摘要
背景与目的:人乳头瘤病毒(humanpapillomavirus,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。而放射治疗是目前宫颈癌治疗的标准和有效的方法。本研究旨在探讨抗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,Northern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<0.01);CaSKi-R细胞p53、bax蛋白表达较CaSKi-P、CaSKi明显升高,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,且对放射治疗的敏感性增加?
Background &Objective:Human papillomavirus(HPV)are the most important etiologic fac tor for cervical carcinoma.E6virus gene is one of the most important oncogene an d expression levels of E6transformi ng oncoproteins of high risk HPV genoty pes,such as HPV16,appear to be necessary for maintaining the malignant phenotype.Radiation treatment represents a standardized and effective modality f or contemporary cervical carcinoma therapy.The goal of this study was to investigate the r adiation sensitizing effect of anti-HPV16E6-ribozyme on cervical carc inoma cell line.Methods :With the method of lipofectin transfection,the anti-HPV16E6ribozyme a nd empty eucaryotic expressing plasmids were transfected into CaSKi cell lin e,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 E6mRNA in the three kinds of cells were detecte d by Northern blot.The growth rates of the CaSKi and transfected cells were examined by cell count and their sensitivity to radiotherapy were examined by colon y formation test.The apoptosis rates of each cell was dete rmined 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 expre ssed stably in transfected CaSKi-R cells.Northern blot showed that E6mRNA was less in CaSKi-R than in CaSKi a nd 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 abili ty of colony formation decreased(P<0.05),while the apoptosis rates of CaSKi-R cells increased more than that of CaS Ki and CaSKi-P cells(P<0.01).Anti-HPV16E6-ribozyme did signif icantly upregulate expression of p53,bax protein,and d ownregulate the expression of bcl-2protein before and after radiothera py(P<0.01).Conclusion:CaSKi-R cells transfected with Anti-HPVE 6-rivozyme showed growth inhi bition and increased sensitivity to radiotherapy.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2002年第2期149-152,共4页
Chinese Journal of Cancer
基金
广东省自然科学基金资助项目(编号96058)
