Anti-Aging -Klotho Gene-Activated Scaffold Txhawb Nqa Rejuvenative Wound Healing Response hauv Tib Neeg Adipose-Derived Stem Cells

3. Kev sib tham

Txoj kev tshawb no tsom los tshawb xyuas qhov cuam tshuam kev ua haujlwm ntawm kev tiv thaiv kev laus -Klotho gene-activated scaffold ntawm tib neeg ADSCs rau kev txhim kho qhov txhab kho mob. Zuag qhia tag nrho, peb pom tias gene-activated scaffold muaj kev tswj kom ua rau ADSCs 'regenerative peev xwm raws li tau piav qhia hauv daim duab5. Tshwj xeeb, gene-activated scaffold ncua sijhawm txhim kho ADSCs 'stemness los ntawm kev ua kom muaj kev hloov pauv hloov Oct-4. Lub gene-activated scaffold kuj txhawb kev ua kom ntxov ntawm cov tshuaj tiv thaiv kab mob gene TGF- 3, qhov tseem ceeb tshaj plaws rau kev tswj kev kho mob nrog txo qis caws pliav. Tsis tas li ntawd, qhov qhib ADSCs ib ntus tswj hwm endothelial angiogenesis thiab txhawb nqa dermal fibroblast kho los ntawm paracrine signaling. Txog rau kev loj hlob, ADSCs kuj tau tswj kev ua kom cov lus teb pro-fibrotic hauv dermal fibroblasts. Lub caij no, ADSCs ntawm cov noob caj noob ces scaffold tau zoo rov tsim dua cov ntaub so ntswg hauv qab los ntawm kev txhim kho laminin thiab collagen IV deposition. Cov lus teb no tau txuas ntxiv nrog txo qhov caws pliav -SMA protein qhia thiab txhim kho zoo elastin matrix deposition. Peb txoj kev tshawb fawb ua ke txiav txim siab tias qhov - Klotho gene-activatedCistanchemuaj peev xwm loj heev raukho qhov txhabthiab ua tauua ntej stem cell-based therapy rau rejuvenating kho daim ntawv thov.

Daim duab 5. Ib qho schematic ntawm kev ua haujlwm cuam tshuam ntawm B-Klotho gene-activated scaffold ntawm tib neeg ADSCs rau daim ntaub ntawv kho qhov txhab. Qhov kev tshawb pom tseem ceeb ntawm txoj kev tshawb fawb no yog (1) kev hloov pauv hloov pauv ntawm cov qia cell pluripotency thiab cov lus teb los tiv thaiv fibrotic, (2) txhim kho paracrine tswj ntawm angiogenesis, thiab pro-fibrotic collagen remodeling hauv dermal fibroblasts thiab thaum kawg, (3) nce maturation ntawm lub hauv paus daim nyias nyias nrog kev tswj hwm ntawm caws pliav-txuas nrog cov proteinsexpressionDotted kab rau elastin matrix implies txhim kho zoo deposition.

Nyem qhov no Kom Paub Yuav Ua Li Cas CistancheQhov mob kho qhov teebmeem

Hauv cov tswv yim kho qhov txhab, plasmid-based gene therapy yog siv los hloov kho cov lus teb ntawm tes kom txog thaum kho qhov txhab ua tiav [37]. Peb gene-activated scaffold kuj qhia tau hais tias cov kev kho mob -Klotho noob tau hloov pauv mus rau 14 hnub. Qhov tseem ceeb txhim kho (170-fold) qhia ntawm qhov -Klotho mRNA nyob rau hnub 3 tuaj yeem raug ntaus nqi rau qhov tam sim-thaum ntxov CMV tus txhawb nqa uas paub txog kev txhim kho kev hloov pauv ntawm kev hloov pauv hloov pauv [38]. Txawm li cas los xij, hauv MSCs, CMV tuaj yeem dhau mus rau DNA methylation lossis histone deacetylation uas tuaj yeem txo qhov kev hloov pauv hloov pauv [39]. Tsis tas li ntawd, cov plasmids feem ntau yog cov tsis-replicating episomes, txwv kev hloov ntawm transgene mus rau faib hlwb, thiab ua rau ib tug poob ntawm transgene qhia nyob rau hauv lub sij hawm.40]. Lub xub ntiag ntawm heterochromatic cim keeb kwm los ntawm cov kab mob kab mob ntawm cov kab mobplasmid tuaj yeem cuam tshuam ntxiv kev nthuav qhia transgene [41], tag nrho ua rau muaj kev hloov pauv noob neej.

