猫脂肪间充质干细胞及其条件培养基对庆大霉素致损猫肾细胞增殖和凋亡的影响

doi:10.16431/j.cnki.1671-7236.2021.07.041

摘要/Abstract

摘要： 试验旨在探究猫脂肪间充质干细胞（adipose mesenchymal stem cells，AD-MSCs）及其条件培养基（adipose mesenchymal stem cells conditioned medium，AD-MSCs-CM）对庆大霉素致损的猫肾细胞（crandell rees feline kidney，CRFK）增殖和凋亡的影响及机制。试验分为空白组、庆大霉素（gentamicin，GM）组、AD-MSCs组和AD-MSCs-CM组；用Ⅰ型胶原酶消化法分离猫AD-MSCs，并用流式细胞术鉴定分离的细胞；以0、2、4、8 mmol/L GM完全培养基处理CRFK，并于12、24和48 h用CCK-8法检测细胞活性，筛选最适造模浓度；致损的CRFK分别与猫AD-MSCs及AD-MSCs-CM共培养，于24、48和72 h后用CCK-8法检测细胞增殖活性，24 h后通过Annexin V流式细胞术检测细胞凋亡率，最后通过实时荧光定量PCR检测细胞周期蛋白D1（cyclin d1，CCND1）、细胞增殖核抗原（proliferative nuclear antigen，PCNA）、Bax和Bcl-2基因的表达水平。结果显示，分离培养的猫AD-MSCs在体外培养条件下呈典型的梭型，高表达MSCs表面标记物CD44、CD90和CD105，不表达白细胞表面标记物CD45；AD-MSCs和AD-MSCs-CM均能促进GM致损CRFK的增殖并抑制其凋亡，其中AD-MSCs和AD-MSCs-CM可通过上调增殖相关基因CCND1、PCNA和抗凋亡基因Bcl-2的表达并降低促凋亡基因Bax的表达而发挥促增殖、抑凋亡作用。试验成功分离得到猫AD-MSCs，并证实AD-MSCs及其条件培养基在体外能促进GM致损CRFK的增殖和迁移，为猫AD-MSCs治疗猫急性肾损伤提供依据。

关键词: 猫; 间充质干细胞; 庆大霉素; 急性肾损伤; 凋亡

Abstract: The aim of the present study was to explore the effect and mechanism of cat adipose mesenchymal stem cells (AD-MSCs) and adipose mesenchymal stem cells conditioned medium (AD-MSCs-CM) on the apoptosis of crandell rees feline kidney (CRFK) damaged by gentamicin.This experiment was divided into 4 groups,which were control group,gentamicin (GM) group,AD-MSCs group and AD-MSCs -CM group.Type Ⅰ collagenase digestion method was used to separate cat AD-MSCs,and identified by flow cytometry identification.CRFK was treated with GM complete medium at concentrations of 0,2,4 and 8 mmol/L,and the cell activity was detected by CCK-8 assay at 12,24 and 48 h to screen the optimal concentration for model making.The damaged CRFK was co-cultured with cat AD-MSCs and AD-MSCs-CM,respectively.After 24,48 and 72 h,the proliferation activity of the cells was detected by CCK-8 assay,and the apoptosis rate was detected by Annexin V flow tometry 24 h later.Finally,the expression levels of cyclin D1 (CCND1),proliferative nuclear antigen (PCNA),Bax and Bcl-2 genes were detected by Real-time quantitative PCR.The results showed that in vitro cultured cat AD-MSCs presented typical long spindle shape,with high expression of MSCs surface markers CD44,CD90 and CD105,but no expression of leukocyte surface marker CD45.The results suggested that AD-MSCs and AD-MSCs-CM enhanced the proliferative activity and decreased the apoptosis rate of damaged CRFK cells by promoting the expression of proliferation gene CCND1,PCNA and anti-apoptotic gene Bcl-2 of GM-damaged CRFK cells and reducing the expression of pro-apoptotic gene Bax.In this experiment,cat AD-MSCs were successfully isolated,and it was confirmed that Ad-MSCs and its conditional medium could promote the proliferation and migration of GM-induced CRFK in vitro,which provided a basis for the treatment of cat AD-MSCs for acute kidney injury.

