Clr-f Kev Qhia Txog Kev Tswj Rau Lub raum Lub Cev Thiab Metabolic Homeostasis Ⅲ
Nov 24, 2023
Kev sib tham
Infammation thiab tiv thaiv cell-mediatedkev puas tsuaj ua lub luag haujlwm tseem ceeb hauv kev txhim kho thiab kev loj hlob ntawm lub raum raug mob thiab ua haujlwm tsis zoo. Hauv txoj kev tshawb no, peb qhia txog qhov yuav tsum tau muaj rau Clr-f hauv kev saib xyuas ntawmlub raum noj qab haus huvthiab ua haujlwmraws li ib tug intrinsic homeostatic tswj tiv thaivtiv thaiv- thiab metabolic-mediated raum puas. Peb qhov kev tshawb pom qhia tias kev tiv thaiv kab mob hauv lub cev, thiab immunoglobulin thiab ntxiv cov protein tso rau hauv lub raum ntawm Clr-f-tsis muaj nas. Cov kab mob tiv thaiv kab mob no ntawm Clr-f-tsis muaj nas yog ua ke nrog cov hnub nyoog sib cuam tshuam ntawm cov kab mob hauv cov hlab ntsha thiab cov kab mob glomerular, ectopic lipid tsub zuj zuj, thiab qhov tsis zoo ntawm ntau cov txheej txheem transcriptional.
Nyem rau cistanche herba rau mob raum
Cov Clr proteins encoded nyob rau hauv NKC ntawm lawv cov caj ces txuas NKR-P1 receptors yog lub hom phiaj ntawm txoj kev tshawb no raws li tsis redundant MHC chav kawm I-ywj siab ntawm tus neeg nruab nrab ntawm ploj-kev lees paub tus kheej los ntawm NKR-P1 qhia lub cev tiv thaiv kab mob 9,27-31. Kev nthuav qhia ntau ntxiv ntawm kev xaiv Clr proteins hauv cov lus teb rau kev ntxhov siab ntawm tes kuj tau tshaj tawm kom ua haujlwm tswj lub cev tiv thaiv kab mob ntawm cov ntaub so ntswg-cov neeg nyob hauv lub cev tiv thaiv kab mob7,12,32. Raws li txoj cai, plab hnyuv Clr-f tau pom zoo los pab txhawb kev tiv thaiv kab mob nyob rau hauv lwm yam kev tiv thaiv kab mob plab ib puag ncig los ntawm nws cov kev cuam tshuam nrog inhibitory NKR-P1G receptor-qhia intraepithelial lymphocytes7. Ua haujlwm los ntawm Leibelt li al. pom tias nas sib tw nrog poly (I: C) nthuav tawm Clr-f qhia ntau ntxiv thiab tias Clr-f / NKR-P1G kev sib cuam tshuam hauv vitro yog qhov cuam tshuam rau NKR-P1G nthuav tawm cov hlwb. Ua ke cov kev tshawb pom no tuaj yeem txhais tau tias yog lub peev xwm txheej txheem uas Clr-f tuaj yeem tiv thaiv kev ua haujlwm ntawm plab hnyuv intraepithelial NKR-P1G zoo subsets ntawm lub epithelial barrier uas yog tas li provoked los ntawm microbial stimuli7.
Cov tiam ntawm Clr-f-tsis muaj nas muab peb lub sijhawm frst los ntsuas qhovkev cuam tshuamntawm Clr-f qhia hauv vivo thiab tso cai rau peb tshawb xyuas lub peev xwm tiv thaiv kab mob homeostatic lub luag haujlwm ntawm Clr-f hauv lub raum. Lub raum puas tau pom nyob rau hauv Clr-f-/- nas ua raws li tus qauv uas qhov kev txo qis ntawm Clr-f ua haujlwm raws li qhov qhia txog kev ntxhov siab ntawm tes thiab kev raug mob uas tso tawm cov kev txwv ntawm NKR-P1G-expressing efector cells. Qhov nce hauv NKR-P1G ntawm T hlwb hauv ob lub raum ntawm Clr-f-/- nas qhia tias NKR-P1G qhia tau nce ntxiv ntawm cov neeg nyob hauv lub raum thaum tsis muaj Clr-f, lossis cov raum ntawm Clr- f−/− nas raug nkag mus rau NKR-P1G-expressing T hlwb. Qhov tsis muaj qhov hloov pauv hauv qhov muaj lossis faib ntawm NKR-P1G-qhia cov hlwb hauv plab ntawm Clr-f-/- nas tuaj yeem qhia tau tias qhov tshwm sim ntawm kev ua haujlwm ntawm NKR-P1G: Clr-f kev cuam tshuam hauv plab yog txwv. rau kev tswj lub cev tiv thaiv kab mob hauv cov ntsiab lus ntawm kev kis kab mob ntau dua li kev tiv thaiv kab mob. Xwb, kev sib cuam tshuam tsis tu ncua kuj tseem tuaj yeem tswj cov haujlwm ntawm NKR-P1G-qhia cov hlwb. Qhov no tau txais kev txhawb nqa los ntawm kev tshawb pom tias NKR-P1G khi tsis yog rau Clr-f nkaus xwb tab sis kuj rau Clr-d thiab Clr-g33, qhov kawg ntawm qhov nruab nrab tau