Ib txoj hauv kev uas tau ua raws li kev kho mob ntawm cov qhov txhab tawv nqaij yog kev siv cov cell-seeded bioactive scaffolds. Ib qho laj thawj yog lawv lub peev xwm los txhawb kev kho qhov txhab sai. Metabolically active hlwb nyob rau hauv graft secrete paracrine yam thiab undergo kev sib txawv, orchestrating complex teeb liab txheej xwm nyob rau hauv lub qhov txhab ib puag ncig [42]. Stem cells, tshwj xeeb tshaj yog, muaj lub peev xwm tshwj xeeb rau kev nkag siab cov stimuli sab nraud thiab hloov kho lawv cov lus teb los kho homeostasis [43]. Txawm li cas los xij, qia hlwb poob lawv cov qia thaum lawv nthuav dav hauv vitro los tsim cov xov tooj ntawm tes loj rau kev cog qoob loo. Peb txoj kev tshawb fawb qhia tau hais tias lub stemness tuaj yeem hloov kho tau yooj yim los ntawm kev siv cov noob caj noob ces nqa cov noob los tiv thaiv kev laus. -Klotho. Tshwj xeeb tshaj yog, peb tau sau tseg tias kev tiv thaiv kev laus ntawm cov noob caj noob ces tau txhim kho qhov kev qhia ntawm cov noob caj noob ceg Oct-4 hauv ADSCs. Kaum Hli Ntuj -4 yog ib qho tseem ceeb ntawm kev hloov pauv hauv cov embryonic qia hlwb thiab yog qhov tseem ceeb rau kev tswj embryonic kev loj hlob [44]. Hauv ADSCs, kev ua kom lub Kaum Hli Ntuj -4 noob tuaj yeem txhawb nqa lawv txoj kev loj hlob thiab muaj peev xwm sib txawv [45]. Yog li, qhov kev nthuav qhia ntau ntxiv ntawm Lub Kaum Hli -4 tuaj yeem pab txhawb kev sib txawv sai ntawm ADSCs ntawm gene-activated scaffold rau hauv cov tswv tsev cell thiab pab txoj kev kho mob.

Tom qab tau soj ntsuam qhov ua kom lub Kaum Hli Ntuj -4 noob hauv ADSCs, peb mam li ntsuam xyuas cov angiogenic potency ntawm ADSCs. Angiogenesis yog qhov tseem ceeb heev rau kev sib koom ua ke ntawm kev cog qoob loo nrog tus tswv tsev ntaub [46]. Cells nyob rau hauv graft ua rau angiogenesis los ntawm secretion ntawm paracrine yam. Angiogenesis kuj yog ib qho kev tshwm sim tseem ceeb rau kev loj hlob ntawm cov ntaub so ntswg granulation thiab nws cov dej num poob qis thaum cov ntaub so ntswg granulation matures [47]. Qhov kev txwv kev txwv ntawm cov kev ua haujlwm angiogenic ntawm kev kho cov theem tom ntej yog qhov tseem ceeb rau kev tswj cov caws pliav thiab hypergranulation uas tuaj yeem cuam tshuam rov qab epithelialization [48]. Peb qhov kev tshawb pom tseem qhia tau tias ADSCs ntawm cov noob caj noob ces tuaj yeem txhim kho endothelial sprouting thiab tswj lawv txoj kev loj hlob los ntawm paracrine signaling. Tsis tas li ntawd, qhov tseem ceeb nce ntxiv hauv cov hlwb endothelial 'kev ua haujlwm metabolic thiab sprouting nyob rau hauv cov lus teb rau hnub 3 CM qhia lub peev xwm ntawm ADSCs-laden gene-activated scaffold los koom nrog cov ntaub so ntswg sai. Tsis tas li ntawd, kev siv cov qia hlwb CM yog ib qho kev siv ntawm tes tsis pub siv los txhim kho qhov txhab zoo [49]. Raws li ib qho kev txwv, peb lees paub tias cov neeg laus dermal endothelial hlwb yuav ua rau cov qauv cell zoo dua rau kev kawm angiogenesis; Txawm li cas los xij, kom muaj kev sib raug zoo nrog peb cov kev tshawb fawb yav dhau los thiab HUVECs tau txais dav los ua "kub-tus qauv" ntawm tes tus neeg sib tw [50], peb tau txais kev ntsuam xyuas HUVECs angiogenesis.

