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目的探讨Puma对于卵巢癌细胞生长及体内成瘤的影响,了解其行使功能的分子机制。方法采用腺病毒载体实现Puma在卵巢癌细胞中的过表达,并通过CCK-8检测细胞增殖情况,通过Annexin-V染色测定细胞凋亡情况,通过小鼠荷瘤实验确定卵巢癌细胞体内成瘤能力。并进一步通过Western blot的方法检测了细胞凋亡相关基因Caspase-9和Bax蛋白的表达及定位情况。结果成功构建hTERT基因启动子介导的Puma基因腺病毒表达载体,并实现Puma基因在卵巢癌细胞中的过表达,过表达Puma后,卵巢癌细胞增殖被明显抑制(P<0.05),细胞凋亡增加,从对照组细胞的7.2%增加为过表达组的29%(P<0.05),且在小鼠体内的成瘤能力下降,所产生肿瘤组织的平均质量由0.53 g降至0.22 g(P<0.05)。结论 Puma通过激活细胞内Caspase-9的活性、促使Bax从胞质转移至线粒体、促进细胞凋亡信号传导诱导细胞凋亡,从而抑制卵巢癌细胞增殖及体内成瘤能力。
Objective To investigate the effect of Puma on ovarian cancer cell growth and in vivo tumorigenesis, and to understand the molecular mechanism of Puma’s function. Methods The adenovirus vector was used to overexpress Puma in ovarian cancer cells. The proliferation of ovarian cancer cells was detected by CCK-8. The apoptosis of ovarian cancer cells was determined by Annexin-V staining. ability. Western blot was used to detect the expression of Caspase-9 and Bax proteins and their localization. Results The hTERT promoter was successfully constructed to express Puma adenovirus vector and overexpression of Puma gene in ovarian cancer cells. After overexpression of Puma, the proliferation of ovarian cancer cells was significantly inhibited (P <0.05) Increased from 7.2% in the control group to 29% in the overexpression group (P <0.05), and decreased in vivo tumorigenicity in mice resulting in a reduction in the average mass of tumor tissue from 0.53 g to 0.22 g P <0.05). Conclusions Puma can promote Bax from cytoplasm to mitochondria through activation of intracellular Caspase-9 activity, promote apoptosis signal transduction and induce apoptosis, thus inhibit ovarian cancer cell proliferation and in vivo tumorigenicity.