nthuav tawm hauv cov ntaub so ntswg ntawm ob lub raum thiab lub plab 6. Thaum peb qhov kev tshawb pom tau qhia txog qhov yuav tsum tau muaj rau Clr-f los tiv thaiv lub raum autoimmune puas tsuaj, NKR-P1G-txhim kho lub luag haujlwm hauv autoimmune raum pathologies ntawm Clr-f-/- nas tsis tiav. Tshwj xeeb, txawm hais tias muaj qhov nce hauv NKR-P1G-qhia T hlwb hauv lub raum ntawm Clr-f-/- nas, qhov sib txawv tsis txaus. Nws yog qhov ua tau tias lwm qhov receptor koom nrog hauv kev soj ntsuam ntawm Clr-f hauv lub raum. Txhawm rau kom daws tau qhov axis ntawm kev tswj hwm hauv lub raum homeostasis, xav tau kev tshawb nrhiav ntxiv
Immune-mediated ob lub raum kev puas tsuaj yog typified los ntawm infiltration ntawm inflammatory hlwb mus rau hauv interstitial qhov chaw los yog mus rau lub glomerulus34. Lub raum tsub zuj zuj ntawm monocytes, B thiab T hlwb, anti-glomerular antibodies, nce IL-12 thiab IFN cytokines, thiab ntau yam glomerular pathologies hauv Clr-f-/- nas zoo ib yam li autoimmune toj roob hauv pes ntawm LN nas qauv ( ie, MRL-Faslpr) 35–37. Txawm hais tias Clr-f-/- ob lub raum nthuav tawm IgA qhov tseem ceeb mesangial deposits nrog txuam IgM thiab IgG deposition, thiab pawg ntawm Clr-f-/- thiab IgAN transcriptional profiles, nws yuav ntxov ntxov los qhia tias cov phenotypes yog qhov qhia txog hom CKD nrog IgAN38. Es tsis txhob, cov kev raug mob no, nrog rau lub xub ntiag ntawm mesangiolysis, GBM thickening, thiab podocyte tuag nyob rau hauv Clr-f-/- ob lub raum thiab tubulointerstitial hloov, rog tsub zuj zuj, thiab qhov tseem ceeb dysregulation nyob rau hauv metabolic txoj kev yog cov ntsiab lus rau ntau yam pathologies. muab tau los ntawm metabolic defects exacerbated los ntawm lub cev tiv thaiv kab mob37,39,40. Txawm hais tias qhov tshwm sim los yog hom kab mob ntawm Clr-f-/- nas lub raum tsis ua haujlwm, Clr-f zoo li ua lub luag haujlwm txo qis kev loj hlob ntawm autoimmunity nrog lub meej meej glomerulonephritis-txog pathology.
Peb qhov kev ntsuam xyuas ntawm Rag1−/−Clr-f−/- nas qhia tau hais tias muaj txiaj ntsig zoo ntawm T thiab B cell-independent mediators rau cov lus teb autoimmune ntawm Clr-f−/- nas. Tshwj xeeb, qhov sib xyaw ntawm periglomerular CD11c+ hlwb, F4/80+ hlwb, thiab NKp46+ hlwb tau tshaj tawm thaum tsis muaj T thiab B hlwb thiab cuam tshuam nrog qhov tseem ceeb ntawm glomerular puas. Qhov no qhia tau hais tias thaum T thiab B hlwb tuaj yeem ua rau muaj kev puas tsuaj rau lub raum thiab autoantibody complexes, hauv Clr-f-/- nas cov no yuav tsis yog qhov tseem ceeb ntawm kev txiav txim siab ntawm pathology, tab sis ob qho tib si pab txhawb rau kev ua haujlwm ntawm lwm lub cev tiv thaiv kab mob lossis muaj peev xwm. ua lub luag haujlwm tiv thaiv kab mob hauv nas uas tsis muaj Clr-f. Qhov no sib piv cov pov thawj los ntawm ddY nas qauv ntawm IgAN uas qhia tau hais tias muaj zog T1 polarized teb, nrog IFN - tsim T hlwb nce nrog hnub nyoog thiab kev loj hlob ntawm glomerular raug mob41. Lub raum infiltration ntawm B hlwb hauv Clr-f-tsis muaj nas kuj tau pom hauv nas qauv ntawm SLE42. Txog rau tam sim no, nws tsis paub meej tias B hlwb hauv lub raum ua rau muaj kev puas tsuaj rau hauv CKD lossis ua si ntau txoj haujlwm tswj hwm43. Cov pov thawj tsis ntev los no ntawm CKD hauv cov neeg mob laus pom tias kev txo qis hauv cov neeg B cell tau cuam tshuam nrog kev mob raum mob44. Cov ntaub ntawv pov thawj los ntawm ob lub hlwb T thiab B tau pom tias muaj kev tiv thaiv kev tiv thaiv kab mob hauv cov kab mob autoimmune raum45,46, thiab yog li qhov uas lawv pab txhawb lossis tswj cov kab mob ntawm Clr-f-/- nas yuav tsum tau tshawb xyuas ntxiv.
