抗HPV16E6核酶对宫颈癌CaSKi细胞放疗影响的研究

目的人乳头瘤病毒（HPV）是官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。点杂交交检测核酶在细胞中的表达，Northem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CaSKi—R细胞中稳定表达，Northern杂交证实CaSKi-R中E6表达较CaSKi—P、CaSKi中明显降低。CaSKi—R细胞生长速度较CaSKi-P、CaSKi明显减慢（P〈0.01）；CaSKi-R细胞对X射线的敏感性较CaSKi—P、CaSKi明显增加，克隆形成率明显下降（P〈0．05），CaSKi-R细胞照射前后的凋亡率较x射线照射后CaSKi—P、CaSKi明显增加（P〈0．（31）；CaSKi—R细胞p53、bax蛋白表达较CaSKi—P、CaSKi明显升高山bcl-2明显减少（P〈0．01）。结论转染抗HPV16E6-Ribozyme的CaSKi—R细胞出现一定程度的生长抑制，且时放射治疗的敏感性增加。

Radiation sensitizing effect of anti-HPV16E6-ribozyme on cervical carcinoma cell line

[Objective] Human papillomavirus (HPV) is the most important etiologic factor for cervical carcinoma. E6 virus gene is one of the most important oncogen and Levels of expression of E6 transforming oncoproteins of high risk HPV genotypes, such as HPV16, appear to be necessary for maintaining the malignant phenotype. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. The goal of this study was to investigate the radiation sensitizing effect of anti-HPV16E6 ribozyme on cervical carcinoma cell line. [Methods] With the method of lipofectin transfection, the anti-HPV16E6-ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell, which named as CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by Northern blot. The growth rates of the CaSKi and transfected cells were examined by cell count and their sensitivity to radiotherapy was examined by colony formation test. The apoptosis rates of each cell were determined by PI/Annexin V stained methods. Expressions of p53, bcl-2, and bax were determined by Flow cytometry analysis. [Results] Anti-HPV16E6 ribozyme can be expressed stably in transfected CaSKi-R cells. Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSKi and CaSKi-P. The growth rate of CaSKi-R was much slower than that of CaSKi and CaSKi-P. The sensitivity of CaSKi-R cells to radiotherapy increased more than that of CaSKi and CaSKi-P cells, the ability to form colony decreased (P <0.05), and the apoptosis rates of CaSKi-R cells increased more than that of CaSKi and CaSKi-P cells(P<0.01). Anti-HPV16E6-ribozyme can significantly upregulated expression of p53, bax protein, and downregulated the expression of bcl-2 protein before and after radiotherapy (P <0.01). [Conclusion] CaSKi-R cells transfected by Anti-HPVE6-rivozyme shows growth inhibition and increased sensitivity to radiotherapy.