摘要
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。
Objectives To investigate whether the human PC-3 cell infected with recombinant Ad-PTEN and Ad-p27Kip1 can steadily produce PTEN and p27Kip1 protein and change the biologic behaviors such as cell proliferation,cell cycle and apoptosis.The synergistic effect of PTEN and p27Kip1 on the therapy for prostate cancer has also been investigated. Methods We constructed recombinant adenovirus vector of human tumor suppressor gene PTEN and p27Kip1.The viral titer was examined by plaque assay and the mRNA and protein expressions of PTEN and p27Kip1 in human prostate cancer cell line PC-3 infected with Ad-PTEN and Ad-p27Kip1 were determined by RT-PCR and Western blot respectively.MTT assay was used to determine the effect of PTEN and p27Kip1 on growth and proliferation of PC-3 cell; the change of cell cycle and apoptosis was examined by flow cytometry,and to compare between the combined therapy group and single gene therapy group. Results The viral titers of Ad-PTEN and Ad-p27Kip1 were 1.8×107 pfu/ml and 1.2×109 pfu/ml respectively.After infected by adenovirus,it had been verified that the mRNA and protein expression of PTEN and p27Kip1 were steady in human PC-3 cell.Ad-PTEN and Ad-p27 Kip1 inhibited the growth and proliferation of PC-3 cells.The progression of cell cycle of PC-3 cell was arrested in G 0-G 1 phase,meanwhile the apoptosis rate of PC-3 was also affected after Ad-PTEN or/and Ad-p27 Kip1 infected.There was significant difference between combined therapy group and single gene therapy group. Conclusion The recombinant Ad-PTEN and Ad-p27Kip1 vector were constructed successfully and the expression of specific PTEN and p27Kip1 was high,steadily in PC-3 cell line.These results suggested that combination of PTEN with p27Kip1 has an application value in treatment of prostate cancer in future.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2004年第10期600-603,共4页
Chinese Journal of Surgery
基金
国家自然科学基金-重大国际合作研究项目(3 0 12 0 12 0 665 )
