav nrog kev tswj hwm ntawm Atipamezole (0.1-1 mg / kg) los ntawm kev txhaj tshuaj intraperitoneal.

4.5. Histopathology

Kev tiv thaiv kab mob hauv cov tsiaj uas tau txais ob qhov kev sib txuas ua ke (ib qho ntawm txhua lub qhov muag) tau soj ntsuam los ntawm histopathology ntawm ob lub qhov muag ntawm hnub 4, hnub 7, hnub 14, thiab hnub 28 ± 1 tom qab txhaj tshuaj thib ob. Cov nas uas tau kho raws li Table 1 tau raug txiav tawm raws li lub sijhawm teev tseg hauv Table 2. Tsiaj txhu tau euthanized los ntawm kev nqus pa carbon dioxide. Cardiac perfusions tau ua rau cov tsiaj sim nrog 2% paraformaldehyde (PFA) hauv phosphate-buffered saline (PBS). Tom qab ntawd, tag nrho cov pob muag raug sau thiab tso cai rau soak hauv 2% PFA / PBS rau 48 teev ntawm 4 ◦C. Cov pob muag ruaj khov tau ua tiav los ntawm sucrose gradient (10% sucrose hauv PBS rau 1 teev ntawm chav tsev kub, 20% sucrose hauv PBS rau 1 h ntawm chav sov, thiab 30% sucrose hauv PBS ntawm 4 ◦C thaum hmo ntuj) ua ntej muab tso rau hauv OCT media rau cryosectioning. Cryosections (10 µm) ntawm ob lub qhov muag tau stained nrog Harrison hematoxylin thiab eosin (H&E) kom pom cov retinal microanatomy thiab nrhiav cov kab mob pub dawb.

Rau cov pab pawg gmRPC-kho, cryosections raug soj ntsuam siv immunofluorescence rau GFP + (tus pub) hlwb. Kev txheeb xyuas cov kab mob tshwj xeeb hauv lub qhov muag tau ua los ntawm kev sau npe nrog cov tshuaj tiv thaiv tshwj xeeb rau cov hauv qab no: CD3 (T lymphocyte marker) [35], CD45R (B lymphocyte marker) [36], Iba-1 (activated macrophage thiab microglia marker) [37], Ly-6G (neutrophil marker) [38], thiab CD49b (natural killer cell marker) [39]. Cryosections raug ntxuav hauv PBS peb zaug ntawm chav tsev kub thiab thaiv nrog 0.03% Triton X-100 thiab 10% tshis cov ntshav hauv PBS (NGS; Jackson ImmunoResearch, West Grove, PA, USA) rau 1 h ntawm chav tsev kub. Rat anti-nas CD3 (1:100 dilution, BD Biosciences, San Jose, CA, USA), nas anti-nas CD45R (1:100, BD Biosciences, San Jose, CA, USA), luav anti-nas Iba{{ 26}} (1:400 dilution, Wako Chemicals, Richmond, VA, USA), nas anti-nas Ly-6G (1:100 dilution, BD Biosciences, San Jose, CA, USA), thiab nas anti -mouse CD49b (1:100 dilution, BD Biosciences, San Jose, CA, USA) tau siv rau cov qauv thiab incubated hmo ntuj ntawm 4 degrees. Cov qauv raug ntxuav dua hauv PBS 3 zaug ntawm chav tsev kub. Alexa-Fluor-conjugated secondary antibodies (tshis anti-rat Alexa-Flour-568 thiab tshis los tiv thaiv luav Alexa-Flour-568, Life Technologies, Carlsbad, CA, USA) tau siv rau cov qauv rau 1 h ntawm chav tsev kub. Tag nrho cov qauv raug ntxuav hauv PBS lwm peb zaug ntawm chav tsev kub. Cov ntawv npog tau muab tso rau siv DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, USA), thiab cov slides raug tso kom qhuav thaum hmo ntuj ntawm chav tsev kub.

