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目的 通过腺病毒介导转染前列腺癌PC 3细胞 ,探讨人 10号染色体上缺失的磷酸酶和张力蛋白类似物 (PTEN)和p2 7Kip1对肿瘤细胞增殖和凋亡等方面的影响及二者的协同作用。方法 构建携带人PTEN和p2 7Kip1基因的腺病毒载体 ,体外转染PC 3细胞 ,通过RT PCR、Westernblot检测目的基因不同水平的表达。采用细胞生长试验、流式细胞仪检测PC 3转染前后细胞增殖、细胞周期和早期凋亡率的变化。结果 病毒滴度Ad PTEN为 1 8× 10 7pfu ml、Ad p2 7Kip1为 1 2× 10 9pfu ml,RT PCR检测有PTEN mRNA(46 2bp)和p2 7Kip1 mRNA (32 0bp) ,Westernblot检测有PTEN蛋白 (6 0KD)和P2 7蛋白 (2 7KD)特异表达 ,可明显抑制PC 3细胞的增殖 ,诱导细胞凋亡 ,联合基因治疗组与单基因组相比差异有显著意义。结论 成功构建携带人PTEN和p2 7Kip1的重组腺病毒载体 ,在前列腺癌细胞株PC 3得到了稳定、特异的高表达 ,联合基因疗法有望成为治疗前列腺癌的有效方法。
Objective To investigate the effect of adenovirus-mediated transfection of prostate cancer PC 3 cells on the proliferation and apoptosis of human prostate cancer cell line PTEN and p27Kip1 Synergy. Methods The adenovirus vector carrying human PTEN and p2 7Kip1 gene was constructed and transfected into PC3 cells in vitro. The expression of target gene was detected by RT PCR and Western blot. Cell growth assay and flow cytometry were used to detect the changes of cell proliferation, cell cycle and early apoptosis before and after PC 3 transfection. Results The viral titer was 18 × 10 7 pfu ml for Ad PTEN and 12 × 10 9 pfu ml for Ad p2 7Kip1. PTEN mRNA (46 2bp) and p2 7 Kip1 mRNA (32 0 bp) were detected by RT PCR. PTEN protein was detected by Western blot 6 0KD) and P2 7 protein (2 7KD), significantly inhibited the proliferation of PC 3 cells and induced apoptosis. The difference between the gene therapy group and the single gene group was significant. Conclusion The recombinant adenovirus vector carrying human PTEN and p2 7Kip1 was constructed successfully and stably and specifically expressed in prostate cancer cell line PC 3. Combined gene therapy is expected to be an effective method for the treatment of prostate cancer.