Key words: cat; mesenchymal stem cells; gentamicin; acute renal injury; apoptosis

中图分类号:

R319

江文康, 罗冬章, 赵明明, 罗惠娜, 樊全宝, 王粹琳, 阮慧敏, 朱海琦, 陈胜锋, 王丙云. 猫脂肪间充质干细胞及其条件培养基对庆大霉素致损猫肾细胞增殖和凋亡的影响[J]. 中国畜牧兽医, 2021, 48(7): 2644-2652.

JIANG Wenkang, LUO Dongzhang, ZHAO Mingming, LUO Huina, FAN Quanbao, WANG Cuilin, RUAN Huimin, ZHU Haiqi, CHEN Shengfeng, WANG Bingyun. Effects of Cat AD-MSCs and Its Conditioned Medium on Apoptosis and Proliferation of CRFK Damaged by Gentamicin[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2644-2652.

参考文献

[1] 罗冬章,罗惠娜,詹小舒,等.猫4种不同来源间充质干细胞的生物学特性比较[J].生物技术通报,2019,35(7):39-45. LUO D Z,LUO H N,ZHAN X S,et al.Comparison of biological characteristics of feline mesenchymal stem cells derived from four different tissues[J].Biotechnology Bulletin,2019,35(7):39-45.(in Chinese)
[2] ARZI B,MILLS-KO E,VERSTRAETE F J,et al.Therapeutic efficacy of fresh,autologous mesenchymal stem cells for severe refractory gingivostomatitis in cats[J].Toxicology and Industrial Health,2016,5(1):75-86.
[3] TRZIL J E,MASSEAU I,WEBB T L,et al.Intravenous adipose-derived mesenchymal stem cell therapy for the treatment of feline asthma:A pilot study[J].Journal of Feline Medicine and Surgery,2016,18(12):981-990.
[4] PENHA E M,AGUIAR P H,BARROUIN-MELO S M,et al.Clinical neurofunctional rehabilitation of a cat with spinal cord injury after hemilaminectomy and autologous stem cell transplantation[J].International Journal of Stem Cells,2012,5(2):146-150.
[5] VILLATORO A J,CLAROS S,FERNANDEZ V,et al.Safety and efficacy of the mesenchymal stem cell in feline eosinophilic keratitis treatment[J].BMC Veterinary Research,2018,14(1):116.
[6] WEBB T L,WEBB C B.Stem cell therapy in cats with chronic enteropathy:A proof-of-concept study[J].Journal of Feline Medicine and Surgery,2015,17(10):901-908.
[7] THOMSON A L,BERENT A C,WEISSE C,et al.Intra-arterial renal infusion of autologous mesenchymal stem cells for treatment of chronic kidney disease in cats:Phase Ⅰ clinical trial[J].Journal of Veterinary Internal Medicine,2019,33(3):1353-1361.
[8] LIM C Y,HAN J I,KIM S G,et al.Evaluation of autologous bone marrow-derived mesenchymal stem cells on renal regeneration after experimentally induced acute kidney injury in dogs[J].American Journal of Veterinary Research,2016,77(2):208-217.
[9] MONTEIRO B S,SANTOS B,ALMEIDA B L,et al.Adipose tissue derived mesenchymal stem cell transplantation in the treatment of ischemia/reperfusion induced acute kidney injury in rats.Application route and therapeutic window1[J].Acta Cirurgica Brasileira,2018,33(11):1016-1026.
[10] QI S,WU D.Bone marrow-derived mesenchymal stem cells protect against cisplatin-induced acute kidney injury in rats by inhibiting cell apoptosis[J].International Journal of Molecular Medicine,2013,32(6):1262-1272.
[11] ZHANG J B,WANG X Q,LU G L,et al.Adipose-derived mesenchymal stem cells therapy for acute kidney injury induced by ischemia-reperfusion in a rat model[J].Clinical and Experimental Pharmacology & Physiology,2017,44(12):1232-1240.
[12] REIS L A,BORGES F T,SIMOES M J,et al.Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats[J].PLoS One,2012,7(9):e44092.
[13] 杞少华.骨髓间充质干细胞对顺铂所致大鼠急性肾损伤的保护作用和分子机制[D].武汉:武汉大学,2013. QI S H.Bone marrow derived mesenchyml stem protect against cisplatin induced acute kidney injury in rats by inhibiting cell apoptosis[D].Wuhan:Wuhan University,2013.(in Chinese)
