Ntu Ⅱ Smad3 Deficiency Txhim Kho Islet-based Therapy Rau Mob Ntshav Qab Zib Thiab Mob Ntshav Qab Zib Raum Kev Kho Mob Los Ntawm Kev Txhawb Cell Proliferation Ntawm E2F3-dependent Mechanism
May 25, 2023
Cov txheej txheem
1. Tsiaj
Smad3-null (Smad3KO) nas yog ib qho khoom plig zoo los ntawm Prof. Chuxia Deng thiab khaws cia hauv heterozygotes (Smad3 ntxiv /- ) hauv C57BL/6 keeb kwm yav dhau [20]. Smad3KO nas tau tsim los ntawm kev sib tshuam ntawm Smad3 ntxiv /- nas. Cov nas db / db nrog kev hloov pauv ntawm Lepr (Leptin receptor) hauv C57BLKS keeb kwm yav dhau tau yuav los ntawm Tsiaj Chaw Tshawb Fawb ntawm Suav University of Hong Kong (CUHK). Txhua tus tsiaj tau khaws cia rau hauv qhov chaw tshwj xeeb uas tsis muaj kab mob (SPF) nrog kev nkag tau dawb rau zaub mov thiab dej ntawm ib hnub / hmo lub voj voog ntawm 12/12 teev. Tag nrho cov kev tswj xyuas tsiaj hauv qhov kev tshawb fawb no tau ua raws li cov kev cai ntawm thiab pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Tsiaj Kev sim ntawm CUHK nrog rau Ref. No. 18-177-MIS.
2. Streptozocin (STZ)-induced mob ntshav qab zib nas qauv
Txiv neej C57BL/6 nas thaum muaj hnub nyoog 8-10 lub lis piam tau txhaj tshuaj intraperitoneally nrog 50 mg / kg / hnub STZ txhua hnub rau 5 hnub tas mus li. 5 hnub tom qab kev txhaj tshuaj STZ zaum kawg, cov nas uas muaj cov ntshav qabzib ruaj khov ntawm ntau dua 16 mM rau 2 hnub sib law liag tau suav tias yog kev txhim kho cov ntshav qab zib tau zoo thiab siv los ua cov nas tau txais kev hloov pauv hauv islet.
3. Islet rho tawm, hloov pauv, thiab kev soj ntsuam ntawm cov ntshav qab zib phenotype
Islets raug cais los ntawm txiv neej lossis poj niam Smad3 knockout (KO) lossis cov nas qus (WT) thaum muaj hnub nyoog 8-10 lub lis piam raws li tau piav qhia yav dhau los [21]. Txhawm rau ua cov islet hloov pauv, qhia tias ntau lub islets tau hloov pauv hauv lub raum tshuaj ntawm cov txiv neej db / db nas thaum muaj hnub nyoog 4 lossis STZ-vim cov nas mob ntshav qab zib hnub 7 tom qab txhaj tshuaj STZ zaum kawg ua raws li cov txheej txheem piav qhia los ntawm Szot et al [ 22] ib. Luv luv, tus neeg txais nas tau tshuaj loog los ntawm cov tshuaj loog Combo (1.5 feem pua ketamine thiab 0.15 feem pua xylazine, 6 μL / g lub cev hnyav). Tom qab tsis nco qab, ib qho kev txiav ntawm 1 cm tau ua rau ntawm daim tawv nqaij npog sab laug raum. Tshem tawm lub raum maj mam thiab ua kom nws ntub nrog tsis muaj ntsev ntsev thaum lub sijhawm phais. Nrog 25G koob, ua ib qho me me ntawm cov tshuaj ntsiav los tsim ib lub npe. Cov lej ntawm cov islets raug xa mus rau hauv lub raum tshuaj ntsiav los ntawm PE50 tubing. Tom qab tag nrho, cov islets tau transplanted, kaw lub nick los ntawm cauterization nrog tsawg tshav kub. Hloov lub raum thiab kaw lub peritoneum nrog sutures. Seal daim tawv nqaij nrog staples. 100 μL Penicillin-streptomycin tov (Gibco, cat# 15140122, USA) tau txhaj tshuaj intraperitoneally los tiv thaiv kab mob. 100 μL Tegmic tau txhaj tshuaj intramuscularly los txo qhov mob. Tus tsiaj tau muab tso rau hauv qhov chaw sov kom txog thaum rov zoo tag nrho. Tib txoj kev ua haujlwm kuj tau ua nyob rau hauv sham tswj nas tab sis tsis muaj islet infusion. Ob qho tib si random thiab 6 h-fasting ntshav qabzib (RBG thiab FBG) qib ntawm tus Tsov tus tw ntshav tau kuaj txhua lub limtiam nrog lub ntsuas ntshav qabzib (Roche, ACCU-CHEK® Performa). Cov tshuaj insulin tsis kam (IPITT), glucose tolerance test (IPGTT), qib HbA1c, thiab cov tshuaj insulin hauv cov ntshav tau ntsuas raws li tau piav qhia dhau los [9].
