【作者】 王丹青；

【导师】 彭芝兰；

【作者基本信息】 四川大学 ， 妇产科学， 2005， 博士

【摘要】 背景与研究目的 高危型HPV感染在宫颈癌的发生发展中起着至关重要的作用,特别是E6/E7癌基因,已被证实有恶性转化能力,因此它们已成为宫颈癌基因治疗的理想靶点。RNA干扰(RNA interference,RNAi)是新近发展起来的一种封闭基因表达的有效方法,它比反义寡核苷酸和核酶技术能更高效的抑制目的基因的表达。已有研究证实化学合成的siRNA(small interfering RNA)能高效地介导RNAi作用,但作用持续时间短,而采用siRNA表达载体(small interfering RNA expression vector)的方法可能延长作用时间。因此本研究旨在探讨HPV16E7特异性siRNA表达载体对宫颈癌Caski和SiHa细胞E7基因的沉默作用及作用持续时间,以及体内外对肿瘤细胞生长的影响,探索siRNA表达载体作为宫颈癌基因治疗新策略的可行性。 方法 1.利用计算机辅助设计软件,设计合成了含不同HPV16E7特异性序列的56nt寡核苷酸片段,并将其定向克隆入真核表达载体psiRNA,得到重组质粒P1、P2、P3;2.利用脂质体将重组质粒转染CaSki和SiHa细胞,以荧光定量RT-PCR检测不同时间点转染前后E7 mRNA的变化,以免疫荧光标记法、western blot检测转染前后E7蛋更多还原

【Abstract】 Background & Objective High-risk human papillomavirus (HPV) infection is believed to play a central in the carcinogenesis and development cervical carcinoma. Specifically, two viral oncogene, E6 and E7, possess cancer transforming ability, have been became ideal targets for gene therapy of cervical carcinoma. RNA interference is an effective method of blocking gene expression at the post-transcriptional level and is several orders of magnitude more efficient than antisense or ribozyme treatment. The study has identified that the objective gene expression could be selective silencing with synthetic siRNA and silencing was not sustained more than 7 days. But a vector-based siRNA expression system might exhibit a comparatively long-term RNA interference (RNAi) effect. So the study was designed to investigate the specificity and efficiency of HPV16 E7 gene silence in CaSki cells and SiHa cells of cervical cancer in vitro and in vivo by vector-based RNAi technique, and to research the effect on these cell proliferations, and to explore the possibility of siRNA expression vector in treatment of cervical carcinoma.Methods 1. According to the computer aided design, 56nt oligonucleotide fragments containing different HPV16E7-specific sequences were synthesized and cloned into the expression vector psiRNA, and three recombinant vectors P1, P2 and P3 were constructed.2. The vectors were transfected into CaSki cells and SiHa cells by liposome. The expression level of HPV16E7 mRNA and protein were detected by real-time RT-PCR, western blot analysis and immunofluorescence using fluorescein isothiocyanate label at different times after transfection.3. The cell cycles and cell proliferations of CaSki cell and SiHa cell were measured with flow cytometry and MTT method after transfection.4. Human cervical cancer CaSki cells were transfected by vector P1 and were seeded hypopercutaneously on nude mice. The in vivo carcinogenic and growth activities of cancer cells were observed. E7 protein expression in tumor tissues was detected by immunohistochemisty.Results 1. PCR analysis and DNA sequencing confirmed that the HPV16 E7-specific siRNA expression vectors were constructed successfully.2. HPV16 E7 mRNA expression could be inhibited 92.86%, 54.24%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of CaSki cellular clone, and the HPV16 E7 protein was reduced by 84.21%, 44.66%, 83.46% respectively. So the inhibition effect of siRNA expression vector P1 was more effective.3. HPV16 E7 mRNA expression could be inhibited 92.15%, 54.51%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of SiHa cellular clone, and the HPV16 E7 protein was reduced by 84.30%, 41.65%, 82.91% respectively. So the inhibition effect of siRNAexpression vector PI was more effective.4. HPV16 E7 mRNA expression could be inhibited 68.95%, 65.69% respectively by vector PI into CaSki cells and SiHa cells at four weeks after the formation of cellular clone, and the HPV16 E7 protein was reduced by62.50%, 59.11% respectively.5. After transfected by vector PI, the ratio of Gl phase of CaSki cell andSiHa cell was improved and the ratio of S phase was descended significantly at one week after the formation of cellular clone, and the cell proliferations of CaSki cell and SiHa cell were inhibited.6. After transfected by vector PI, the carcinogenic activity of CaSki cell was decreased in nude mice, the volume and the weigh of tumors were reduced, the rate of inhibition was 50.61%, and E7 protein level in tumor tissues was reduced by 74.75%.Conclusions The expression of E7 gene in CaSki cells and SiHa cells can be inhibited specifically and high efficiently by HPV16E7-specific siRNA expression vector, and inhibition effect was sustained for at least four weeks after the formation of cellular clone. E7-specific siRNA expression vector could control the cell cycle, inhibit the cancer cell proliferation and partly reversed the malignant phenotype of tumor. So it might be one of the ideal strategies for gene therapy of cervical cancer.更多还原