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目的:建立稳定转染人宫颈癌癌基因(human cervical cancer oncogene,HCCR)siRNA真核表达质粒的人胰腺癌PANC1细胞株,探讨siRNA干扰HCCR的表达后对人胰腺癌PANC1细胞增殖、凋亡和侵袭的影响。方法:通过脂质体转染法将含HCCRsiRNA的真核表达质粒pGCsi-HCCR稳定转染至人胰腺癌细胞株PANC1,抗生素G418筛选获得稳转细胞株;Western blot检测PANC1细胞中HCCR的表达,同时检测肿瘤相关基因p53蛋白表达的变化;流式细胞仪检测PANC1细胞的细胞周期和凋亡率变化;MTT比色法检测siRNA干扰后对PANC1细胞增殖能力的影响;Transwell侵袭实验观察siRNA干扰后对PANC1细胞侵袭能力的影响。结果:Western blot证实siRNA稳转组的PANC1细胞株和空载体稳转组比较HCCR蛋白表达水平下调,稳转细胞株建立成功。siRNA稳转组p53蛋白表达下降。siRNA稳转组的S期细胞数目减少而G0/G1期细胞数目增加,细胞凋亡增加。MTT结果显示siRNA稳转组1和稳转组2细胞吸光度分别为空载体组细胞的0.65倍和0.68倍,细胞增殖能力下降。Transwell侵袭实验显示siRNA稳转组细胞和空载体组细胞穿膜数分别为24.4±9.9和49.1±15.4(P<0.01),稳转组细胞侵袭能力下降。结论:siRNA干扰HCCR的表达后能抑制胰腺癌PANC1细胞增殖和侵袭,促进其凋亡。
OBJECTIVE: To establish a human pancreatic cancer PANC1 cell line stably transfected with the human cervical cancer oncogene (HCCR) siRNA eukaryotic expression plasmid to investigate the effect of siRNA on the proliferation and apoptosis of pancreatic cancer PANC1 cells after it interfered with the expression of HCCR. Impact of invasion. Methods: The eukaryotic expression plasmid pGCsi-HCCR containing HCCRsiRNA was stably transfected into human pancreatic cancer cell line PANC1 by lipofection method. The stable cell lines were screened by antibiotic G418. The expression of HCCR in PANC1 cells was detected by Western blot, The changes of p53 protein expression in tumor-related genes were also detected. The cell cycle and apoptosis rate of PANC1 cells were detected by flow cytometry. The effects of siRNA on the proliferation of PANC1 cells were detected by MTT colorimetric assay. On the invasiveness of PANC1 cells. Results: Western blot confirmed that the expression of HCCR protein was down-regulated in PANC1 cell line stably transfected with siRNA compared with that in blank control group, and the stable cell line was established successfully. p53 stable expression of p53 protein decreased. The number of S phase cells in siRNA stable transfected group decreased while the number of cells in G0 / G1 phase increased and apoptosis increased. The results of MTT showed that the absorbance of 2 cells in stable transfected group 1 and stable transfected group were 0.65 and 0.68 times that of empty vector group respectively, and the cell proliferation ability decreased. Transwell invasion assay showed that the numbers of transmembrane cells in the cells transfected with siRNA were 24.4 ± 9.9 and 49.1 ± 15.4, respectively (P <0.01), and the invasive ability of cells in the stable transfection group was decreased. CONCLUSIONS: siRNA interfering with the expression of HCCR can inhibit the proliferation and invasion of pancreatic cancer PANC1 cells and promote its apoptosis.