4.6. ELISPOT thiab MLR Kev Tshawb Fawb

RPC-cov cim xeeb tshwj xeeb T hlwb raug saib xyuas ntawm 2 thiab 4 lub lis piam tom qab thawj qhov kev tso tawm ntawm cov enzyme-linked immunosorbent chaw (ELISPOT) kev soj ntsuam. Cov pab pawg sim tau teeb tsa raws li qhia hauv Cov Lus 1 thiab 2 rau txhua lub sijhawm taw tes. ELISPOT assay captures secreted proteins on a specific antibody-coated microplate and can be used to txiav txim siab T cell activation los ntawm kev kuaj T cell IFN secretion. Qhov kev sim nyeem ntawv yog tus naj npawb ntawm IFN qhov chaw ntawm lub microplate. Tus naj npawb ntawm IFN qhov chaw tam sim no yog qhia txog tus naj npawb ntawm T hlwb tau qhib. Qhov zaus ntawm alloreactive T hlwb raug soj ntsuam los ntawm kev ua 48 h sib xyaw leukocyte cov tshuaj tiv thaiv (MLR) ntawm 96-zoo Multiscreen-IP daim hlau (Millipore, Burlington, MA, USA). Cov hlwb teb yog cov lymphocytes / splenocytes uas muaj T hlwb purified los ntawm lub ncauj tsev menyuam qog (CLNs) thiab spleens ntawm cov tsiaj sim, thiab stimulator hlwb yog cov gmRPCs Ameslikas muab los ntawm C57BL/6 nas. Qhov kev ntsuam xyuas ELISPOT tau ua raws li tus qauv txheej txheem. 96-zoo Multiscreen-IP daim hlau tau pre-wetted nrog 15 µL ntawm 35% ethanol ib qhov dej nyob rau hauv tsis muaj menyuam. Cov phaj raug ntxuav nrog tsis muaj menyuam PBS peb zaug ua ntej 100 µL ntawm IFN ntes cov tshuaj tiv thaiv (clone AN-18, eBioscience, San Diego, CA, USA) diluted hauv PBS (2 µg / mL) tau siv rau cov phiaj. Cov phiajcim tau ua rau hmo ntuj ntawm 4 degrees ua ntej MLR kev soj ntsuam tau nqa ntawm daim phiaj. Cov ntawv ntes cov tshuaj tiv thaiv-coated tau ntxuav peb zaug nrog PBS thiab tom qab ntawd thaiv nrog RPMI1640 rau 2 teev ntawm 37 ◦C.

Txhua daim ntawv tshuaj ntsuam xyuas suav nrog cov kev tswj hauv qab no: qhov dej uas tsis muaj hlwb, qhov dej uas muaj cov hlwb tsis muaj kev txhawb nqa, thiab cov dej uas muaj cov hlwb nrog kev kho mob ntawm phorbol {{0}}myristate 13-acetate (PMA) thiab Ca ionophore raws li kev tswj zoo. PMA kev kho mob ua haujlwm zoo los ntawm kev ua raws li tus xa xov thib ob, DAG, txhawm rau qhib T cell receptor txoj hauv kev thiab ua rau T cell activation, nrog Ca ionophore pab txhawb kev nkag ntawm calcium ions rau hauv hlwb. Cov phaj tau incubated rau 36 h ntawm 37 ◦C ua ntej cov tshuaj tiv thaiv xim. Cov phaj raug ntxuav nrog PBS uas muaj {{10}}}01% Tween 20 rau lub sij hawm thiab tom qab ntawd 100 µL nrhiav tshuaj tiv thaiv kab mob (clone R64A2, eBioscience, San Diego, CA, USA), diluted Hauv PBS (0.5 µg / mL) tau siv rau txhua qhov dej. Cov phiajcim tau muab tso rau ntawm 37 ◦C rau 2 teev ntxiv. Cov phaj tau rov qab ntxuav nrog 0.01% Tween 20 hauv PBS. 100 µL ntawm Streptavidin-AP (1: 1000 dilution, Invitrogen) rau ib lub qhov dej tau siv thiab cov phaj tau incubated rau 45 min ntawm chav tsev kub. Txhua daim hlau raug ntxuav peb zaug dua nrog 0.01% Tween 20 hauv PBS thiab PBS ib leeg rau lwm peb zaug. Thaum kawg, 100 µL BCIP / NBT (Sigma Aldrich, Munich, Lub teb chaws Yelemees) tau ntxiv rau txhua qhov dej kom muaj xim. Tag nrho cov phaj tau muab ntxuav nrog dej ntau thiab qhuav ua ntej kev soj ntsuam cov ntaub ntawv.