Ib qho kev nce hauv fibroblast remodeling kev ua si yog ib qho kev nthuav dav ntawm granulation cov ntaub so ntswg maturation [51]. Thaum lub sij hawm no, lub collagen III matrices yog maj mam hloov los ntawm muaj zog collagen kuv fibers [51]. Collagen fibers ces txhawb qhov txhab contraction [52], thiab qhov txhab contraction yog qhov tseem ceeb rau kev kaw qhov txhab kom tiav [53]. Txawm li cas los xij, qhov hnyav nce ntxiv hauv collagen I deposition tuaj yeem nce caws pliav [54]. Kev tswj cov caws pliav yog qhov tseem ceeb vim nws tuaj yeem cuam tshuam kev loj hlob ntawm lwm cov tawv nqaij appendages xws li cov hauv paus plaub hau, qog sebaceous, thiab qog hws, piv txwv li, hauv qhov txhab kub hnyiab [54]. Peb qhov kev tshawb pom pom tau tias cov hnub nyoog (hnub 14) CM los ntawm pawg gene-activated scaffold tuaj yeem tswj cov kev qhia ntawm pro-fibrotic collagen I hauv cov neeg laus dermal fibroblasts. Qhov no tswj cov nyhuv ntawm cov fibroblasts tshwm sim yam tsis muaj kev cuam tshuam ntxiv rau ntawm fifibroblast txoj kev tsiv teb tsaws los yog lawv cov anti-fibrotic collagen III kab lus hais txog cov noob tsis muaj scaffold pawg. Yog li, peb xav tias ADSCs-laden gene-activated scaffold yuav muab kev tswj zoo dua hauv kev txwv tsis pub caws pliav hauv vivo los ntawm kev tswj hwm collagen I qhia. Li et al., kuj tau qhia tias ADSCs CM tuaj yeem txo qis collagen I qhia hauv fibroblasts los ntawm hypertrophic nti [55]. Lwm txoj kev tshawb fawb los ntawm Wang li al., tau pom qhov txo qis hauv collagen I qhia hauv keloid-derived fibroblasts [56], sib sau ua qauv qhia txog qhov muaj peev xwm tiv thaiv kab mob ntawm ADSCs 'CM.

Ib qho ntawm cov kev tshawb pom tseem ceeb uas peb tau sau tseg nrog rau kev siv ntawm cov -Klotho gene-activated scaffold yog qhov kho kom zoo dua qub ntawm cov ntaub hauv qab daus hauv ADSCs. Kev tsim kho ntawm lub hauv paus membrane yog qhov tseem ceeb rau kev loj hlob ntawm cov hlab ntsha thiab ua tiav rov qab epithelialization [57,58]. Qhov tseem ceeb, qhov sib txawv ntawm cov proteins laminin(2.59]. Dhau li ua raws li kev tiv thaiv kev laus [60], cov beta klotho ua haujlwm los txhim kho kev nkag siab zoo rau fibroblast kev loj hlob zoo (FGF) los ntawm FGF receptors [61]. Tsis tas li ntawd, qhov nce hauv FGF signaling tau pom los txhawb kev loj hlob hauv qab daus los ntawm kev tso tawm ntawm laminin thiab collagen IV [62]. Yog li ntawd, xav txog beta klotho lub luag hauj lwm raws li ib tug co-receptor ntawm FGF, qhov nce hauv qab daus maturation nyob rau hauv lub gene-activated scaffold pab pawg neeg muaj feem cuam tshuam los ntawm kev nce hauv ADSCs 'FGF signaling.

