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目的比较慢病毒转染绿色荧光蛋白(GFP)的骨髓间充质干细胞(BMSCs)和GFP转基因的BMSCs在大鼠肝缺血再灌注损伤修复中修复时效性及荧光稳定性的差异。方法常规体外培养2种BMSCs,MTT法检测二种细胞生长曲线间的异同;将40只SD大鼠随机分为假手术组、模型组、慢病毒转染GFP组(LV-GFP组)和GFP转基因组(GFP-BMSCs组),造模后LV-GFP组及GFP-BMSCs组于门静脉立即注入相应细胞悬液200μl(数量约1×106个),模型组注入等体积的PBS溶液。于术后1、2、3、4周检测4组大鼠天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)及血清白蛋白(ALB)水平;于术后1~5 d切取实验组肝脏组织检测BMSCs入肝情况;于术后4周切取实验组肝脏组织检测BMSCs的荧光稳定性及其肝角蛋白18(CK18)的表达情况。结果 GFP转基因大鼠的BMSCs在对数期的增殖能力明显强于慢病毒转染GFP的BMSCs(P<0.05);术后1、2周GFP-BMSCs组AST及ALT水平明显低于LV-GFP组(P<0.05),术后2、3周GFP-BMSCs组ALB水平明显高于LV-GFP组(P<0.05);GFP-BMSCs组与LV-GFP组分别于术后3、5 d在肝区内见到GFP标记的BMSCs细胞;GFP-BMSCs及LV-GFP组都可于术后4周在肝区内见到已分化为肝细胞的BMSCs细胞,但GFP-BMSCs组BMSCs的荧光强度明显优于LV-GFP组。结论GFP转基因大鼠的BMSCs在大鼠肝缺血再灌注损伤修复中较慢病毒转染GFP的BMSCs展现出较好的修复时效性及荧光稳定性。
OBJECTIVE: To compare the timeliness and fluorescence stability of BMSCs transfected with green fluorescent protein (GFP) and BMSCs transfected with GFP gene in liver ischemia-reperfusion injury in rats. Methods Two kinds of BMSCs were cultured in vitro. MTT assay was used to detect the difference between the two cell growth curves. Forty SD rats were randomly divided into sham operation group, model group, lentivirus-transfected GFP group (LV-GFP group) and GFP In the GFP-BMSCs group, LV-GFP group and GFP-BMSCs group were injected with 200μl of the appropriate cell suspension (about 1 × 106 cells) in the portal vein. The model group was injected with an equal volume of PBS solution. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and serum albumin (ALB) were measured at 1, 2, 3, The liver tissues of the experimental group were cut out for 5 days to detect the infiltration of BMSCs into the liver. The liver tissues of the experimental group were cut off for 4 weeks to detect the fluorescence stability and the expression of hepatic keratin 18 (CK18). Results The proliferation of BMSCs in GFP transgenic mice was significantly stronger than that of GFP-transfected BMSCs in logarithmic phase (P <0.05). The levels of AST and ALT in GFP-BMSCs were significantly lower than those in LV-GFP (P <0.05). The levels of ALB in GFP-BMSCs were significantly higher than those in LV-GFP groups at 2 and 3 weeks after operation (P <0.05). The levels of ALB in GFP-BMSCs group and LV- GFP-labeled BMSCs were seen in the liver area. BMSCs differentiated into hepatocytes were seen in the liver area in GFP-BMSCs and LV-GFP group at 4 weeks after operation. However, the fluorescence intensity of BMSCs in GFP-BMSCs group Obviously superior to LV-GFP group. Conclusion The BMSCs transfected with GFP in transgenic GFP transgenic rats showed a better recovery time and higher fluorescence stability in the repair of liver ischemia-reperfusion injury in rats.