Peb qhov kev tshawb pom ua ke qhia txog qhov yuav tsum tau muaj rau Clr-f qhia nyob rau hauv tubular epithelium ntawm proximal convoluted tubules, podocytes, thiab / los yog txoj hnyuv epithelium. Kev poob ntawm Clr-f qhia ua rau muaj kev tawm tsam autoimmune thiab inflammatory cascade uas ua rau lub raum puas thiab cuam tshuam adiposity. Cov tshuaj tiv thaiv kab mob uas tau txais los ntawm qhov poob ntawm Clr-f tshwm sim yog T thiab B-cell-ywj siab. Zuag qhia tag nrho, lub luag haujlwm ua haujlwm rau Clr-f hauv lub raum tuaj yeem pom tau tias yog ib qho inhibitory molecule rau kev soj ntsuam cov tshuaj tiv thaiv kab mob hauv lub raum raws li tso rau hauv plab, lossis ua ib qhointrinsic regulator ntawm tubular cell muaj nuj nqi.
Cov ntaub ntawv thiab txoj kev nas.
Txhua tus nas tau khaws cia rau hauv qhov chaw tsis muaj kab mob tshwj xeeb. C57BL/6 (WT) thiab B6.129S7- Rag1tm1Mom /J (Rag1−/−) nas tau txais los ntawm Jackson Laboratory (Bar Harbor, ME, USA). Cov nas nqa cov kev hloov pauv tsis sib xws hauv Rag1 thiab Clr-f (Rag1−/−; Clr-f −/−) tau tsim los ntawm hla Clr-f-defcient (Clr f −/−) nas nrog (Rag1−/−) nas. . Txhua qhov kev yug me nyuam thiab kev ua haujlwm ntawm cov tsiaj tau ua raws li tsev kawm ntawv cov lus qhia thiab pom zoo los ntawm Dalhousie University Animal Ethics Committee thiab Institutional Animal Care and Use Committee (IACUC) ntawm University of Ottawa. Txhua qhov kev tshawb fawb tsiaj tsim, kev sim thiab kev tshaj tawm tau ua raws li ARRIVE cov lus qhia.
Generation ntawm Clr-f-defcient nas. Clr-f-deficient (Clr-f −/−) nas tau tsim los ntawm homologous recombination ntawm lub hom phiaj tsim uas muaj foxed phosphoglycerate kinase (PGK)-neomycin cassette thiab genomic sequence spanning exons 3, 4, thiab ib feem ntawm exon 5. Clr-f noob. Lub hom phiaj ua tiav yog confrmed los ntawm kev sib txuas ntawm Ottawa Hospitals Research Institute (OHRI) ua ntej electroporation rau hauv C57BL/6 Bruce-4 embryonic qia (ES) hlwb. ES clone xaiv los ntawm G{{10}} tawm tsam tau ua los ntawm IRCM (Montreal Clinical Research Institute) Transgenic Core Facility (Montreal, QC, Canada). Lub caij no, 5′ thiab 3′ Clr-f sojntsuam tau sim nyob rau hauv kev txwv tsis pub ncua ntev polymorphism (RFLP) kev tshuaj xyuas siv thymic genomic DNA los ntawm tus nas WT B6, uas tau zom nrog BamHI thiab PstI, feem. Digested DNA tawg tau daws ntawm 1% agarose gel thiab xa mus rau nylon membrane blots. Clr-f cDNA probes tau sau npe nrog cov xov tooj cua 32P-dCTP (Perkin Elmer, Guelph, ON, Canada) siv NEB ntau cov khoom siv (New England Biolabs, USA). Blots raug soj ntsuam nrog 32P-labeled Clr-f cDNA sojntsuam hauv cov tshuaj hybridization (10% (w/v) dextran sulfate; 1 M NaCl; 1% SDS). Hybridization tau ua thaum hmo ntuj ntawm 65 degree thiab tom qab hybridization ntxuav tau ua nrog cov tshuaj uas muaj 2 × SSC thiab 1% SDS pre-warmed rau 65 degree. Te blots tau nthuav tawm cov duab yeeb yaj kiab los nthuav qhia cov cim hybridization (Figs. S1A, S3B). Cov membranes tau stripped nrog 0.2% SSC / 0.2% SDS rau 30 min ntawm 85 degree ntawm txawv hybridizations. Neomycin-resistant clones tau soj ntsuam los ntawm Southern blot tsom, thiab pom qhov ua tau zoo ntawm ~ 15%. Xaiv ES clones tau microinjected rau hauv blastocysts ntawm IRCM Microinjection Service Facility los tsim cov chimeric Clr-flox tus tsim nas. Cov nas tau raug tshuaj xyuas los ntawm Southern blot tsom xam thiab tom qab ntawd muab cov poj niam B6 los tsim Clr-fwt / lox heterozygous nas. Cov nas heterozygous tau muab sib xyaw kom tau Clr-flox / lox nas. Txhawm rau tshem tawm cov neomycin cassette, Clr-flox / lox nas tau bred nrog homozygous CMV-cre transgenic nas ntawm B6 keeb kwm (Jackson Laboratory). Qhov tshwm sim ob npaug heterozygous CMV-CreTg/0; Clr-f-/+ nas tau sib xyaw ua ke los tsim Clr-f-/- nas uas tsis muaj CMV-cre transgene. Cov nas tau genotyped siv cov primers tshwj xeeb (Table 1, Fig. S1B, C, thiab Fig. S3C, D). PCR amplification tau ua los ntawm AccuStart II Taq DNA polymerase (Quanta Biosciences, Gaithersburg, MD, USA). PCR sib tov tau npaj ua raws li cov chaw tsim khoom cov lus qhia. Phenol / Chloroform rho tawm pob ntseg DNA tau siv los ua cov qauv. Cov nram qab no PCR cycling tsis tau siv: 30 amplification cycles ntawm 94 degree rau 40 s, 60 degree rau 45 s, 72 degree rau 60 s. WT thiab Clr-f −/- littermates tau siv rau hauv txhua qhov kev sim tshwj tsis yog qhia lwm yam.