Lub hauv paus membrane tivthaiv remarkably upregulated nyob rau hauv lub gene-activated scaffold pawg yog cov collagen IV protein. Lub collagen IV protein sawv cev rau 50 feem pua ​​​​ntawm tag nrho cov hauv qab daus daim nyias nyias [63] thiab muab cov qauv kev ruaj ntseg rau hauv qab daus membrane [59]. Txawm li cas los xij, kev laus tseem ceeb txo qis collagen IV qhia hauv dermal-epidermal hlws ris [58,64] thiab qhov tsis muaj collagen IV tuaj yeem txhawb kev caws pliav pathogenesis [65]. Yog li, peb cov txiaj ntsig tau qhia tias qhov nce hauv qab daus membrane regenerative potency ntawm ADSCs-laden gene-activated scaffold tuaj yeem pab txhawb kev kho kom zoo rau cov neeg laus. Lwm cov kev tshawb fawb kuj tau pom tias cov ntaub so ntswg adipose-derived ECM muaj qhov kho tau zoo heev rau kev kho tawv nqaij [66]. Tsis tas li ntawd, cov ntaub so ntswg-engineered grafts nplua nuj nyob rau hauv laminin thiab collagen IV kuj yog qhov txaus siab rau kev kho mob ntawm cov hlab ntsws epithelium [67]. 

Ntxiv nrog rau kev tswj hwm cov fibrotic rau cov fibroblasts, peb txoj kev tshawb fawb ntxiv qhia tau tias ADSCs nyob rau hauv cov noob caj noob ces scaffold kuj qhia ib tug 2-fold qis qhia ntawm cov caws pliav-kwv yees protein. -SMA [68]. Txawm hais tias peb tsis tau ntsuas nws qhov cuam tshuam hauv vivo, qhov txo qis hauv zos -SMA kev qhia yog qhov tseem ceeb rau kev kho kom zoo [69]. Siv cov tshuaj fibrotic xws li bleomycin [70] tuaj yeem yog ib txoj hauv kev los ntsuas qhov zoo ntawm cov khoom tiv thaiv fibrotic ntawm ADSCs / gene-activated scaffold tsim hauv vitro; Txawm li cas los xij, peb lub hom phiaj yog los tsim kom muaj kev kho mob ib ntus ntawm kev tsim kho.

Lwm qhov qhia tau hais tias ADSCs-laden gene-activated scaffold tuaj yeem txhim kho kev kho kom zoo yog qhov tso tawm ntawm fibrous elastin matrix es tsis txhob muaj cov kab mob ntxiv rau hauv cov gene-dawb scaffold pawg. Elastin deposition yog ib qho ntawm cov haujlwm tseem ceeb uas ua rau lub plab tsis muaj qhov txhab kho [71]. Txawm li cas los xij, cov tawv nqaij laus tsis muaj qhov tso tawm ntawm elastin [71]. Yog li ntawd, tropoelastin, ib tug precursor ntawm elastin, feem ntau muab tso rau hauv biomaterial scaffolds los tswj caws pliav nyob rau hauv lub qhov txhab kho [72]. Ua ke, peb qhov kev tshawb nrhiav pom tau hais tias ADSCs / -Klotho gene-activated scaffold construct yuav ua kom muaj zog tiv thaiv kab mob hauv vivo.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Kev npaj ntawm Gene-Activated Scaffold

Lub gene-activated scaffold tau tsim los siv 2- cov txheej txheem kauj ruam. Ua ntej, cov khoom ntxeem tau ntawm collagen-chondroitin sulfate scaffolds tau tsim los ntawm khov-drying slurry ntawm bovine tendon hom 1 collagen thiab chondroitin-6-sulfate muab tau los ntawm shark pob txha mos (Sigma, UK). Ib qho kev ua kom zoo khov-drying txheej txheem tsim los tsim cov pores sib xws tau siv los tsim cov scaffolds [73]. Raws li peb cov txheej txheem luam tawm [74], cov Scaffolds khov-dried ces dehydrothermally (DHT) kho ntawm 105◦C nyob rau hauv lub tshuab nqus tsev rau kom tsis muaj menyuam thiab kho tshuab. Cov sterilized scaffolds ces chemically cross-linked nrog 14 mM N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride thiab 5.5 mM N-Hydroxysuccinimide (2.5 molar ratios EDC/NHS) (Sigma, UK) tov rau ntxiv txhim kho cov mechanical stability [75]. Cov kab txuas txuas txuas txuas tau raug ntxuav nrog PBS (Gibco, Paisley, UK) kom tshem tawm cov tshuaj seem. Thaum cov scaffolds tau npaj lawm, polyplexes ntawm N / P10 piv (nitrogen rau phosphate ratio) tau npaj los ntawm kev sib xyaw ua ntej ntim ntawm branched polyethyleneimine (PEI) tov nrog plasmid DNA (pDNA) encoding rau tib neeg beta-Klotho noob ( -Klotho), tau los ntawm SinoBiological, Beijing, Suav. N / P10 piv tau raug xaiv los ntawm peb cov kev tshawb fawb yav dhau los uas pom tias polyplexes tsim ntawm N / P 10 piv tuaj yeem tsim cov cationic nanoparticles me me, ruaj khov nrog plasmids loj li GLuc (5.76 kb) [21,23]. Ua ntej siv, plasmids tau diluted hauv dej tsis muaj endotoxin kom tau txais kev ua haujlwm siab.ntawm 0.5µg/µL. Cov plasmid muaj tib neeg txhim kho tam sim ntawd-thaum ntxov cytomegalovirus(CMV3) tus neeg txhawb nqa los txhawb cov qib siab ruaj khov thiab ntu ntu ntawm kev nkag musgene nyob rau hauv mammalian hlwb.

