两种示踪方式的骨髓间充质干细胞在促进肝缺血再灌注损伤修复中的对比研究

doi:10.13418/j.issn.1001-165x.2016.03.016

中国临床解剖学杂志 ›› 2016, Vol. 34 ›› Issue (3): 312-317.doi: 10.13418/j.issn.1001-165x.2016.03.016

两种示踪方式的骨髓间充质干细胞在促进肝缺血再灌注损伤修复中的对比研究

杨青，　秦书俭，　包翠芬，　单伟

锦州医科大学人体解剖与组织胚胎教研室，辽宁锦州 121000

收稿日期:2015-07-27 出版日期:2016-05-25 发布日期:2016-06-17
通讯作者: 秦书俭，教授，博士生导师，E-mail:qinshujian　@lnmu.edu.cn
作者简介:杨青(1988-),男，辽宁朝阳人，在读研究生，研究项目：骨组织工程，Tel:15142647105, E-mail:734562938@qq.com
基金资助:
国家自然基金项目（31170930）; 辽宁省科技计划项目（2011408004）

The comparative study on two tracing ways of bone marrow mesenchymal stem cells in repairing of the hepatic ischemia reperfusion injury

YANG Qing, QIN Shu-jian, BAO Cui-fen, SHAN Wei

Department of Human Anatomy and Histology and Embryology, Jinzhou Medical University, Jinzhou, Liaoning Province 121000, China

Received:2015-07-27 Online:2016-05-25 Published:2016-06-17

摘要/Abstract

摘要：

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200 μl (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。

关键词: , 骨髓间充质干细胞, 绿色荧光蛋白, 肝脏缺血再灌注损伤, 慢病毒

Abstract:

Objective　To compare the difference of the time-validity of repairing and the fluorescence stability between BMSCs infected by a lentiviral vector carrying GFP and the BMSCs in GFP rats in treatment of hepatic ischemia reperfusion injury. 　Method　The two kinds of BMSCs were cultured and the method of MTT was used to detect the differences of the two cell growth curves. Healthy SD rats were randomly divided into a sham operation group、a lentiviral transfection GFP group(LV-GFP group)、a GFP transgene group(GFP-BMSCs group) and a model group. After operation, the rats of the LV-GFP group and the GFP-BMSCs group were immediately injected with the corresponding cell suspension of 200 μl (the number of 1×106) by portal vein, and the model group was injected with the same volume of PBS. After 1, 2, 3 and 4 weeks，the levels of AST、ALT and ALB were tested by the blood sampling in different groups. From 1 to 5 days after operation, the hepatic tissue from the experience groups were transected to observe the migrating of the GFP positive cells in the liver; At week 4, the hepatic tissue was transected from the experience groups to detect the fluorescence stability of the BMSCs and its expression of the keratin 18 (CK18) in the liver.　Results　The proliferation of the BMSCs in GFP rats was significantly stronger than that of BMSCs infected by a lentiviral vector carrying GFP in logarithmic phase. At weeks 1 and 2, the levels of the aspartate amino transferase (AST) and alanine amino transferase (ALT) in GFP-BMSCs group were lower than those in LV-GFP group (P<0.05), and the level of serum albumin (ALB) in GFP-BMSCs group was higher than that in LV-GFP group at weeks 2 and 3 (P<0.05). BMSCs labeled GFP in hepatic lobules were successively found in the GFP-BMSCs group and the LV-GFP group on the third and 5th day, respectively. The hepatic cells from BMSCs cells in the liver were found in the GFP-BMSCs group and the LV-GFP group at week 4 after operation, but the fluorescence intensity in GFP-BMSCs group was better than that in LV-GFP group.　Conclusion　The BMSCs in GFP rats show the better time-validity of repairing and fluorescence stability than the BMSCs infected by a lentiviral vector carrying GFP in treatment of hepatic ischemia reperfusion injury.

Key words: Bone marrow mesenchymal stem cells, Green fluorescence protein, Hepatic ischemia reperfusion injury, Lentiviral

杨青,秦书俭,包翠芬,单伟. 两种示踪方式的骨髓间充质干细胞在促进肝缺血再灌注损伤修复中的对比研究[J]. 中国临床解剖学杂志, 2016, 34(3): 312-317.

YANG Qing, QIN Shu-jian, BAO Cui-fen, SHAN Wei. The comparative study on two tracing ways of bone marrow mesenchymal stem cells in repairing of the hepatic ischemia reperfusion injury[J]. Chinese Journal Of Clinical Anatomy, 2016, 34(3): 312-317.

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两种示踪方式的骨髓间充质干细胞在促进肝缺血再灌注损伤修复中的对比研究

The comparative study on two tracing ways of bone marrow mesenchymal stem cells in repairing of the hepatic ischemia reperfusion injury

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