Cistanche deserticola-improve immunity -

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

5. Cov lus xaus

Txoj kev tshawb no qhia tau hais tias kev sib txuas ntawm binocular grafts ntawm allogeneic RPCs rau cov kab noj hniav vitreous tsis provoke classical tiv thaiv kab mob hauv nas. Thaum nws tau lees paub tias cov kev tshawb fawb allogeneic histological tsis tuaj yeem ua nrog tib neeg RPC cov khoom siv kho mob, cov ntaub ntawv tshwm sim raws li cov txiaj ntsig ntawm kev soj ntsuam kev nyab xeeb (jCyte, JC-01E, cov ntaub ntawv tsis tau tshaj tawm) uas cov neeg mob retinitis pigmentosa tau txais kev txhaj tshuaj tsis tu ncua ntawm ob sab yam tsis muaj kev tiv thaiv kab mob. Txawm hais tias nws nyuaj rau kev sib piv ob txoj kev tshawb fawb ncaj qha, nws yog qhov tsim nyog tias tag nrho cov txiaj ntsig ntawm ob qho tib si pom zoo sib xws hauv cov ntsiab lus ntawm allograft ciaj sia taus.

Cov ntaub ntawv

1. Hori, J.; Ng, TF; Shatos, M.; Klassen, H.; Streilein, JW; Hluas, MJ Neural progenitor hlwb tsis muaj kev tiv thaiv kab mob thiab tiv thaiv kev puas tsuaj li allografts. Stem Cells 2003, 21, 405–416. [CrossRef] [PubMed]

2. Klassen, H.; Schwartz, PH; Zias, B.; Nethercott, H.; Young, MJ; Bragadottir, R. Tullis, GE; Warfvinge, K.; Narfstrom, K. Neural precursors cais los ntawm kev tsim cov miv lub hlwb qhia txog kev sib koom ua ke ntawm retinal tom qab hloov mus rau retina ntawm dystrophic miv. Vet. Ophthalmol. 2007, 10, 245–253. [CrossRef] [PubMed]

3. Salado-Manzano, C.; Perpina, UA; Straccia, M. Molina-Ruiz, FJ; Koj, E.; Rosser, AW; Canals, JM Puas yog Kev Tiv Thaiv Kev Tiv Thaiv Kev Tiv Thaiv Ib Lub Cev rau Kev Kho Mob Cell hauv Cov Kab Mob Neurodegenerative? Pem hauv ntej. Cell Neurosci. 2020, 14, 250. [CrossRef]

4. Petrus-Reurer, S.; Romano, M.; Howlett, S.; Jones, JL; Lombardi, G.; Saeb-Parsy, K. Immunological kev txiav txim siab thiab cov teeb meem rau kev kho cov cellular regenerative. Pawg. Biol. 2021, 4, 798. [CrossRef]

5. Bucher, K.; Rodriguez-Bocanegra, E.; Dauletbekov, D.; Fischer, MD Immune teb rau retinal gene therapy siv ade tsis cuam tshuam tus kab mob vectors-Kev cuam tshuam rau kev kho mob kom zoo thiab kev nyab xeeb. Prog. Retin. Qhov muag Res. 2021, 83, 100915. [CrossRef]