Nyob rau hauv hybridization.
Nyob rau hauv situ hybridization kev soj ntsuam ntawm Clr-f tau ua tiav los ntawm cloning lub full-length sense strand CDS thiab Clr-f-spanning antisense strand rau hauv pGEM-T Ib qho yooj yim vector raws li tau piav yav tas los. Digoxi genin-labeled antisense thiab kev nkag siab RNA sojntsuam tau luam tawm los ntawm linearized plasmids encoding Clr-f noob nrog T7 polymerase siv DIG RNA labeling mix (Roche, Basel, Switzerland). Cov ntaub so ntswg tau sau los ntawm cov neeg laus B6 nas thiab tau kho nyob rau hauv 10% formalin ntawm RT los yog hauv 4% paraformaldehyde ntawm 4 degree nrog maj mam co mus txog 24 teev. Cov ntaub so ntswg tau muab tso rau hauv paraffin thiab 4 µm ntu tau npaj rau ntawm cov iav slides. Cov ntaub so ntswg tau decarbonate los ntawm kev kho mob nrog 0.2 N HCl rau 15 feeb thiab 30 µg/mL ntawm proteinase K ntawm 37 degree rau 20 min. Cov ntaub so ntswg raug kho hauv 4% PFA rau 10 feeb tom qab kho nrog 0.25% acetic anhydride hauv 0.1 M triethanolamine ob zaug rau 5 feeb txhua. Cov swb tau ua ntej hybridized nrog hybridization tov (50% (v/v) formamide/5 × SSC, pH 4.5; 2% (w/v) thaiv hmoov (Roche); 0.05% (w/v) CHAPS; 5 mM EDTA; 50 µg / mL heparin; 1 µg / mL poov xab RNA) nyob rau hauv qhov cub 58 degree rau tsawg kawg yog 1 h, thiab ces incubated nrog hybridization tov uas muaj 500 ng / mL digoxigenin-labeled sojntsuam hmo ntuj ntawm 58 degree. Tom qab hybridization ntxuav tau ua nrog 50% formamide / 2 × SSC, pH 4.5 ntawm 55 degree 3 × rau 20 min txhua, thiab cov slides tau stained nrog yaj anti-digoxigenin alkaline phosphatase-conjugated antibody ntawm 1: 1000 dilution hauv thaiv kev daws ( Roche). Kev tsim xim yog ua los ntawm kev ntxiv nitro xiav tetrazolium/5-Bromo, 4-chloro, thiab 3-indoylphosphate (NBT/ BCIP, Roche) substrates rau cov swb. Slides tau khaws cia hauv qhov tsaus ntuj ntawm RT kom txog thaum pom cov cim pom tshwm. Kev tswj cov ntawv sau npe tau ua los ntawm kev sojntsuam kev nkag siab-strand thiab kev sib tw sib tw nrog kev nkag siab thiab kev sojntsuam antisense confrmed tshwj xeeb. Tom qab ntawd cov slides tau counterstained nrog lub teeb ntsuab los yog methyl ntsuab thiab pom nyob rau hauv lub teeb lub teeb.
RT-PCR. Lub raum cov ntaub so ntswg los ntawm lub hnub nyoog- thiab kev sib deev-match WT thiab Clr-f −/− nas raug tshem tawm, yaug nrog PBS, thiab khov ntawm -80 degree . RNA raug cais tawm ntawm cov ntaub so ntswg khov siv RiboZol RNA extraction reagent (AMRESCO Inc., West Chester, PA, USA) ua raws li cov chaw tsim khoom cov lus qhia. Kwv yees li ntawm 1 ug ntawm RNA tau hloov mus rau cDNA siv Verso cDNA cov khoom siv (Thermo Scientific, Waltham, MA, USA). PCR amplification tau ua tiav ntawm 1 μL ntawm cDNA cov khoom siv tshwj xeeb primers (Table 1). PCR cov xwm txheej siv yog 94 degree rau 30 s, 58 degree rau 30 s, 72 degree rau 60 s, thiab 35 amplification cycles. PCR cov khoom raug pom ntawm 1% agarose gel stained nrog ethidium bromide (Fig. 1C, Fig. S3A).
Immunohistochemistry.
