[肝细胞生长因子基因对骨髓间充质干细胞的修饰及活性观察]

[Modification and activity observation of hepatocyte growth factor gene on bone marrow mesenchymal stem cells].

作者信息

Ha Xiaoqin, Dong Fang, Lv Tongde

机构信息

Medical Experiment Center, Stem Cells and Gene Medicine Key Laboratory of Gansu Province, Lanzhou General Hospital, Lanzhou Command of Chinese PLA, Lanzhou Gansu 730050, PR China.

出版信息

Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 May;24(5):613-7.

PMID:20540271

Abstract

OBJECTIVE

To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors.

METHODS

Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multiplicity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the proliferation effect of HGF expression supernatant on BMSCs.

RESULTS

Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% +/- 0.04%, 40.72% +/- 0.81%, 61.72% +/- 1.04%, 85.33% +/- 0.83%, and 17.91% +/- 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the proliferation of BMSCs. The proliferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg).

CONCLUSION

BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the proliferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.

摘要

目的

通过将携带肝细胞生长因子（HGF）基因的重组腺病毒（Ad-HGF）转染至骨髓间充质干细胞（BMSCs）中，评估其转染效率及HGF的表达水平，并探讨表达上清对BMSCs的体外作用，为制备高效表达细胞因子的基因药物奠定基础。

方法

采用Percoll密度梯度法分离大鼠BMSCs，并根据其贴壁特性进行培养。通过免疫组织化学检测c-Met的表达。以不同感染复数（MOI，0、25、50、100和200 pfu/细胞）用携带绿色荧光蛋白基因的重组腺病毒（Ad-GFP）感染BMSCs。转染48小时后，分别通过流式细胞术和MTT法检测转染效率及细胞损伤程度，以选择最佳MOI。用ELISA法检测以最佳MOI的Ad-HGF转染的BMSCs中HGF的表达。采用MTT法评估HGF表达上清对BMSCs的增殖作用。

结果

免疫组织化学染色显示BMSCs表达HGF的c-Met受体。用不同MOI（0、25、50、100和200 pfu/细胞）的Ad-GFP转染48小时后，转染效率分别为0.34%±0.04%、40.72%±0.81%、61.72%±1.04%、85.33%±0.83%和17.91%±0.63%；其中在MOI为100 pfu/细胞时转染效率最高。当MOI为200 pfu/细胞时，明显观察到细胞损伤。以100 pfu/细胞MOI的Ad-HGF转染BMSCs 48小时后，HGF表达达到最高水平。表达产物可刺激BMSCs的增殖。BMSCs的增殖随HGF蛋白增加而逐渐升高，并在10%（320 pg）时达到最高水平。