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目的人乳头瘤病毒(HPV)是宫颈癌最主要的致病因素,E6是主要的致癌基因之一,高危HPV基因型,如HPV16的E6蛋白表达水平是维持宫颈癌恶性表型的必要条件;放射治疗是目前宫颈癌治疗的标准和有效的方法;该研究旨在探讨抗HPV16E6核酶(ribozyme)对宫颈癌CaSKi细胞放疗敏感性的影响。方法以脂质体法将抗HPV16E6-ribozyme、空载体质粒分别导入CaSKi细胞,命名为CaSKi-R、CaSKi-P细胞。点杂交检测核酶在细胞中的表达,N orthern 杂交检测 3 种细胞中 E6 基因的表达。用克隆形成试验检测 3 种细胞对放疗的敏感性,Annexine/PI双标法检测细胞凋亡率, 流式细胞术检测 bcl-2、p53、bax 蛋白表达。结果点杂交证实核酶能在 C aSKi-R 细胞中稳定表达,N orthern 杂交证实 C aSKi-R 中 E6 表达较 C aSKi-P、C aSKi中明显降低。C aSKi-R 细胞生长速度较 C aSKi-P、C aSKi明显减慢( P <0.01);C aSKi-R 细胞对 X 射线的敏感性较C aSKi-P、C aSKi明显增加,克隆形成率明显下降(P <0.05),C aSKi-R 细胞照射前后的凋亡率较 X 射线照射后C aSKi-P、C aSKi明显增加( P <0.01);C aSKi-R 细胞 p53、bax 蛋白表达较 C aSKi-P、C aSKi明显升高,bcl-2 明显减少( P <0.01)。结论转染抗 H PV16E6-R ibozym e的 C aSKi-R 细胞出现一定程度的生长抑制,且对放射治疗的敏感性增加。
Objective Human papilloma virus (HPV) is the most important causative factor of cervical cancer. E6 is one of the major oncogenes. The high-risk HPV genotypes such as HPV16 E6 protein expression are the necessary conditions to maintain the malignant phenotype of cervical cancer. Radiation therapy is the standard and effective method for the treatment of cervical cancer. The aim of this study was to investigate the effect of anti-HPV16E6 ribozyme on the radiosensitivity of cervical cancer CaSKi cells. Methods The anti-HPV16E6-ribozyme and empty plasmid were introduced into CaSKi cells by lipofectamine respectively and named as CaSKi-R and CaSKi-P cells. The expression of ribozyme in the cells was detected by dot blot hybridization and the expression of E6 gene in the three kinds of cells was detected by N orthern hybridization. The sensitivity of the three kinds of cells to radiotherapy was tested by colony formation assay. The apoptosis rates of the three kinds of cells were detected by Annexin / PI double-labeled method. The expressions of bcl-2, p53 and bax were detected by flow cytometry. Results Dot blotting confirmed that ribozyme was stably expressed in C aSKi-R cells. N orthern hybridization confirmed that E6 expression in C aSKi-R was significantly lower than that in C aSKi-P and C aSKi. Compared with C aSKi-P and C aSKi, the growth rate of C aSKi-R cells was significantly slower than that of C aSKi-P and C aSKi (P <0.01). The sensitivity of C aSKi-R cells to X-rays was significantly higher than that of C aSKi-P and C aSKi (P <0.05). The apoptotic rates of C aSKi-R cells before and after irradiation were significantly higher than those of C aSKi-P and C aSKi after X-ray irradiation (P <0.01) aSKi-P, C aSKi significantly increased, bcl-2 significantly reduced (P <0.01). Conclusions C aSKi-R cells transfected with anti-H PV16E6-R ibozym e showed some degree of growth inhibition and increased sensitivity to radiotherapy.