Lub raum cov ntaub so ntswg ntawm lub hnub nyoog- thiab kev sib deev-match WT thiab Clr-f −/- nas tau deparaffinized los ntawm peb 5 min incubations hauv xylene, ob 5 min incubations hauv 100% ethanol, ua raws li 90 % ethanol, ces 80% ethanol, thiab thaum kawg 70% ethanol. Cov zawv zawg tau rov ua dej los ntawm kev ntxuav hauv cov dej distilled (20 dips), thiab qhov kub hnyiab ntawm cov epitope retrieval tau ua los ntawm kev siv lub siab cooker hauv citrate buffer (pH 6.0). Endogenous peroxidases tau thaiv nrog 3% hydrogen peroxide hauv dej tsis muaj menyuam rau 10 min, thiab cov proteins raug thaiv siv Background Sniper (Biocare Medical, Pacheco, CA, USA). Tom qab ntawd cov ntu tau muab tso nrog nas los tiv thaiv nas monoclonal anti-Clr-f antibodies7 ntawm 4 degree thaum hmo ntuj. Hnub tom qab, cov slides tau muab yaug nrog 0.1 M PBS thiab incubated rau 1 h nyob rau hauv lwm yam tshuaj tiv thaiv. Staining tau tsim los siv cov khoom siv tshis-on-Rodent HRP-Polymer (Biocare Medical) thiab Betazoid DAB Bufer (Biocare Medical), ua raws li hematoxylin counter-staining. Cov txheej txheem tib yam tau siv los ua rau lub raum cov ntaub so ntswg rau IHC daim ntawv lo ntawm NKp46+ thiab CD{28}} hlwb, siv cov tshuaj tiv thaiv NKp46 (eBioscience, Santa Clara, CA, USA, clone 29A1.4, CAT# 48-3351-82) thiab anti-CD3 (BioLegend CAT # 100,327) raws li thawj cov tshuaj tiv thaiv. IHC daim ntawv lo ntawm F4/80 tau ua nyob rau hauv lub raum khov seem siv tshuaj tiv thaiv-F4/80 nas monoclonal BM8 antibody. Ib tug tshis anti-rat IgG H&L HRP-conjugated secondary antibody (Abcam, Cambridge, UK CAT#ab97057) tau siv raws li saum toj no thiab IHCs tau tsim nrog, horseradish peroxidase (HRP) substrate tau siv DAB Substrate Kit (Abcam, CAT# ib 64238).
Immunofluorescence.
Lub raum thiab/los yog cov ntaub so ntswg ntawm lub hnub nyoog- thiab kev sib deev-matched WT thiab Clr-f −/- nas tau cryopreserved nyob rau hauv 30% sucrose ntawm −80 degree nyob rau hauv daim ntaub-Tek optimal cutting temperature compound (Fisher Scientific, Pittsburgh, PA, USA). 20 μm cryostat seem tau kho nrog acetone hauv lub fume hood ntawm chav tsev kub rau 16 min thiab airdried tsawg kawg yog 30 min, tom qab ntxuav hauv 0.01 M PBS, pH 7.4. Nonspecific binding tau thaiv nrog 10% luav ntshav. Cov ntu tau muab tso rau hmo ntuj ntawm 4 degree nrog cov tshuaj tiv thaiv thawj zaug: anti-Clr-f7, anti-NKR-P1G (tus nas anti-nas monoclonal antibody tau piav qhia yav dhau los7), anti-IgA (tshis anti-nas IgA alpha saw (Abcam, Cambridge). , UK CAT # ab97235), anti-IgG-PE (BioLegend, San Diego, CA, USA CAT # 405324, anti-IgM-FITC (Lub Neej Technologies, Carlsbad, CA, USA CAT # A21042), Complement C3 Anti lub cev (11H9 ) (Novus Biologicals, Littleton, CO, USA Cat# NB200-540), anti-IFN -PE (BD-Biosciences, Franklin Lakes, NJ, USA CAT#554411), anti-IL-12-PE (BD-Pharmingen CAT# 554479), anti-CD45-FITC (BioLegend CAT#103108), anti-CD11c-FITC (eBioscience CAT#11-0114-85), anti-F4/80- APC (BioLegend CAT#123116), thiab anti-NKp46-eFluor 450 (eBioscience, CAT#48-3351-82) ntawm 1:100 concentration nyob rau hauv 5% ib txwm cov ntshav nyob rau hauv ib tug humidified chamber. Secondary antibody labeling ntawm anti -Clr-f thiab anti-NKR-P1G cov tshuaj tiv thaiv uas siv FITC conjugated polyclonal antibodies tau ua ntawm RT rau 1 h incubation. Cov qauv yog DAPI nuclear stained ntawm RT rau 5 min, ntxuav nrog PBS, thiab mounted rau iav slides nrog ib tee ntawm VECTASHIELD PLUS antifade mounting media (Vector Laboratories, San Francisco, CA, USA) thiab npog nrog iav npog. Slides tau tshuaj xyuas siv Zeiss LSM 510 laser scanning confocal microscope.
Histology thiab pathology qhab nia.