6. Singh, MS; Park, SS; Albini, TA; Canto-Soler, MV; Klassen, H.; MacLaren, RE; Takahashi, M. Neeb, A.; Schwartz, SD; Bharti, K. Retinal qia cell transplantation: Ntsuas kev nyab xeeb thiab muaj peev xwm. Prog. Retin. Qhov muag Res. 2020, 75, 100779. [CrossRef]

7. Kamao, H.; Mandai, M.; Okamoto, S.; Sakai, N.; Suga, A.; Sugita, S.; Kiryu, J.; Takahashi, M. Tus cwj pwm ntawm tib neeg induced pluripotent qia cell-derived retinal pigment epithelium cell nplooj ntawv aiming rau kev kho mob daim ntawv thov. Stem Cell Rep. 2014, 2, 205–218. [CrossRef]

8. Sugita, S.; Kamao, H.; Iwasaki, Y.; Okamoto, S.; Hashiguchi, T.; Ib, K.; Hayashi, N.; Mandai, M.; Takahashi, M. Inhibition ntawm T-cell activation los ntawm retinal pigment epithelial hlwb muab tau los ntawm induced pluripotent qia hlwb. Tshawb xyuas. Ophthalmol. Vis. Sci. 2015, 56, 1051–1062. [CrossRef]

9. McGill, TJ; Stoddard, J.; ib. Renner, LWM; Mesaoudi, I.; Bharti, K.; Mitalipov, S.; Lauer, A.; Wilson, DJ; Neuringer, M. Allogeneic iPSC-Derived RPE Cell Graft Failure Tom qab hloov pauv mus rau qhov chaw subretinal hauv Nonhuman Primates. Tshawb xyuas. Ophthalmol. Vis. Sci. Xyoo 2018, 59, 1374–1383. [CrossRef]

10. Sugita, S.; Mandai, M.; Kamao, H.; Takahashi, M. Immunological yam ntawm RPE cell transplantation. Prog. Retin. Qhov muag Res. 2021, 84, 100950. [CrossRef]

11. Nair, DSR; Thomas, BB Stem Cell-based Treatment Strategies for Degenerative Diseases of Retina. Curr. Stem Cell Res. Ther. 2022, 17, 214–225. [CrossRef] [PubMed]

12. Martinez Velazquez, LA; Ballios, BG The Next Generation of Molecular and Cellular Therapeutics for Inherited Retinal Disease. Int. J. Mol. Sci. 2021, 22, 11542. [CrossRef]

13. Liu, Y.; Chen, SJ; Li, SY; Qu, LH; Meng, XH; Wang, Y.; Xu, HW; Liang, ZQ; Yin, ZQ Kev nyab xeeb ntev ntawm tib neeg retinal progenitor cell transplantation hauv cov neeg mob retinitis pigmentosa. Stem Cell Res. Ther. 2017, 8, 209. [CrossRef] [PubMed]

14. Wang, Y.; Tang, Z.; Gu, P. Stem/progenitor cell-based transplantation for retinal degeneration: Kev tshuaj xyuas cov kev sim tshuaj. Cell Tuag Dis. 2020, 11, 793. [CrossRef] [PubMed]

15. German, OL; Vallese-Maurizi, H.; Soto, TB; Rotstein, NP; Politi, LE Retina qia hlwb, kev cia siab thiab teeb meem. World J. Stem Cells 2021, 13, 1446–1479. [CrossRef]

16. Karamali, F.; Behtaj, S.; Babaei-Abraki, S.; Hadady, H.; Atefi, A.; Savoj, S.; Soroushzadeh, S.; Najafian, S.; Nasr Esfahani, MH; Klassen, H. Muaj peev xwm kho cov tswv yim rau photoreceptor degeneration: Txoj kev los kho qhov muag. J. Transl. Med. 2022, 20, 572. [CrossRef]

17. Seo, M.; Haamedi, N.; Stevanato, L. Carter, D.; Brooke, G.; Young, M.; Coob, P.; Sinden, J.; Patel, S.; Vugler, A. Kev ua tau zoo thiab kev nyab xeeb ntawm tib neeg Retinal Progenitor Cells. Txhais lus. Vis. Sci. Tshuab. 2016, 5, 6. [CrossRef]

