Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (41): 6585-6590.doi: 10.3969/j.issn.2095-4344.2014.41.005
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Cao Zhi-qiang, Wang Yang, Liu Long
Revised:
2014-09-18
Online:
2014-10-01
Published:
2014-10-01
Contact:
Liu Long, Master, Chief physician, Department of Urinary, General Hospital of Shenyang Military Command, Shenyang 110840, Liaoning Province, China
About author:
Cao Zhi-qiang, M.D., Associate chief physician, Department of Urinary, General Hospital of Shenyang Military Command, Shenyang 110840, Liaoning Province, China
Supported by:
the Natural Science Foundation of Liaoning Province, No. 2013020199
CLC Number:
Cao Zhi-qiang, Wang Yang, Liu Long. Recombinant CXCR4 gene lentivirus transfection of rat bone marrow mesenchymal stem cells in vitro [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6585-6590.
2.1 转染效率分析 骨髓间充质干细胞转染靶基因CXCR4或GFP。两组实验方案、实验条件完全一致,所以,转GFP组在荧光显微镜下观察绿色荧光作为转染效率的定性检查,见图1A,Western Blot方法在蛋白水平验证各组GFP和CXCR4蛋白的表达情况,见图1B。RT-PCR法在mRNA水平上再次验证转染效率成功(图1 C),转染CXCR4组表达CXCR4最高,转染GFP组表达GFP最高(P < 0.05)。 2.2 转染后骨髓间充质干细胞增殖及凋亡分析 经慢病毒转染不同基因进入骨髓间充质干细胞后,为了验证病毒转染是否影响骨髓间充质干细胞自身生物特性,实验测定了转染CXCR4组和未转染组细胞的细胞周期和凋亡率(图2)。结果发现,转染CXCR4而过表达CXCR4后的骨髓间充质干细胞生长状态良好,流式细胞仪检测处于复制期的骨髓间充质干细胞明显增多,而凋亡细胞明显减少,提示CXCR4具有促增殖和抗凋亡作用(P < 0.05)。 2.3 转染后骨髓间充质干细胞上清液分泌蛋白变化 经慢病毒转染不同基因进入骨髓间充质干细胞后,为了验证病毒转染是否影响骨髓间充质干细胞的蛋白分泌,实验测定了各组细胞培养上清中分泌蛋白水平变化,过表达CXCR4后的骨髓间充质干细胞分泌蛋白水平发生变化,测定的白细胞介素2,白细胞介素10,转化生长因子β,Fas-Ligand,血管内皮生长因子,吲哚胺-2,3-二加氧酶水平均呈上调趋势(P < 0.05,图3)。"
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