發(fā)布時(shí)間：2018-06-10 03:24

  本文選題：肝癌 + hIL-12　； 參考：《遵義醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的：構(gòu)建真核表達(dá)載體pIRES2-EGFP-hIL-12-HSP70,并觀察其轉(zhuǎn)染7402肝癌細(xì)胞后的表達(dá)情況及對(duì)7402肝癌細(xì)胞生長(zhǎng)的影響,旨在獲得同時(shí)具有hIL-12和HSP70活性的雙功能融合蛋白分子。 方法：應(yīng)用聚合酶鏈反應(yīng)(PCR)技術(shù),從pBIl21-hIL-12質(zhì)粒中擴(kuò)增hIL-12基因,從pShuttle-CMV-HSP70質(zhì)粒中擴(kuò)增HSP70基因,應(yīng)用基因定向重組技術(shù)構(gòu)建重組真核表達(dá)載體pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70,并通過PCR、酶切和DNA測(cè)序進(jìn)行鑒定。用Lipofectamine2000將以上各重組真核表達(dá)載體及pIRES2-EGFP(空白對(duì)照)質(zhì)粒轉(zhuǎn)染7402肝癌細(xì)胞,同時(shí)設(shè)空細(xì)胞組,各組分別命名為7402/hIL-12,7402/HSP70,7402/hIL-12-HSP70,7402/PE,7402/0。通過倒置熒光顯微鏡檢測(cè)各組細(xì)胞的綠色熒光表達(dá)情況；Real-t- ime PCR檢測(cè)各重組外源基因在7402肝癌細(xì)胞中的表達(dá)差異；MTT法檢測(cè)細(xì)胞生長(zhǎng)情況；用AnnexinV-FITC/PI雙染法檢測(cè)細(xì)胞凋亡率。 結(jié)果：真核表達(dá)載體pIRES2-EGFP-hIL-12.pIRES2-EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70構(gòu)建成功,通過PCR擴(kuò)增、酶切及DNA測(cè)序證實(shí)各插入基因及聯(lián)合基因的大小、位置、方向均正確無誤；各轉(zhuǎn)染組細(xì)胞在轉(zhuǎn)染后24h、48h、72h均可在倒置熒光顯微鏡下觀察到綠色熒光；Real-time PCR證明各重組外源基因在已轉(zhuǎn)染重組真核表達(dá)載體的7402肝癌細(xì)胞中均有表達(dá)。MTT法結(jié)果顯示7402/hIL-12-HSP70組細(xì)胞的細(xì)胞抑制率最高,各處理組細(xì)胞48h抑制率明顯高于24h和72h,7402/hIL-12-HSP70組細(xì)胞的細(xì)胞抑制率與7402/hIL-12組、7402/HSP70組、7402/PE組的差異均有顯著性(P0.05).AnnexinV-FITC/PI雙染法結(jié)果顯示7402/hIL-12-HSP70組細(xì)胞較其余各組細(xì)胞凋亡數(shù)量增多,7402/hIL-12-HSP70組細(xì)胞凋亡數(shù)量與7402/hIL-12組、7402/HSP70組、7402/PE組的差異均有顯著性(P0.05)。 結(jié)論：成功構(gòu)建了真核表達(dá)載體pIRES2-EGFP-hIL-12.pIRES2一EGFP-HSP70及pIRES2-EGFP-hIL-12-HSP70；各重組真核表達(dá)載體均能轉(zhuǎn)染進(jìn)7402肝癌細(xì)胞并有效表達(dá)；聯(lián)合hIL-12和HSP70基因能明顯地促進(jìn)7402肝癌細(xì)胞凋亡,并具有協(xié)同增強(qiáng)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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

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