Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (19): 2953-2960.doi: 10.3969/j.issn.2095-4344.2014.19.001
Hui Chun-ying1, Xiao Hong-yan2, He Xin-ling1, Wang Wei3
Revised:
2014-02-26
Online:
2014-05-07
Published:
2014-05-07
Contact:
Wang Wei, Master, Associate professor, Department of Rehabilitation, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
About author:
Hui Chun-ying, Department of Cardiocerebral Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001, Liaoning Province, China
Supported by:
the Funded Project of the Social Development Department of Liaoning Science and Technology Bureau, No. 2012408002
CLC Number:
Hui Chun-ying, Xiao Hong-yan, He Xin-ling, Wang Wei. In vitro transfection of triple-point mutants of hypoxia-inducible factor 1 alpha into bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 2953-2960.
2.2 腺病毒包装结果 经3种病毒重组体转染的HEK293A细胞在荧光显微镜下均观察到有大量绿色荧光蛋白表达(图3A-C),同时在普通倒置显微镜下观察均出现相似的细胞病变效应:细胞触角回收、肿胀变圆,一部分细胞脱落,悬浮于视野中(图3D-F);病毒滴度检测结果依次为2.3×108 pfu/mL,2.6×108 pfu/mL和1.8×108 pfu/mL,符合后续转染实验要求。 2.3 病毒转染骨髓间充质干细胞表达结果 3种包装成功的病毒液在高倍数荧光显微镜下均观察到较强绿色荧光蛋白表达并且表达绿色荧光蛋白的大鼠骨髓间充质干细胞也较多,转染效率较高;而未转染任何病毒液的骨髓间充质干细胞在荧光显微镜下并未显现出任何荧光效应(图4)。"
2.4 RT-PCR结果 含突变基因病毒液组、含未突变基因病毒液组细胞内低氧诱导因子1α mRNA表达相对吸光度值分别为0.85±0.06,0.83±0.07,两组差异无显著性意义(P > 0.05);空病毒液组、空白组低氧诱导因子1α mRNA的表达量非常低,几乎可以忽略,差异也无显著性意义(P > 0.05);而含突变基因病毒液组、含未突变基因病毒液组细胞内低氧诱导因子1α mRNA表达量均明显高于空病毒液组、空白组,差异有显著性意义(P < 0.05,图5A)。 2.5 Western blot结果 含突变基因病毒液组细胞低氧诱导因子1α蛋白及血管内皮细胞生长因子蛋白表达相对吸光度值分别为0.79±0.08和0.61±0.05,显著高于含未突变基因病毒液组、空病毒液组、空白组(P < 0.05);而含未突变基因病毒液组、空病毒液组、空白组3组差异均无显著性意义(P > 0.05,图5B,C)。结果显示,三点突变型低氧诱导因子1α不仅可以在常氧条件下高效表达,还能够促进其下游基因血管内皮细胞生长因子的高效表达。"
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