Lub raum ntu ntawm hnub nyoog- thiab kev sib deev-match WT, Clr-f −/−, Rag1−/− thiab Rag1−/−Clr-f −/− nas (3 μm) yog Periodic acid-Schif (PAS) stained. Lub raum cov ntaub so ntswg raug soj ntsuam rau mesangial cellularity (txhais tau tias yog 4 los yog ntau dua nuclei ib cheeb tsam mesangial), endocapillary proliferation, crescents, thiab glomerular sclerosis. Cov ntu tseem raug soj ntsuam rau qhov muaj tubulopathy, interstitial fibrosis, tubular atrophy, thiab tubulointerstitial cell infiltrates. Rau glomeruli pathology tau qhab nia ib nrab-quantitative qhab nia tau siv los ntsuas qhov degree ntawm glomerular puas. Yam tsawg kawg ntawm 50 glomeruli hauv txhua pab pawg tau kuaj xyuas thiab qhov hnyav ntawm qhov txhab raug muab qhab nia los ntawm 0 mus rau +3: a+1 qhov txhab tau sawv cev qhov kev koom tes tsawg dua lossis sib npaug li 30% ntawm glomerulus, +2 ntawm 30–60%, thaum ib qho +3 lesion qhia ntau dua lossis sib npaug li 60% ntawm cov glomerulus tau koom nrog. Kev ntsuas ntawm GBM thickness tau ua tiav ntawm glomerular electron micrographs ntawm WT thiab Clr-f −/- siv duab J V1.53a47. PAS-stained raum cov ntaub so ntswg los ntawm ob Rag1−/− thiab ob Rag1−/−Clr-f −/- nas tau ntxiv qhab nia qhov muag tsis pom kev rau qhov muaj cov yam ntxwv ntawm cov kab mob saum toj no, thiab cov txiaj ntsig zoo tau suav los ntawm qhov nruab nrab ntawm plaub lub raum ib feem. mus.
Electron microscopy.
Ua cov ntaub so ntswg rau kev xa hluav taws xob microscopy ua raws li cov txheej txheem luam tawm 48. Luv luv, me me ntawm lub raum cov ntaub so ntswg los ntawm 12-lub lim tiam WT thiab Clr-f −/− nas tau kho nyob rau hauv 2.5% glutaraldehyde tov thiab ces ua tiav siv 1% osmium tetroxide/uranyl acetate txoj kev. Cov qauv tau muab tso rau hauv Epon Araldite resin ua ntej txiav siv Reichert-Jung ultra txiav E ultramicrotome. Cov qauv tau pom ntawm JEOL JEM 1230 kis tau tus mob electron microscope thiab cov duab raug ntes siv Hamamatsu ORCA-HR cov koob yees duab digital.
Cov zis thiab ntshav plasma tshuaj pov tseg thiab ntsuas electrolyte.
Kwv yees li ntawm 150 μL ntawm cov zis tau sau txhua hnub los ntawm 12- cov txiv neej hnub nyoog WT thiab Clr-f −/− nas nyob rau tib lub sijhawm ntawm hnub rau 5 hnub sib law liag. Cov zis tau centrifuged ntawm 10, 000 rpm kom tshem tawm cov khib nyiab thiab tom qab ntawd khaws cia ntawm −80 degree . Cov ntshav ntawm lub ntsej muag tau sau los ntawm tib cov nas nyob rau hauv heparinized hlab thiab cais los ntawm centrifugation ntawm 3000g rau 5 min thiab khaws cia ntawm -80 degree. Cov zis thiab ntshav kuaj tau raug xa mus rau Idexx Laboratories (Markham, Ontario) rau cov protein, creatinine, thiab electrolyte concentration ntsuas.
Kev ntsuas ntshav siab ntawm cov nas muaj hnub nyoog 12 thiab 24 lub lis piam. Systolic thiab diastolic ntshav siab (BPs) tau ntsuas los ntawm tail-cuff plethysmography ntawm 12 thiab 24-cov txiv neej hnub nyoog WT thiab Clr-f −/- nas nyob ruaj khov siv BP2000 Visitech qauv. BP tau ntsuas txhua hnub hauv tib chav thiab tib lub sijhawm ntawm ib hnub rau 5 hnub sib law liag. BP qhov tseem ceeb ntawm 3 qhov kev ntsuas sib law liag tau siv los suav cov txiaj ntsig BP qhov nruab nrab raws li frst 2 hnub tau suav tias yog qhov tseem ceeb rau kev yoog rau nas.
Kidney RNA sequencing. For kidney RNA-seq, total RNA was isolated from freshly frozen kidneys of three age- and sex-matched WT and Clr-f −/− mice at 7, 13, and 24 weeks of age using TRIzol (Thermo Fisher Scientific) and RNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA, USA). RNA samples were sent to Genome Quebec Innovation Centre, McGill University, Montreal, QC for library preparation and RNA-seq. RNA-seq was performed on the Illumina HiSeq 4000 platform, with paired-end reads, at>30 × 106 nyeem qhov tob rau ib qho qauv.