Nyem qhov no kom tau txaiscov txiaj ntsig Cistanche
4. Tag nrho cov zis protein thiab ntshav creatinine
24 teev cov zis tau sau rau hauv lub tawb metabolic. 24 teev tag nrho cov zis tso zis tau suav los ntawm kev sib npaug ntawm cov zis ntim thiab cov zis muaj protein ntau uas tau txiav txim siab los ntawm Quick Start Bradford Protein Assay Kit (Bio-Rad, cat# 5000201, USA). Serum creatinine tau txiav txim siab nrog cov khoom siv creatinine kom muaj nuj nqis (Stanbio Laboratory, cat# 0430-120, USA).
5. Histopathology
Lub raum histopathological hloov pauv tau raug tshuaj xyuas hauv paraformaldehyde-taw, paraffin-embedded seem (4 µm) nrog Periodic Acid-Schiff's (PAS) reagent raws li tau piav qhia yav dhau los [23]. Mesangial matrix expansion tau qhab nia siv qhov kev pab cuam duab ntau (Image-Pro Plus 7.0, Media Cybernetics) raws li tau piav qhia yav dhau los [23].
6. Immunohistochemistry
Islets-cov kabmob raum lossis txiav txiav tau kho nyob rau hauv 4 feem pua ntawm paraformaldehyde, embedded hauv paraffin, thiab ntu ntawm 4 μm. Tom qab rov ua dej, cov ntu tau kho nrog microwave-mediated antigen retrieval hauv 0.01 M citrate (pH 6.0) rau 10 min thiab thaiv hauv 5 feem pua BSA rau 1 h . Thawj cov tshuaj tiv thaiv kab mob incubation tau ua thaum hmo ntuj ntawm 4 degree. Rau fluorescent staining, fluorescent Secondary antibody yog siv nyob rau hauv chav tsev kub (RT) rau 1 h. Rau DAB-mediated thermogenesis, ib kauj ruam ntxiv ntawm 3 feem pua H2O2 thaiv rau 15 min tau suav nrog ua ntej cov tshuaj tua kab mob. Tom qab thawj cov tshuaj tiv thaiv kab mob incubation, HRP-conjugated polymer (Envision plus System, Dako, USA) tau ntxiv. Rau staining ntawm E2F3, antigen retrieval tau ua nyob rau hauv EDTA-Tris tov (pH 9.0). Cov tshuaj tiv thaiv uas siv hauv immunohistochemistry tau piav qhia hauv Table S2. Qhov chaw staining-zoo yog quantitated nrog Image J software (v1.47, NIH, USA).
