骨髓间充质干细胞在溃疡性结肠炎大鼠模型体内的示踪研究

摘要

Objective To trace the homing of bone mesenchymal stem cells(BMSCs) in the ulcerative colitis (UC) rat mod-el in order to provide the experimental basis for transplantation of BMSCs to treat UC.Methods BMSCs of Sprague-Dawley (SD) male rats were cultured ,propagated in vitro and identified by flow cytometry.They were transfected with lentivirus-medi-ated green fluorescent protein (GFP) in order to mark the cells (Ad-GFP-BMSCs).SD female rats were randomly divided into three groups :control group ,model group ,Ad-GFP-BMSCs group.The UC rat model was induced by using 2 ,4 ,6-trinitro-benzene sulfonic acid (TNBS) ,and the disease activity index (DAI) was employed to score the rats in each group.Ad-GFP-BM-SCs were then transplanted to the female rats by tail intravenous injection.Colon specimens were collected a week later for path-ological assessment.The expression of GFP was measured by immunofluorescence and the expression of sex-determining region on the Y chromosome (SRY) gene by PCR.Results UC rat models were successfully established by using TNBS.The DAI sig-nificantly down-regulated in Ad-GFP-BMSCs group compared with model group (P<0.05).BMSCs steadily expressed the GFP after lentivirus-mediated GFP transduction.One week after tail intravenous injection ,immunofluorescence showed the expres-sion of GFP ,and PCR revealed the expression of SRY gene in Ad-GFP-BMSCs group.Conversely ,the expressions of GFP and SRY gene were not detectable in control group and model group.Conclusion Transplanted BMSCs can be tracked by lentivirus-mediated GFP transfection technology and by determining SRY gene.BMSCs can home to damaged colon tissue through tail in-travenous injection.These findings may provide an experimental basis for BMSCs transplantation to treat UC.%目的：追踪骨髓间充质干细胞（bone mesenchymal stem cells ，BMSCs）在溃疡性结肠炎大鼠模型体内的归巢情况，为BMSCs移植治疗溃疡性结肠炎提供实验基础。方法体外培养扩增Sprague-Dawley（SD）雄性大鼠BMSCs ，流式细胞术鉴定BMSCs ，采用慢病毒载体介导的绿色荧光蛋白（green fluorescent protein ，GFP）技术（lentivirus-GFP）对BMSCs进行荧光蛋白标记（Ad-GFP-BMSCs）。SD雌性大鼠被随机分为3组：空白组、模型组、Ad-GFP-BMSCs组。2，4，6-三硝基苯磺酸（2，4，6-trinitrobenzene sulfonic acid ，TNBS）诱导溃疡性结肠炎，疾病活动指数（disease activity index ， DAI）对各组大鼠进行评估，采用性别交叉移植的方法，尾静脉注射Ad-GFP-BMSCs于雌性大鼠体内，1周后收集结肠标本，对结肠标本进行病理学评估，免疫荧光检测结肠组织GFP表达，PCR检测Y染色体的性别决定区（sex-determining region on the Y chromosome ，SRY）基因。结果 TNBS能成功诱导溃疡性结肠炎，与模型组相比，Ad-GFP-BMSCs组DAI评分显著下降（P＜0.05）。Lentivirus-GFP成功标记SD大鼠的BMSCs ，转染后BMSCs能够稳定表达GFP蛋白，尾静脉注射1周，Ad-GFP-BMSCs组免疫荧光可检测到GFP表达，PCR可检测出SRY基因，空白组和模型组无GFP和SRY基因表达。结论慢病毒载体介导的GFP转染技术和SRY基因可以追踪移植的BMSCs ，而且BMSCs通过尾静脉注射可以归巢于受损的结肠组织，这可能为BMSCs移植治疗溃疡性结肠炎提供实验基础。