RNA‑seq analysis. Te quality of the sequencing reads was inspected using FastQC tool V0.11.5 (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/) and reads with a Phred quality score of 30 over 90% of the reads were retained utilizing the Fastx-toolkit V0.0.13 (http://hannonlab.cshl.edu/). Next, the high-quality reads were mapped to the mouse GENCODE M25 annotation database (GRCm38.p6)49 using the STAR aligner V2.7.5a50. STAR aligner was then used to remove duplicates, create transcriptome hits count tables, and generate wiggle fles. Subsequently, DESeq2 package V1.28.151 was used to remove hits with CPM values less than 1 and calculate differentially expressed genes with FDR-transformed P-value>{{0}}.05 thiab log hloov pauv txiav tawm ntawm 1.5. FPKM qhov tseem ceeb rau txhua lub sijhawm cov ntsiab lus thiab cov qauv raug muab (Cov Ntaub Ntawv Ntxiv Cov Ntaub Ntawv 1). Functional enrichment tsom xam. Gene ontology thiab Functional Enrichment Analysis tau ua tiav siv g: Profiler (version e102_eg49_p15_7}a9b4d6) nrog g: SCS ntau yam kev ntsuas kho txoj kev siv qhov tseem ceeb ntawm 0.05 (https: //biit.cs.ut.ee/gprofiler/gost) 52 thiab PANTHER Classification System nrog Fisher's Exact testing thiab FDR qhov pib ntawm<0.05 (http://pantherdb.org/citePanther.jsp) 53.
Cov kev sib txuas ua kom muaj txiaj ntsig zoo tau tsim los siv cov noob ntawm GO Biological Txheej Txheem (GO: BP) rau Mus Musculus (GRCm38. p6) BioMart tso tawm: 2020-12-08 rub tawm los ntawm g: Profiler (https://biit.cs.ut.ee /gprofiler/gost). Gene Set Enrichment Analysis (GSEA) tau ua tiav siv GSEA V4.0354,55 siv Network enrichment raws li FDR<0.05. Networks were assembled, curated, and visualized using Cytoscape V3.7.156. Heatmaps were generated and clustered by Euclidian distance using Morpheus (https://sofware.broadinstitute.org/morpheus).
Kev tsom xam ntawm plab thiab ectopic lipid. Cov rog rog tau txiav txim siab los ntawm qhov hnyav ntawm plab rog tawm los ntawm cov nas ib leeg. Kev ntsuas lub cev hnyav tau muab coj los ua ntej cov roj rho tawm. Rau Roj Liab O (ORO) staining, khov raum seem raug txiav ntawm 10 μm siv lub cryostat thiab tom qab ntawd cua qhuav rau 30 min. ORO staining tau ua los ntawm kev kho cov ntu nrog 3 ntxuav ntawm 60% isopropanol, incubating lawv nrog kev ua haujlwm siab (66%) ntawm ORO tov (Sigma-Aldrich, St. Louis, MO, USA) staining rau 15 min. Cov swb tau raug ntxuav sai sai nrog 60% isopropanol, sai sai nrog Mayer's hematoxylin kom pom cov nuclei, thiab tom qab ntawd mounted siv VectaMount AQ Aqueous Mounting Media (Vector Laboratories).
Kev tsom xam ntawm pathotype. Kev soj ntsuam Pathtype tau ua tiav siv Diferentially Expressed Genes (DEGs) nrog q<0.05 identified from RNA-seq of WT and Clr-f −/− 13-week-old mouse kidneys. Signifcant Clr-f −/− DEGs with identifiable ENSEMBL human orthologs were compared with DEGs of 15 human kidney disease expression sets (disease versus healthy kidney)57–64 downloaded from Nephroseq database at (http://www.nephroseq.org) (Supplementary Dataset File 2)65. Clr-f −/− mouse and human kidney disease DEG sets were hierarchically clustered by Euclidean distance and graphed in a similarity matrix using the Morpheus software (https://sofware.broad institute.org/morpheus). Functional enrichment using g: Profler, (using parameters described above) was performed on four groups of expression clusters with corresponding increased and decreased DEGs and non-corresponding DEGs from the compared expression profiles of Clr-f −/− and IgA nephropathy (IgAN) kidney disease. Flow cytometry. Kidneys from age- and sex-matched WT and Clr-f −/− mice were harvested, homogenized in a Petri dish on ice with RPMI media containing collagenase type 4 (2 mg/mL) (Fisher), and incubated at 37 °C for 30 min with gentle agitation.
Cov ntaub so ntswg zom tau dhau los ntawm 70-μm nylon lim, PBS ntxuav, thiab cov hlwb liab ntxiv tau lysed nrog ACK lysing buffer rau 5 min rau dej khov. Lub raum lub cev tiv thaiv kab mob raug cais los ntawm RT centrifugation ntawm 2000 rpm rau 30 min hauv 40% Percoll. Lub cell pellet tau rov ua dua hauv PBS thiab cov xov tooj ntawm tes tau txais los ntawm hemocytometer. Ntau mAbs tau siv los txheeb xyuas cov cell-nto protein qhia, cov hlwb tau stained rau fow cytometry tsom nrog fluorochrome-labeled nas antibodies: anti-CD45 (eBioscience, clone 30-F11), anti-TCR (eBioscience, clone H{ {12}}), anti-NK1.1 (eBioscience, clone PK136), anti-CD19 (BioLegend, clone 1D3/CD19), anti-CD11b (BioLegend, clone M1/70), anti-F4/80 (BioLegend, clone BM8), anti-MHCII (IA/IE) (BioLegend, clone M5/114.15.2), anti-Ly6G (BioLegend, clone 1A8), anti-NKp46 (eBioscience, clone 29A1.4), thiab isotype tswj. Cell viability tau txiav txim siab siv Fixable Viability Dye (eBioscience, Cat# 65-0865-14). Txhawm rau txheeb xyuas thiab txheeb xyuas cov neeg tiv thaiv kab mob hauv lub cev WT thiab Clr-f −/− ob lub raum, multiparametric fow cytometry tsom xam siv cov tshuaj tiv thaiv kab mob ntawm tes tshwj xeeb tau siv los txheeb xyuas cov neutrophils (CD11b + Ly6G +), macrophages (CD11b + Ly6G-F4 / { {57}}MHCII+), NK cells (CD11bloLy6G-TCR −NK1.1+), thiab NKp46+ cells (CD11bloLy6G-TCR −NKp46+), T cells (Ly6G-NK1 .1-TCR +) thiab B hlwb (CD11b-Ly6G-TCR −CD19+). Tag nrho cov qauv tau txais ntawm BD-Fortessa fow cytometer.