18. Mochizuki, M.; Sugita, S.; Kamoi, K. Immunological homeostasis ntawm lub qhov muag. Prog. Retin. Qhov muag Res. Xyoo 2013, 33, 10–27. [CrossRef]

19. Yamasaki, S.; Sugita, S.; Horiuchi, M.; Masuda, T.; Fujii, S.; Mas, K.; Kawasaki, UA; Hayashi, T.; Kev, A.; Kisino, A.; ua al. Tsawg Immunogenicity thiab Immunosuppressive Properties ntawm Tib Neeg ESC- thiab iPSC-Drived Retinas. Stem Cell Rep. 2021, 16, 851–867. [CrossRef]

20. Klassen, H.; Schwartz, MR. Bailey, AW; Cov tub ntxhais hluas, MJ Surface cov cim qhia los ntawm ntau tus tib neeg thiab nas neural progenitor hlwb suav nrog tetraspanins thiab tsis muaj protein ntau epitopes. Neurosci. Lett. 2001, 312, 180–182. [CrossRef]

21. Klassen, H.; Zias, B.; Kirov, II; Young, MJ; Schwartz, PH Kev rho tawm ntawm retinal progenitor hlwb los ntawm post-mortem tib neeg cov ntaub so ntswg thiab kev sib piv nrog autologous hlwb progenitors. J. Neeb. Res. 2004, 77, 334–343. [CrossRef]

22. Ingulli, E. Mechanism of cellular rejection in transplantation. Pediatr. Nephrol. 2010, 25, 61–74. [CrossRef]

23. Ib., F.; Schiopu, UA; Ntoo, KJ ​​Lub luag haujlwm ntawm T hlwb hauv kev tsis lees txais graft thiab hloov kev kam rau siab. Kws txawj Rev. Clin. Immunol. 2010, 6, 155–169. [CrossRef] [PubMed]

24. Nasr, M.; Sigdel, T.; Sarwal, M. Advances in diagnostics for transplant rejection. Kws txawj Rev. Mol. Diagnos. 2016, 16, 1121–1132. [CrossRef] [PubMed]

25. Klassen, H.; Imfeld, KL; Rau, J.; Young, MJ; Gage, FH; Berman, MA Cov cuab yeej tiv thaiv kab mob ntawm cov neeg laus hippocampal progenitor hlwb. Vis. Res. 2003, 43, 947–956. [CrossRef] [PubMed]

26. Weinger, JG; Weist, BM; Plaisted, WC; Klaus, SM; Walsh, CM; Txoj kab, TE MHC kev tsis sib haum xeeb ua rau muaj kev tsis lees paub ntawm neural progenitor cell rejection tom qab hloov qaum qaum hauv tus qauv ntawm tus kab mob kis tau los ntawm demyelination. Stem Cells 2012, 30, 2584–2595. [CrossRef] [PubMed]

27. ReNeuron, L. First-in-Human Phase I/IIa, Open-Label, Prospective Study of the Safety and Tolerability of Subretinally Transplanted Human Retinal Progenitor Cells (hRPC) in Patients with Retinitis Pigmentosa (RP). Clinical Trial # NCT02464436. Muaj nyob online: https://clinicaltrials.gov/ct2/show/NCT02464436 ( nkag mus rau 27 Lub Ob Hlis 2023).