Cistanche extract
7. Kev ntsuas ntawm grafted cell loj nyob rau hauv lub islets-cov kabmob raum
Ib qho Performance index ntawm thaj tsam nruab nrab ntawm tes ib ntu tau siv los ua ib nrab kom muaj nuj nqis ntawm cov cell hauv cov islet graft. Luv luv, cov islets-cov kabmob raum (paraffin-embedded) tau muab faib ua ntu zus ntawm 4 μm raws sagittal axis. Nruam ntu ntawm 160 μm ib ntus tau fluorescently immunostained rau insulin. Tsawg kawg yog 6 ntu nrog cov tshuaj insulin-zoo staining tau siv rau kev suav ntawm thaj chaw ntawm tes hauv ib ntu hauv txhua tus nas. Qhov chaw ntawm cov tshuaj insulin-zoo hauv txhua ntu tau suav nrog Image J software (v1.47, NIH, USA). Qhov Performance index ntawm thaj chaw nruab nrab ntawm tes hauv ib ntu hauv txhua tus nas tau suav los ntawm kev faib tag nrho cov tshuaj insulin-zoo rau hauv txhua ntu los ntawm cov lej ntawm cov tshuaj insulin-zoo.
8. Thawj kab lis kev cai ntawm islet cells
Cov islets cais tau muab faib ua ib lub hlwb thiab muab noob rau hauv 96-zoo phaj hauv 200 μL ntawm RPMI 1640 (Gibco, USA) ntxiv rau 10 feem pua FBS (Gibco, USA) ntxiv rau 1 feem pua Penicillin-streptomycin (Gibco, USA) ntxiv nrog 20 mM qabzib. Islet hlwb raug coj mus kuaj rau lub sijhawm teev nyob rau hauv lub incubator teem ntawm 37 degree, 5 feem pua CO2, thiab 100 feem pua cov av noo. Rau adenovirus-mediated knockdown ntawm E2F3, dissociated islet cells raug coj mus rau hauv ib qho nruab nrab uas muaj adenovirus overexpressing luv hairpin RNA (shRNA) targeting nas E2F3 (shE2F3) los yog tswj tsis-specific shRNA (pom) ntawm ib titer ntawm 10 pfu ib cell rau 2. hnub ua raws li qhov nruab nrab hloov. Cov kab ke ntawm shE2F3 duplex yog 5'-CATTCAATGCTCTATTTGTGT-3'. Dispersed hlwb los ntawm 50 islets tau noob ib lub qhov dej rau kev tiv thaiv kab mob thiab RT-PCR, thiab 100 islets ib qhov dej rau Western blot, uas tau ua raws li tau piav qhia yav dhau los [9].
9. Hauv vivo thiab hauv vitro BrdU daim ntawv lo
Txhawm rau ua hauv vivo BrdU labeling, nas tau txhaj tshuaj intraperitoneally nrog 50 mg / kg / hnub BrdU (hauv PBS) rau 7 hnub txij hnub thib 2 tom qab hloov cov islet thiab txi 2 teev tom qab txhaj tshuaj kawg. Rau hauv vitro BrdU labeling, dispersed islet cells tau coj mus kuaj rau 48 h tom qab hloov nrog cov nruab nrab tshiab uas muaj 10 μM BrdU thiab kab lis kev cai ntxiv rau 24 teev. Fluorescent immunostaining tiv thaiv BrdU hauv cov ntaub so ntswg thiab kab mob hlwb tau ua raws li tau piav qhia yav dhau los [9].
Cistanche hmoov
10. RNA-Seq
Islets raug cais los ntawm Smad3WT-db/db, Smad3KO-db/db, Smad3WT-db/m, thiab Smad3KO-db/m nas. Rau txhua tus genotype, cov islets ntawm ntau dua 5 nas tau sib sau ua ke rau RNA cais. RNA tau npaj nrog miRNeasy Mini Kit (Qiagen, Lub teb chaws Yelemees). RNA-seq thiab cov ntaub ntawv downstream tsom xam tau piav qhia yav dhau los [9]. Cov theem qhia ntawm cov noob tau muab nthuav tawm raws li qhov tawg ntawm ib kilobase ntawm cov ntawv sau ib lab tau nyeem (FPKM). Qhov sib txawv ntawm cov noob (DEG) tau txhais tias ntau dua lossis sib npaug rau 2 folds qhov sib txawv ntawm FPKM ntawm ob pawg. Upregulated thiab downregulated DEGs tau ua ke thiab siv rau Gene Ontology (GO) thiab Kyoto Encyclopedia ntawm Genes thiab Genomes (KEGG) txoj kev txhim kho kev txheeb xyuas nrog rau hauv online DAVID bioinformatics cov chaw (v6.8) nrog rau qhov chaw pib. GOTERM_BP_DIRECT sub-category tau ua haujlwm rau GO tsom xam.
