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

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。

Effects of Cat AD-MSCs and Its Conditioned Medium on Apoptosis and Proliferation of CRFK Damaged by Gentamicin

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.