Cov ntaub ntawv
1. Hato, T. & Dagher, PC Yuav ua li cas lub cev tiv thaiv kab mob hauv lub cev muaj kev ntxhov siab thiab ua rau muaj teeb meem.Clin. J. Am. Soc. Nephrol.10, 1459–1469 (2015).
2. Krüger, T.ua al.Kev txheeb xyuas thiab kev ua haujlwm zoo ntawm cov hlwb dendritic hauv lub raum murine noj qab haus huv thiab hauv kev simglomerulonephritis mob.J. Am. Soc. Nephrol.15, 613–621 (2004).
3. Weisheit, CK, Engel, DR & Kurts, C. Dendritic cells thiab macrophages: Sentinels nyob rau hauv lub raum.Clin. J. Am. Soc. Nephrol.10, 1841–1851 (2015).
4. Woltman, AMua al.Kev ntsuas ntawm dendritic cell subsets nyob rau hauv tib neeg lub raum cov ntaub so ntswg nyob rau hauv ib txwm thiab pathological mob.Raum Int.71, 1001–1008 (2007). 5. Gottschalk, ib.ua al.Batf3-dependent dendritic cells nyob rau hauv lub raum lymph node induce kam rau siab tiv thaiv circulating antigens.J. Am.Soc. Nephrol.24, 543–549 (2013).
6. Zhang, Q.ua al.Mouse Nkrp1-Clr gene cluster sequence and expression analytics qhia kev txuag cov ntaub so ntswg tshwj xeeb MHCywj siab immunosurveillance.PLoS Ib7, e50561 (2012).
7. Leibelt, S.ua al.Dedicated immunosensing ntawm nas plab hnyuv epithelium yooj yim los ntawm ib khub ntawm caj ces txuas nrog lectin zoo licov receptors.Mucosal Immunol.
8, 232–242 (2015). 8. Rahim, MMAua al.Te nas NKR-P1B: Clr-b paub qhov system yog qhov tsis zoo ntawm cov tshuaj tiv thaiv kab mob hauv lub cev.Ntshav125, 2217–2227 (2015).
9. Carlyle, JRua al.Tsis lees paub tus kheej ntawm Ocil / Clr-b los ntawm inhibitory NKR-P1 natural killer cell receptors.Proc. Natl. Acad. Sci.U.S.A. 101, 3527–3532 (2004).
10. Rutkowski, E.ua al.Clr-a: Ib qho tshiab tiv thaiv kab mob ntsig txog C-hom lectin-zoo li molecule tshwj xeeb tshaj tawm los ntawm nas plab epithelium.J. Immunol.198, 916–926 (2017).
11. Balaji, GRua al.Kev lees paub ntawm tus tswv Clr-b los ntawm inhibitory NKR-P1B receptor muab lub hauv paus rau kev lees paub tus kheej ploj lawm.Nat.Pawg.9, 4623 (2018)
12. Friede, ME, Leibelt, S., Dudziak, D. & Steinle, A. Xaiv Clr-g qhia ntawm lub hlwb ua kom muaj zog dendritic pab txhawb kev sib txuas lusnrog rau ib qho me me ntawm splenic NK hlwb qhia txog inhibitory Nkrp1g receptor.J. Immunol.200, 983–996 (2018).
13. Abou-Samra, E.ua al.NKR-P1B qhia nyob rau hauv lub plab-kwv yees innate lymphoid hlwb yuav tsum tau rau kev tswj ntawm plab hnyuv.kab mob kab mob.Cell. Mol. Immunol.16, 868–877 (2019).
14. Germain, C.ua al.Cov yam ntxwv ntawm kev hloov pauv hloov pauv hloov pauv ntawm CLEC2D noob.J. Biol. Chem.285, 36207–36215 (2010).
Supportive Service Ntawm Wecistanche-Qhov loj tshaj plaws cistanche exporter nyob rau hauv Tuam Tshoj:
Email: wallence.suen@wecistanche.com
Whatsapp / Tel: +86 15292862950
Khw Muag Khoom Kom Paub Ntxiv Specifications:
https://www.xjcistanche.com/cistanche-shop