[14] MOGHADASALI R,MUTSAERS H A,AZARNIA M,et al.Mesenchymal stem cell-conditioned medium accelerates regeneration of human renal proximal tubule epithelial cells after gentamicin toxicity[J].Experimental and Toxicologic Pathology,2013,65(5):595-600.
[15] RANDJELOVIC P,VELJKOVIC S,STOJILJKOVIC N,et al.Gentamicin nephrotoxicity in animals:Current knowledge and future perspectives[J].EXCLI Journal,2017,16:388-399.
[16] COUTINHO A,BISCAIA S,FERNANDEZ R,et al.The aminoglycoside antibiotic gentamicin is able to alter metabolic activity and morphology of MDCK-C11 cells:A cell model of intercalated cells[J].Brazilian Journal of Medical and Biological Research,2018,51(10):e7417.
[17] 严磊,张绘艳,杨小康,等.庆大霉素致原代大鼠肾小管上皮细胞凋亡的研究[J].畜牧与兽医,2017,49(7):46-49. YAN L,ZHANG H Y,YANG X K,et al.Apoptosis of primary renal epithelial cells in rats induced by gentamicin[J].Animal Husbandry & Veterinary Medicine,2017,49(7):46-49.(in Chinese)
[18] BERNHARDT W M,ECKARDT K U.Physiological basis for the use of erythropoietin in critically ill patients at risk for acute kidney injury[J].Current Opinion in Critical Care,2008,14(6):621-626.
[19] HAMMERMAN M R.Potential role of growth factors in the prophylaxis and treatment of acute renal failure[J].Kidney International Supplements,1998,64:S19-S22.
[20] MARIN-GREZ M,FLEMING J T,STEINHAUSEN M.Atrial natriuretic peptide causes pre-glomerular vasodilatation and post-glomerular vasoconstriction in rat kidney[J].Nature,1986,324(6096):473-476.
[21] 倪雁君.多巴胺在犬急性肾损伤治疗中的疗效验证[D].杨凌:西北农林科技大学,2018. NI Y J.Diagnosis and treatment of acute kidney injury in dogs and analysis of clinical application of dopamine[D].Yangling:Northwest A&F University,2018.(in Chinese)
[22] HUMPHREYS B D,BONVENTRE J V.Mesenchymal stem cells in acute kidney injury[J].Annual Review of Medicine,2008,59:311-325.
[23] TOGEL F,ZHANG P,HU Z,et al.VEGF is a mediator of the renoprotective effects of multipotent marrow stromal cells in acute kidney injury[J].Journal of Cellular and Molecular Medicine,2009,13(8B):2109-2114.
[24] 宋洁.骨髓间充质干细胞对横纹肌溶解致急性肾损伤大鼠肾脏保护作用及机制研究[D].天津:天津医科大学,2016. SONG J.The protective effect and mechanism of bone marrow mesenchymal stem cells against rhabdomyolysis-induced acute kidney injury in rats[D].Tianjin:Tianjin Medical University,2016.(in Chinese)
[25] 董超.骨髓间充质干细胞联合维生素E对急性肾损伤的治疗研究[D].长春:吉林大学,2014. DONG C.Study on therapy effect of bone marrow derived mesenchymal stem cell combined vitamin E against acute kidney injury[D].Changchun:Jilin University,2014.(in Chinese)
[26] CHIABOTTO G,BRUNO S,COLLINO F,et al.Mesenchymal stromal cells epithelial transition induced by renal tubular cells-derived extracellular vesicles[J].PLoS One,2016,11(7):e159163.
[27] BANU K,MITRA P,SUBBARAO N,et al.Role of tyrosine residue (Y213) in nuclear retention of PCNA1 in human malaria parasite Plasmodium falciparum[J].FEMS Microbiology Letters,2018,365(17):fny182.
[28] ASHKENAZI A,FAIRBROTHER W J,LEVERSON J D,et al.From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors[J].Nature Reviews Drug Discovery,2017,16(4):273-284.
[29] LI S Y,QI Y,HU S H,et al.Mesenchymal stem cells-conditioned medium protects PC12 cells against 2,5-hexanedione-induced apoptosis via inhibiting mitochondria-dependent Caspase 3 pathway[J].Toxicology and Industrial Health,2017,33(2):107-118.