28. Lu, B.; Lin, Y.; Tsai, Y.; Girman, S.; Adas, G.; Jones, MK. Shelley, IB; Svendsen, CN; Wang, S. Ib tug tom ntej Human Neural Progenitor Transplant into the Degenerate Retina Does Not Compromise Initial Graft Survival or Therapeutic Efficacy. Txhais lus. Vis. Sci. Tshuab. 2015, 4, 7. [CrossRef] [PubMed]

29. Jones, MK; Lu, B.; Girman, S.; Wang, S. Cell-based therapeutic strategies for replacement and preservation in retinal degenerative disease. Prog. Retin. Qhov muag Res. 2017, 58, 1–27. [CrossRef] [PubMed]

30. Tswb, BA; Kaul, C.; Bonilha, VL; Rayborn, ME; Shadrach, K.; Hollyfield, JG Lub BALB / c nas: Cov txiaj ntsig ntawm cov qauv vivarium teeb pom kev zoo ntawm cov kab mob retina thaum laus. Exp. Qhov muag Res. 2015, 135, 192–205. [CrossRef]

31. Klassen, HJ; Ng, TF; Kurimoto, Y.; Kirov, I.; Shatos, M.; Coob, P.; Cov tub ntxhais hluas, MJ Multipotent retinal progenitors qhia txog kev txhim kho cov cim, sib txawv rau hauv cov paj hlwb, thiab khaws cia tus cwj pwm teeb pom kev zoo. Tshawb xyuas. Ophthalmol. Vis. Sci. 2004, 45, 4167–4173. [CrossRef]

32. jCyte, Inc. Kev nyab xeeb ntawm ib leeg, Intravitreal Txhaj Tshuaj ntawm Tib Neeg Retinal Progenitor Cells (jCell) hauv Retinitis Pigmentosa. Clinical Trial # NCT02320812. Muaj nyob online: https://clinicaltrials.gov/ct2/show/NCT02320812 ( nkag mus rau 24 Lub Ob Hlis 2023).

33. jCyte, Inc. Kev nyab xeeb thiab kev ua tau zoo ntawm kev txhaj tshuaj Intravitreal ntawm tib neeg Retinal Progenitor Cells hauv cov neeg laus nrog Retinitis Pigmentosa. Clinical Trial # NCT03073733. Muaj nyob online: https://clinicaltrials.gov/ct2/show/NCT03073733 ( nkag mus rau 24 Lub Ob Hlis 2023).

34. jCyte, Inc. Kev nyab xeeb ntawm Kev Rov Ua Txhaum Intravitreal Txhaj Tshuaj Ntawm Tib Neeg Retinal Progenitor Cells (cell) hauv Cov Neeg Laus nrog Retinitis Pigmentosa. Clinical Trial # NCT04604899. Muaj nyob online: https://clinicaltrials.gov/ct2/show/NCT04604899 ( nkag mus rau 24 Lub Ob Hlis 2023).

35. Oudejans, JJ; van der Valk, P. Immunohistochemical kev faib tawm ntawm T cell thiab NK cell neoplasms. J. Clin. Pathol. 2002, 55, 892. [CrossRef] [PubMed]

36. Tostanoski, LH; Chiu, YC; Gammon, JM; Simon, T.; Andorko, JI; Bromberg, JS; Jewell, CM Reprogramming lub zos Lymph Node Microenvironment txhawb kev kam rau siab uas yog Systemic thiab Antigen tshwj xeeb. Cell Rep. 2016, 16, 2940–2952. [CrossRef] [PubMed]

37. Jurga, AM; Paleczna, M.; Kuter, KZ Txheej txheem cej luam ntawm General thiab Discriminating Markers ntawm Differential Microglia Phenotypes. Pem hauv ntej. Cell Neurosci. 2020, 14, 198. [CrossRef] [PubMed]

38. Li, JC; Zou, XM; Yang, SF; Jin, JQ; Zhu, L.; Li, CJ; Yang, H.; Zhang, AW; Zhao, TQ; Chen, CY Neutrophil extracellular ntxiab koom nrog kev txhim kho mob qog noj ntshav hauv cov neeg mob qog noj ntshav. Ntiaj teb J. Gastroenterol. 2022, 28, 3132–3149. [CrossRef]

39. Kee, BL Kev Loj Hlob Ntawm Ntuj Killer Cells thiab ILC1. Encycl. Immunobiol. 2016, 1, 140–148.


Koj Tseem Yuav Zoo Li