Cov plasmid / PEI sib xyaw tau tso cai rau kev sib haum xeeb rau 30 feeb kom nws tus kheej sib sau ua ke hauv polyplexnanoparticles. Tom qab ntawd cov nanoparticles tau tsau-loaded mus rau hauv lub scaffolds los ntawm pipettingsib npaug ntawm cov tshuaj polyplex rau ib sab ntawm scaffold. Tag nrho ntawm 2µg pDNA uasiv ib scaffold los tsim cov noob-activated scaffold.

4.2. Cell Seeding rau -Klotho Gene-Activated Scaffold

Tib neeg ADSCs (iXCells Biotechnologies, San Diego, CA, USA) tau nthuav dav mus rau nqe 4 hauv ADSCs kev loj hlob nruab nrab (Cat no. MD0003) muab los ntawm lub tuam txhab. Tag nrho ntawm 5× 105 ADSCs (2.5× 105 ntawm ib sab) ces noob tau noob rau ib lub noob tsis muaj scaffold (tswj,n = 3) lossis gene-activated scaffold (test,n = 3). Tom qab tso cov cell nyob rau li 20 min, 2 mL ntawm transfection media OptiMEM (Gibco, UK) tau ntxiv, thiab cov cellularized scaffolds tau incubated ntawm 37◦C rau 24 h. Tom qab 24 teev incubation, cov cellularized gene-dawb los yog gene-activated scaffolds tau pauv mus rau hauv tshiab 12- zoo daim hlau, thiab pub nrog 2 mL ntawm ADSCs loj hlob nruab nrab. Kev hloov pauv xov xwm tau ua txhua txhua 3-4 hnub mus txog rau hnub 14 los ntawm kev sau 1 mL ntawm cov xov xwm txias (CM) thiab hloov nrog qhov sib npaug ntawm cov xov xwm tshiab. Tag nrho CM tau khaws cia ntawm−80 ◦C mus txog rau kev tsom xam.

4.3. qRT-PCR Analyzes los txiav txim -Klotho Gene Overexpression thiab ua kom muaj nuj nqi Genes

Txhawm rau txiav txim siab cov kev cai ncua sij hawm ntawm lub hom phiaj cov noob, cov hlwb tau sau rau hnub 3 (thaum ntxov) thiab 14 (lig) tom qab cov noob rau ntawm cov noob tsis pub dawb lossis cov noob qoob loo. Cov hlwb tau ua ntej lysed siv Qiazol lysis reagent (Qiagen, Germantown, MD, USA). Chloroform tau ntxiv rau cais cov cell lysate rau hauv cov protein, DNA, thiab RNA theem. Siv cov khoom siv RNeasy (Qiagen, Manchester, UK), RNA tau muab rho tawm, thiab lawv qhov zoo thiab qhov ntau tau txiav txim siab siv Multiskan Go phaj nyeem (Thermo Scientific, UK) nrog lub absorbance teem ntawm 260 nm. Genomic DNA raug tshem tawm los ntawm kev sib xyaw RNA nrog genomic DNA wipeout tsis (Qiagen, Manchester, UK) thiab cua sov rau 42.◦C rau 2 min. Tom qab ntawd, thim rov qab tau ua tiav los npaj cDNA. Duplicates ntawm cDNA ib replicate (n = 3) tau muab tso rau hauv qRT-PCR daim hlau thiab tom qab ntawd qhov kev ntsuam xyuas tau ua haujlwm siv cov primers teev nyob rau hauv Table1. Fold hloov hauv mRNA qhia txheeb ze rau cov hlwb ntawm cov noob tsis muaj scaffold tau suav nrog 2−∆∆CTtxoj kev los ntawm qhov nruab nrab ntawm peb replicates ib pab pawg. Tib neeg GAPDH (Hs{0}}GAPDH_1_SG, Cat. No. QT00079247) tau siv los ua cov noob hauv tsev.

