谷氨酰胺联合脐血间充质干细胞移植在大鼠肠缺血再灌注损伤中的作用

Effects of glutamine in combination with umbilical cord blood mesenchymal stem cell transplantation on intestinal ischemia reperfusion injury in rats

目的探讨谷氨酰胺联合脐血间充质干细胞(MSCs)移植在大鼠肠缺血再灌注损伤中作用。方法体外复苏并培养脐血间充质干细胞移植前备用,观察CM-Di I荧光标记后脐血间充质干细胞的去向。80只SD大鼠随机分为正常对照组,缺血再灌注损伤组,谷氨酰胺组,MSCs移植组及联合组每组各15只。对照组采用生理盐水灌肠,损伤组采用TNB(S乙醇稀释)灌肠,在TNBS建模后1 h,谷氨酰胺组于尾静脉输入谷氨酰胺0.45 g/kg、MSCs移植组于尾静脉输入1×10^(10)/L脐血间充质干细胞悬液,联合组尾静脉输入谷氨酰胺0.45 g/kg+脐血间充质干细胞悬液1×10^(10)/L。通过ELISA法检测各组大鼠血清中肠脂肪酸结合蛋白(IFABP)、白介素-6(IL-6)、超氧化物歧化酶(SOD)的含量;各组于再灌注1 h、3 h后检测肠组织含水率;通过RT-PCR、Western blot观察大鼠肠黏膜上皮细胞caspase-3、NF-k B、Bcl-2在谷氨酰胺联合MSCs移植后的mRNA和蛋白的表达情况。结果通过荧光示踪法观察到移植的MSCs细胞分布于肠粘膜淋巴组织内和腺上皮细胞间,表明MSCs可能参与了肠缺血再灌注损伤的修复过程。各组大鼠血清中SOD、IFABP、IL-6的含量变化比较,损伤组血清中IFABP、IL-6的含量较对照组显著增加,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著减少,联合组减少更为明显,损伤组血清中SOD的含量较对照组显著减少,而谷氨酰胺组,MSCs移植组及联合组与之比较,则显著增高,联合组增高更为明显(P<0.05)。再灌注1 h和3 h,损伤组肠组织含水率均明显高于对照组;与损伤组相比,谷氨酰胺组、MSCs移植组及联合组肠组织含水率值均显著降低,联合组降低更为明显,而谷氨酰胺组、MSCs移植组差异无统计学意义(P>0.05)。与对照组比较,损伤组肠黏膜上皮细胞caspase-3、NF-k B的mRNA和蛋白表达明显上调,Bcl-2的mRNA和蛋白表达明显下调(P<0.05),而谷氨酰胺组、MSCs移植组及联合组与之比较,caspase-3、NF-k B的mRNA和蛋白表达明显下调,Bcl-2的mRNA和蛋白表达明显上调(P<0.05),谷氨酰胺组及MSCs移植组之间无统计学差异(P>0.05),但两组与联合组比较,差异明显(P<0.05)。结论谷氨酰胺组及MSCs移植后,明显减轻了大鼠肠缺血再灌注损伤程度,其可能通过抑制caspase-3、NF-k B表达和促进Bcl-2表达减轻肠黏膜缺血再灌注损伤。

Objective To investigate the effect of glutamine in combination with umbilical cord bloodmesenchymal stem cells（ MSCs） transplantation on intestinal ischemia-reperfusion injury in rats. Methods Umbilical cord blood mesenchymal stem cells were isolated,and were labeled with CM-Di I fluorescent dye. Eighty Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion injury group, glutamine group, MSCs transplantation group and combined group with 15 rats in each group. The control group received saline enema. The injury group was treated with TNBS（ ethanol dilution） enema. The glutamine group at 1 h after TNBS received intravenous injection of 0. 45 g / kg glutamine. The rats of MSCs transplantation group had tail vein injection of 1 × 10-（10）/ L umbilical cord blood mesenchymal stem cell suspension,and the combined group received intravenous injection of glutamine 0. 45 g /kg and 1 × 10-（10）/ L umbilical cord blood mesenchymal stem cell suspension. ELISA was used to detect the midgut fatty acid binding protein（ i FABP）,interleukin 6（ IL-6）,and superoxide dismutase（ SOD） content in the rat serum. The water content of intestinal tissue was detected at 1 h and 3 h after reperfusion in each group. The expressions of NF-k B,Bcl-2and caspase-3 mRNA and proteins in the rat intestinal epithelial cells after treated with glutamine in combination with MSCs were detected by RT-PCR and Western blot assays. Results The fluorescent tracer method revealed that the transplanted MSCs cells were distributed in the intestinal mucosal lymphoid tissues and glandular epithelial cells,indicating that MSCs might be involved in the repair process of intestinal ischemia-reperfusion injury. The content of serum IFABP and IL-6 in the injured group was significantly higher than that in the control group,while significantly reduced in the glutamine group,MSCs transplantation group and combined group,with the most obvious in the combined group. The content of SOD in the injury group was significantly lower than that in the control group,and significantly increased than that in the glutamine group,MSCs transplantation group,with the most striking in the combined group（ P〈0. 05 for all）. The water content of intestinal tissue in the injury group at 1 and 3 hours after reperfusion was significantly higher than that in the control group,significantly lower in the glutamine group,MSCs transplantation group and the combined group,with the most decreased in the combination group,and there was no significant difference between the glutamine group and MSCs transplantation group（ P〈0. 05）. Compared with the control group,the caspase-3 and NF-k B mRNA and protein expressions in the intestinal mucosal epithelial cells of the injury group were significantly increased,and the expressions of Bcl-2 mRNA and protein were significantly reduced（ P〈0. 05）,the expressions of caspase-3 and NF-k B mRNA and protein were significantly reduced in the glutamine group,MSCs transplantation group and combined group. The expressions of Bcl-2 mRNA and protein were significantly increased（ P〈0. 05）,while no significant difference was shown between the glutamine group and MSCs transplantation group（ P〈0. 05）,but there was a significant difference between these two groups and the combined group（ P〈0. 05）. Conclusions After treated with glutamine and MSCs transplantation,the degree of intestinal ischemia reperfusion injury is obviously reduced in rats. It may be mediated through inhibiting the expression of caspase-3 and NFk B and promoting the expression of Bcl-2.