摘要
目的宫颈癌发病率在全球妇女恶性肿瘤中居第二位。PUMA(p53 up-regulatedmodulator of apoptosis)是p53的下游基因,具有强大的促凋亡作用。本研究以腺病毒为载体,将PUMA基因导入HPV阳性宫颈癌细胞HeLa和CaSki,分析PUMA对宫颈癌细胞的作用以及与放疗联合应用后的效果。
方法应用MTT法比较Ad-PUMA和Ad-p53对HeLa和CaSki细胞增殖的影响,平板克隆方法检测Ad-PUMA与X射线联合应用后对细胞生长的影响,并通过DAPI染色、流式细胞检术检测细胞凋亡情况。Western Blot法检测X射线处理后HeLa细胞PUMA表达。
结果RT-PCR和Western Blot证实Ad-PUMA感染HeLa细胞3h后PUMA即有表达。MTT结果显示Ad-PUMA能够明显抑制HeLa和CaSki细胞增殖,而Ad-p53对细胞生长无明显抑制作用。DAPI染色和流式细胞术证实Ad-PUMA可诱导HeLa细胞凋亡。Western Blot显示X射线能够诱导HeLa细胞PUMA表达升高,具有时间和剂量效应。Ad-PUMA与X射线联合应用后可显著增加细胞凋亡,减少平板克隆形成数,但Ad-PUMA对X射线引起的G2期阻滞无明显影响。
结论PUMA在放射线诱导肿瘤细胞凋亡的过程中发挥作用。Ad-PUMA抑制宫颈癌细胞增殖,诱导细胞凋亡。与X射线联合应用后,Ad-PUMA显著提高宫颈癌细胞放疗敏感性,促进放疗诱导的肿瘤细胞凋亡。
Objective:Cervical cancer is the second most common malignancy in women worldwide.Human papilloma virus(HPV) infection is the most important risk factor of cervical cancer.PUMA was recently identified as a mediator of DNA damage-induced and p53-dependent apoptosis.In this study,we investigated whether exogenous PUMA suppresses the growth of HPV positive cervical cancer and sensitizes HPV positive cervical cancer cells to irradiation.
Methods:An adenovirus expressing PUMA(Ad-PUMA),alone or in combination with irradiation,was used to treat HPV positive cervical cancer cells HeLa and CaSki. The growth inhibitory and pro-apoptotic effects of PUMA in vitro were examined.The efficacy of PUMA and p53 in suppressing the growth of HeLa and CaSki were also compared.The expression of PUMA was examined in HeLa cell treated with irradiation.
Results:RT-PCR and Western Blot revealed that the expression of exogenous PUMA was elevated 3 hours after infection.Ad-PUMA suppressed the growth of HPV positive cervical cancer cells,and was more efficient than Ad-p53.Combination of Ad-PUMA and irradiation significantly suppressed the cell growth,and increased the percentage of apoptotic cells than separate treatment.Irradiation induced PUMA expression in HeLa cell.
Conclusions:Our data indicate that transfection of cervical cancer cells with Ad-PUMA is a potential novel approach to the therapy of HPV positive cervical cancer.
引文
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