4.4. Bioactivity Analyzes ntawm Secreted Factors los ntawm ADSCs rau -Klotho Gene-Activated Scaffold

4.4.1. Pro-Angiogenic Bioactivity Analyzes

4.4.2. Dermal Fibroblasts Kho thiab Maturation Analyzes

Tom qab qhov kev ntsuam xyuas angiogenesis, peb tau tshawb xyuas CM lub zog rau kev kho mob ntawm daim tawv nqaij los ntawm kev siv cov tshuaj txhuam hniav ntawm cov neeg laus dermal fibroblasts (iXcells, Cat no. 10 HU-014). Rau txoj kev tshawb no, peb tshwj xeeb xaiv cov hnub nyoog CM los ntawm hnub 14 los kawm txog nws txoj kev cuam tshuam ntawm fibroblast qhov txhab kaw thaum lub sijhawm loj hlob. Tag nrho ntawm 1× 104 cell/zoo nyob rau hauv ib tug 96-zoo phaj tau noob thiab incubated hmo ntuj nyob rau hauv lub fibroblast loj hlob nruab nrab. Hnub tom qab, ib lub qhov txhab raug tsim ntawm lub monolayer los ntawm kab rov tav khawb 200 ul pipette taub hla lub qhov dej. Lub monolayer tau muab yaug nrog PBS kom tshem tawm cov khib nyiab thiab pub nrog CM. Kev kaw qhov txhab tau kaw ntawm 12-16 teev tom qab khawb. Immunostaining tau siv los kawm txog kev qhia ntawm pro-fibrotic collagen I (1: 100, Novusbio, UK) thiab anti-fibrotic collagen III (1: 100, Novusbio, UK) hauv fibroblasts. Cov fibroblasts tau counterstained nrog rhodamine (1:800, Abcam, UK) rau F-actin imaging. Immunostaining tau ua raws li tau piav qhia hauv Tshooj4.5, tshwj tsis yog cov ntaub so ntswg ua thiab deparaffinization. Ob leeg HUVECs thiab cov fibroblasts tau siv ntawm nqe 4.

4.5. Immunoflfluorescence Imaging ntawm Extracellular Matrix Proteins

Tom qab 14 hnub ntawm kab lis kev cai, cellularized gene-dawb lossis gene-activated scaffolds tau sau rau kev kuaj pom ntawm matrix deposition siv immunofluorescence. Kev ua haujlwm ntawm cov qauv tau ua raws li tau piav qhia dhau los. Luv luv, cov scaffolds tau xub ntxuav nrog PBS thiab kho nyob rau hauv 10 feem pua ​​​​ntxhiab buffered formalin rau 20 min. Cov qauv ruaj khov tom qab ntawd tau ua tiav siv cov txheej txheem txheej txheem rau deparaffinization. Cov blocks tau txiav rau hauv 7-µm tuab slices thiab sau rau ntawm nqi slides. Cov ntu tom qab ntawd tau deparaffinized siv xylene ua raws li kev rov ua dej ntawm ntu nrog kev txo qis ntawm ethanol. Tom qab ntawd, cov hlwb tau permeabilized nrog 0.2 feem pua ​​Tween®20 (Sigma-Aldrich, Fabkis) tov hauv PBS rau 30 min (10 min ntxuav× 3) thiab thaiv kev siv 10 feem pua ​​NGS (Normal Goat Serum, Invitrogen, Rockford, IL, USA)/5 feem pua ​​BSA/0.3 M Glycine (npaj hauv cov tshuaj permeabilizing) rau 1 teev. Tom qab thaiv, cov swb tau muab yaug hauv PBS thiab tom qab ntawd incubated ntawm 4◦C thaum hmo ntuj nrog cov tshuaj tiv thaiv rau lub hom phiaj matrix proteins teev nyob rau hauv Table2. Hnub tom qab, cov slides tau muab yaug hauv PBS peb zaug rau 2-3 feeb txhua txhua kom tshem tawm cov tshuaj tiv thaiv tsis sib xws. Tom qab ntawd, cov slides tau incubated nyob rau hauv ob Alexa 488-conjugated tshis anti-nas IgG (A32723, Invitrogen, UK) los yog Alexa 594-conjugated tshis tiv thaiv luav IgG (A11012, Invitrogen, UK) ntawm 1: 800 dilution ntawm chav tsev kub rau 1 h hauv qhov tsaus ntuj. Cov kauj ruam yaug tau ua raws li ua ntej thiab counterstained rau nuclei siv qhov nruab nrab mounting nrog DAPI (ab104139, Abcam, UK). Tom qab ntawd cov slides tau yees duab siv lub fluorescence microscope (Olympus BX43, Nyiv) ntawm 20× Lub hom phiaj. Cov qauv incubated nrog cov tshuaj tiv thaiv theem nrab tsuas yog siv los tswj.