11. Immunoprecipitation
Txhawm rau ua cov tshuaj tiv thaiv kab mob hauv nas islets, kwv yees li 500 cov islets tshiab uas tau cais tawm tau lysed hauv 500 mL ntawm RIPA tsis. Ib tug luav anti-Smad3 antibody (2 ug) los yog luav IgG isotype tau ntxiv thiab incubated ntawm 4 degree ib hmos nrog maj mam tig. Tom qab ntawd, 20 μL ntawm cov protein A agarose hlaws (Beyotime, miv # P2051, Tuam Tshoj) tau ntxiv thiab incubated rau 2 h ntawm 4 degree. Tom qab ntxuav ribustly nrog tshiab RIPA tsis, cov bounded protein tau tso tawm los ntawm denaturing nyob rau hauv 1xSDS loading tsis thiab raug rau Western blot tsom xam. Txhawm rau kom tsis txhob cuam tshuam ntawm IgG cov saw hnyav, lub teeb saw hlau tshwj xeeb nas tiv thaiv luav IgG (HRP conjugated, Abmart, miv # M21006, Tuam Tshoj) tau siv los paub txog cov protein ntau Smad3 precipitated.
12. Chromatin immunoprecipitation-ChIP
Txhawm rau ua CIP hauv nas islets, kwv yees li 2000 islets tau sau los ntawm 8-10 lub lis piam qub C57BL/6 nas thiab siv los ua ib qho kev npaj ChiP. Cov islets tshiab uas raug rho tawm tau raug txhawb nrog 10 ng/mL TGF- 1 (Gibco, USA) rau 15 feeb hauv RPMI 1640 ntxiv rau 1 feem pua FBS ntxiv rau 1 feem pua Penicillin-streptomycin. Chip tau ua nrog SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST, USA) ua raws li cov lus qhia pom zoo. Cov tshuaj tiv thaiv kab mob thiab cov tshuaj primers siv nyob rau hauv qhov kev ntsuam xyuas CIP tau piav qhia hauv Table S1 thiab S2.
Standardized Cistanche
13. Dual-luciferase reporter assay
Mouse E2F3 tus txhawb nqa (-1000 rau ntxiv rau 451 bp) tau cloned rau hauv pGL3-cov vector yooj yim (pGL3-E2F3.Pro). Concurrently, taw tes hloov pauv ntawm Smad3 khi qhov chaw tau ua los tsim cov pGL3- E2F3.Pro. Mut. Txhawm rau ua qhov kev ntsuam xyuas dual-luciferase tus neeg sau xov xwm, HEK293T hlwb tau muab noob rau hauv 24-zoo phaj ntawm qhov ntom ntawm 5 x 104 hlwb/zoo. Hnub tom qab, cov hlwb tau hloov nrog 500 ng pGL3-Basic lossis pGL3-E2F3.pro lossis pGL3-E2F3.pro.mut ua ke nrog pcDNA3.1-Smad3, uas overexpresses nas Smad3, los yog tsis (raws li qhia nyob rau hauv daim duab 10F) los ntawm phosphate calcium txoj kev. PEGFP-C1 vector tau ntxiv kom sib npaug ntawm cov tshuaj hloov pauv hauv txhua qhov kev kho mob. 10 ng pGL4.73.huc/SV40 vector qhia renilla luciferase tau suav nrog hauv txhua qhov kev kho mob kom normalize qhov kev hloov pauv tau zoo. Tom qab 6 teev tom qab hloov pauv, qhov nruab nrab tau hloov pauv tom qab kev tsim kho rau 48 teev ntxiv. Cov hlwb tau lysed, thiab cov kev ua ntawm luciferase tau txiav txim siab nrog Dual-Luciferase ® Reporter Assay System (Promega, USA) thiab muab xam rau hauv lub luminometer (VICTORTM X4 2030 Multilabel Reader, PerkinElmer, USA). Cov kev ua haujlwm ntawm cov cloned E2F3 txhawb nqa tau nthuav tawm raws li tus nqi piv ntawm firefly luciferase rau renilla luciferase. Txhua qhov kev kho mob tau ua hauv triplicates.
