pIRES2-EGFP-hIL-12-HSP70真核表达载体的构建及对7402肝癌细胞生长的影响

发布时间：2018-06-10 03:24

本文选题：肝癌 + hIL-12　；参考：《遵义医学院》2011年硕士论文

【摘要】：目的：构建真核表达载体pIRES2-EGFP-hIL-12-HSP70,并观察其转染7402肝癌细胞后的表达情况及对7402肝癌细胞生长的影响,旨在获得同时具有hIL-12和HSP70活性的双功能融合蛋白分子。方法：应用聚合酶链反应(PCR)技术,从pBIl21-hIL-12质粒中扩增hIL-12基因,从pShuttle-CMV-HSP70质粒中扩增HSP70基因,应用基因定向重组技术构建重组真核表达载体pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70,并通过PCR、酶切和DNA测序进行鉴定。用Lipofectamine2000将以上各重组真核表达载体及pIRES2-EGFP(空白对照)质粒转染7402肝癌细胞,同时设空细胞组,各组分别命名为7402/hIL-12,7402/HSP70,7402/hIL-12-HSP70,7402/PE,7402/0。通过倒置荧光显微镜检测各组细胞的绿色荧光表达情况；Real-t- ime PCR检测各重组外源基因在7402肝癌细胞中的表达差异；MTT法检测细胞生长情况；用AnnexinV-FITC/PI双染法检测细胞凋亡率。结果：真核表达载体pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70构建成功,通过PCR扩增、酶切及DNA测序证实各插入基因及联合基因的大小、位置、方向均正确无误；各转染组细胞在转染后24h、48h、72h均可在倒置荧光显微镜下观察到绿色荧光；Real-time PCR证明各重组外源基因在已转染重组真核表达载体的7402肝癌细胞中均有表达。MTT法结果显示7402/hIL-12-HSP70组细胞的细胞抑制率最高,各处理组细胞48h抑制率明显高于24h和72h,7402/hIL-12-HSP70组细胞的细胞抑制率与7402/hIL-12组、7402/HSP70组、7402/PE组的差异均有显著性(P0.05).AnnexinV-FITC/PI双染法结果显示7402/hIL-12-HSP70组细胞较其余各组细胞凋亡数量增多,7402/hIL-12-HSP70组细胞凋亡数量与7402/hIL-12组、7402/HSP70组、7402/PE组的差异均有显著性(P0.05)。结论：成功构建了真核表达载体pIRES2-EGFP-hIL-12.pIRES2一EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70；各重组真核表达载体均能转染进7402肝癌细胞并有效表达；联合hIL-12和HSP70基因能明显地促进7402肝癌细胞凋亡,并具有协同增强作用。
[Abstract]:Aim: to construct the eukaryotic expression vector pIRES2-EGFP-hIL-12-HSP70 and to observe the expression of pIRES2-EGFP-hIL-12-HSP70 in 7402 hepatoma cells and its effect on the growth of 7402 hepatoma cells. Methods: hIL-12 gene was amplified from pB21-IlhIL-12 plasmid and HSP70 gene was amplified from pShuttle-CMV-HSP70 plasmid by polymerase chain reaction (PCR). Recombinant eukaryotic expression vectors pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70 and pIRES2-EGFP-hIL-12-HSP70 were constructed by gene directed recombination technique. The recombinant eukaryotic expression vector and pIRES2-EGFP (blank control) plasmid were transfected into 7402 hepatoma cells with Lipofectamine 2000. The cells were divided into empty cell group and named 7402% hIL-127402% HSP70402% hIL-12-HSP702% PE7402% 0 / 0 respectively. The green fluorescence expression of each group was detected by inverted fluorescence microscope. Real-time ime was used to detect the difference of the expression of the recombinant foreign genes in 7402 hepatoma cells. The growth of the cells was detected by MTT assay. Results: the eukaryotic expression vector pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70 and pIRES2-EGFP-hIL-12-HSP70 were successfully constructed. The size, position and direction of each inserted gene and associated gene were correct by PCR amplification, restriction endonuclease digestion and DNA sequencing. In each transfection group, at 24 h after transfection, 48 h and 72 h after transfection, green fluorescent real-time PCR was observed under inverted fluorescence microscope to demonstrate that all recombinant foreign genes were expressed in 7402 hepatoma cells transfected with recombinant eukaryotic expression vector. MTT assay showed that 7402% hIL-12-HSP70 was detected by MTT assay. The cell inhibition rate of the group was the highest. The cell inhibition rate in each treatment group was significantly higher than that in 24 h and 72 h / 7402% hIL-12-HSP70 group and 7402% hIL-12 / HSP70 group respectively. There were significant differences in cell apoptosis between 7402% hIL-12 / HSP70 group and 7402% HSP12 / HSP70 group. The results of Annexin V-FITCPI double staining showed that 7402 hIL-12-HSP70 cells increased the number of apoptosis in 7402 hIL-12-HSP70 group compared with the other groups. There was significant difference in apoptosis between 7402% HSP70 group and 7402% HSP70 group. Conclusion: the eukaryotic expression vectors pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70 and pIRES2-EGFP-hIL-12-HSP70 were successfully constructed and each recombinant eukaryotic expression vector could be transfected into 7402 hepatoma cells and expressed effectively. The combination of hIL-12 and HSP70 gene can obviously promote apoptosis of 7402 hepatoma cells and has synergistic effect.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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