Duab Quantification

Duab] software (Image, NIH, MD, USA) tau siv los txiav txim siab ntau npaum li cas cov proteins uas tau qhia. Rau txhua tus cim, tus nqi pib tsis tu ncua yog thawj zaug txiav txim siab los ntawm kev ntsuas ua ntej ntawm ntau ntu. Siv cov txheej txheem pib tus nqi sib xyaw ua ke (stained area x txhais tau tias grey tus nqi) ntawm cov duab tau txiav txim siab thiab tom qab ntawd normalized rau cov xov tooj ntawm tes (DAPI suav) kom muab qhov kawg txhais tau tias fluorescence ntom ntawm ib lub xov tooj. Qhov nruab nrab tau suav los ntawm 8-10 random dluab ib replicate nrog tsawg kawg yog peb replicates ib pab pawg. Qhov nruab nrab tau txais los ntawm peb qhov sib txawv / pab pawg tau siv los ntsuas cov txheeb ze ntawm cov pab pawg.

4.6. Kev txheeb cais

Tag nrho cov txiaj ntsig tau nthuav tawm raws li qhov txhais tau tias ntxiv rau tus qauv sib txawv. Unpaired, ob-tailed t-test feem ntau yog siv los xam qhov tseem ceeb ntawm cov pab pawg, qhov twg p < 0.05 raug suav hais tias yog qhov tseem ceeb

Txoj kev tshawb no qhia tau hais tias lubanti-aging - Klotho gene-activated cistanchemuaj kev tswj xyuas Ua kom muaj peev xwm rov ua dua tshiab ntawm ADSCs. Cov txiaj ntsig tseem ceeb ntawm txoj kev tshawb fawb no yog (1)Kev hloov pauv hloov pauv ntawm qia cell pluripotency thiab cov lus teb los tiv thaiv fibrotic, (2)txhim kho paracrine tswj ntawm angiogenesis, thiab pro-fibrotic collagen remodelingnyob rau hauv dermal fibroblasts, thiab thaum kawg, (3)nce maturation ntawm lub hauv paus membranenrog kev tswj hwm ntawm caws pliav-txuas nrog cov proteins cov lus qhia. Qhov tseeb, cov txiaj ntsig no qhiauas ADSCs-laden -Klotho gene-activated scaffold tuaj yeem muaj peev xwm ua tau zookho ntau yam kab mob dermal.

cov peev txheej, TXHUA, FJO thiab MBK; cov ntaub ntawv curation, TXHUA thiab MBK; sau ntawv- thawj daim ntawv npajtion, TXHUA; kev sau ntawv— tshuaj xyuas thiab kho, TXHUA, FJO thiab MBK; kev pom, TAG NRHO; kev saib xyuas,FJO thiab MBK; project tswj, MBK; Kev nrhiav nyiaj txiag, FJO thiab MBK Txhua tus kws sau ntawvtau nyeem thiab pom zoo rau cov ntawv luam tawm ntawm cov ntawv sau.

Kev tsis sib haum xeeb ntawm kev txaus siab:Cov kws sau ntawv tshaj tawm tias tsis muaj qhov tsis sib haum xeeb ntawm kev txaus siab.

Cov ntaub ntawv

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