结肠炎大鼠炎症黏膜提取液对骨髓间质干细胞增殖和表面分化抗原的影响

摘要

背景:动物实验和临床试验均表明间质干细胞(MSCs)对受损肠道组织有一定修复作用,然而目前尚不清楚肠道炎症微环境对MSCs移植治疗炎症性肠病(IBD)有何影响.目的:通过体外实验观察结肠炎大鼠模型肠道炎症黏膜提取液对骨髓MSCs增殖和表面分化抗原的影响.方法:以全骨髓贴壁法分离和原代培养大鼠MSCs并行传代扩增.取三硝基苯磺酸(TNBS)结肠炎大鼠模型肠道炎症黏膜提取液(0、1、2、3 ml)与MSCs共培养,倒置相差显微镜观察各组细胞贴壁和生长情况,绝对计数法绘制细胞生长曲线,流式细胞术检测细胞表面分化抗原表达.结果:全骨髓贴壁法可成功分离MSCs,所获细胞CD29、CD44表达阳性,CD105表达低度阳性,CD34、CD45表达阴性.3 ml炎症黏膜提取液处理组MSCs接种6h后见细胞贴壁,36 h开始克隆性增生,此后细胞增殖速度较空白对照组明显加快,第6d即达100％融合,但表面分化抗原表达与空白对照组相比无明显变化.结论:结肠炎大鼠炎症黏膜提取液可促进骨髓MSCs增殖,但对细胞表面分化抗原无明显影响.%Previous animal and clinical studies demonstrated that mesenchymal stem cells ( MSCs) may enhance the recovery of damaged intestinal mucosa, however, the influence of gut inflammatory microenvironment on MSCs transplanted for treatment of inflammatory bowel disease (IBD) is not yet clarified. Aims: To investigate the effect of intestinal inflammatory mucosa extracts taken from experimental colitis model rats on proliferation and surface differentiation antigens of bone marrow-derived MSCs in vitro. Methods; Rats bone marrow-derived MSCs were isolated by whole bone marrow adherent method and then cultured primarily and passaged. The passaged MSCs were co-cultured with intestinal inflammatory mucosa extracts (0, 1 , 2, and 3 ml) taken from trinitrobenzenesulfonic acid (TNBS)-induced colitis model rats. Adherence and proliferation of MSCs were observed by inverted phase contrast microscope, cell growth curve was plotted by absolute counting method, and the expressions of surface differentiation antigens were analyzed by flow cytometry. Results; MSCs could be obtained successfully by whole bone marrow adherent method, and the MSCs obtained were CD29 + CD44 + CD105 + (low-grade) CD34- CD45-. MSCs co-cultured with 3 ml inflammatory mucosa extracts began to adhere to the culture flask 6 hours after inoculation, clonal proliferation was observed after 36 hours and then these MSCs proliferated more rapidly than blank control MSCs an d grew to 100% confluence on the 6th day. No significant difference was observed in expressions of surface differentiation antigens between 3 ml inflammatory mucosa extracts-treated MSCs and blank control MSCs. Conclusions; Inflammatory mucosa extracts from colitis rats can promote the proliferation of bone marrow-derived MSCs, but cannot affect the surface differentiation antigens.