14. Kev txheeb cais
Cov ntaub ntawv tau nthuav tawm raws li qhov txhais tau tias ± SD thiab txheeb xyuas nrog SPSS software (16.0). Lub normality ntawm cov ntaub ntawv raug kuaj los ntawm ib tug qauv Kolmogorov-Smirnov xeem. Qhov sib txawv ntawm qhov sib txawv ntawm cov pab pawg tau sim los ntawm Levene statistic. Rau kev sib piv ntawm ob pawg, Cov Tub Ntxhais Kawm T-test tau siv. Rau kev sib piv ntawm ntau pawg, ib-txoj kev ANOVA nrog LSD xeem tau siv yog tias qhov sib npaug sib npaug. Rau qhov sib npaug sib npaug uas tsis tau xav, lwm qhov kev ntsuas Welch tau siv los ntawm ntau qhov kev sib piv nrog Tamhane's T2 xeem. P < 0.05 tau suav tias yog qhov tseem ceeb.
Cistanche ntxiv
Cov lus xaus
Smad3 deficiency feem ntau txhim kho islet hloov kho rau ob qho tib si T1DM thiab T2DM thiab tiv thaiv kev mob ntshav qab zib raum. Enhanced E2F3-dependent cell proliferation tej zaum yuav yog ib qho tseem ceeb ntawm Smad3KO islet therapy nyob rau hauv ob qho tib si T1DM thiab T2DM. Yog li, Smad3KO cell replacement therapy tej zaum yuav yog ib qho kev kho mob zoo dua rau kev kho mob ntshav qab zib.
Cov ntaub ntawv
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Hong-Lian Wang1,2, Biao Wei2, Hui-Jun He2, Xiao-Ru Huang2,3, Jing-Yi Sheng2,4, Xiao-Cui Chen2,5, Li Wang1, Rui-Zhi Tan1, Jian-Chun Li1, Jian Liu 1, Si-Jin Yang6, Ronald CW Ma2, Hui-Yao Lan 2,7
1 Lub Chaw Tshawb Fawb Txog Kev Siv Tshuaj thiab Lub Tsev Haujlwm ntawm Nephrology, Affiliated Traditional suav Tshuaj Tsev Kho Mob ntawm Southwest Medical University, Luzhou, Sichuan, 646000, Suav.
2. Department of Medicine and Therapeutics, thiab Li Ka Shing Institute of Health Sciences, Suav University of Hong Kong, Hong Kong, 999077, Suav.
3. Guangdong-Hong Kong Joint Laboratory on Immunological and Genetic Kidney Diseases, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, 510080, Suav teb.
4. State Key Laboratory ntawm Bioelectronics, Jiangsu Key Laboratory rau Biomaterials thiab Devices, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing, Suav.
5. Lub Tuam Txhab Tseem Ceeb ntawm Kev Tiv Thaiv thiab Kev Tswj Xyuas Kab Mob Raum Kab Mob ntawm Zhanjiang City, Lub Tsev Kawm Ntawv ntawm Nephrology, Affiliated Tsev Kho Mob ntawm Guangdong Medical University, Zhanjiang, Guangdong, 524001, Tuam Tshoj.
6. National Traditional Chinese Medicine Clinical Research Base, Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, 646000, Suav.
7. CUHK-Guangdong Provincial People's Hospital Joint Research Laboratory on Immunological and Genetic Kidney Diseases, Suav University of Hong Kong, Hong Kong, 999077, China.
