| 尹强兵# 王晓捷# 马晓姣邹飞陈雪梅.慢病毒介导的GFP-Hsp90αE47A 基因在HepG2 细胞表达及对
细胞增殖性影响的研究[J].,2011,11(9):1643-1646 |
| 慢病毒介导的GFP-Hsp90αE47A 基因在HepG2 细胞表达及对
细胞增殖性影响的研究 |
| Expression of Recombinant GFP-Hsp90P E47A Gene by Lentiviral VectorTransfection and Its Effect on Cell Proliferation in HepG2 Cells |
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| DOI： |
| 中文关键词: 热休克蛋白90 ATP 酶活性 慢病毒载体 绿色荧光蛋白 |
| 英文关键词: Hsp90α ATPase activity Lentiviral vector Green fluorescence protein |
| 基金项目:国家自然科学基金资助项目(No.305000580, No.30971193)；广东省自然科学基金资助（No.05300465） |
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| 中文摘要: |
| 目的：建立真核细胞表达的GFP-Hsp90αE47A 基因重组慢病毒载体三质粒包装细胞系统,并检测其对细胞增殖性的影响，
为进一步研究HSP90 分子伴侣功能奠定基础。方法：制备完整的重组慢病毒载体三质粒系统:转移质粒(Hsp90αE47A/psin-GFP),
包装质粒(ΔNRF)及包膜蛋白质粒(VSV-G)。磷酸钙法将三质粒共转染293T 包装细胞,48 h 后收集病毒上清。将制备好的慢病毒
颗粒感染HepG2 细胞,在荧光显微镜下观察报告基因GFP 的表达情况, Western blot 检测HepG2 细胞GFP-Hsp90α 表达。MTT 法
检测细胞增殖情况。结果：转染后的293T 和感染后的HepG2 细胞能观察到较强的绿色荧光，培养液上清病毒滴度约为3.0×103
ifu /μl,HepG2 细胞中有GFP-Hsp90α 蛋白的表达。内源性Hsp90α 表达无明显上升（为对照组的1.05 ± 0.15 倍，P<0.05，t 检验），
有明显外源性GFP-Hsp90αE47A 蛋白的表达，为对照组内源性Hsp90α 的0.68± 0.12 倍。外源性GFP-Hsp90αE47A 蛋白的表达
HepG2 细胞增殖活性于第4d 有明显抑制。(1.051±0.03 vs 1.349±0.05, P<0.05，t 检验)。结论：成功建立重组慢病毒载体的三质粒
包装细胞系统，并将GFP-Hsp90αE47A 基因在HepG2 细胞中稳定表达，且并未引起细胞明显的热休克反应而导致的内源性
Hsp90α 增高；且能明显抑制细胞增殖，为后期Hsp90α 分子伴侣功能进行研究奠定基础。 |
| 英文摘要: |
| Objective: To construct the three-plasmid packaging cell line of the recombinant lentiviral vector encoding
Hsp90αE47A/psin-GFP gene and investigate the molecular chaperon function of hsp90α. Methods: The three-plasmid recombinant
lentiviral vector, which was made up of the vector plasmid (Hsp90αE47A/psin-GFP), the packaging plasmid (ΔNRF) and the envelop
plasmid encoding the vesicular stomatitis virus-glycoprotein (VSV-G), was isolated and purified. Human embryonic kidney 293T cells
were co-transfected with the three plasmids by calcium phosphate method. 48 hours after the transfection, the viral supernatant was collected
to infect HepG2cells. The expression of reporter gene GFP was detected by fluorescence microscope, and the GFP-Hsp90αE47A
protein expressed in Hepg2 cells was detected by Western blot. Cell proliferation was tested by CCK-8 method. Results: There was
strong expression of GFP s in 293T and Hepg2 cells after transfection. The viral titer was 3.0 ×103 ifu /μl. The expression of
GFP-Hsp90α in Hepg2 cells was confirmed by Western blot. Cell proliferation of GFP-Hsp90αE47A protein expressing Hepg2 cells
was obviously inhibited at 4d. (1.051±0.03 vs 1.349±0.05, P>0.05，t test). Conclusion: The three-plasmid packaging cell line system of
recombinant lentiviral vector was successfully established, cell proliferation was obviously inbited by GFP-Hsp90αE47A gene transfection,
which will provide a basis for exploring the molecular chaperon function of Hsp90α. |
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