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目的通过原代培养HPV阳性的宫颈上皮内瘤变Ⅲ度(CINⅢ)的成纤维细胞与HPV阴性的小儿包皮成纤维细胞,探索HPV E7在DNA损伤应答过程中的作用。方法选取临床病理确认为HPV16阳性的CINⅢ新鲜宫颈组织和HPV16阴性的小儿包皮组织,组织块经胶原酶A消化后培养,用慢病毒E7或pLV感染HPV16-成纤维细胞,用离子射线(X光,2Gy或5Gy)分别照射HPV16阴性与HPV16阳性细胞以及经pLV或E7慢病毒感染的细胞0~8h,诱导细胞DNA双链断裂,采用间接免疫荧光法检测DNA双链断裂应答相关蛋白53BP1、BRCA1、NBS1、RPA32的应答特征。结果成功从宫颈上皮内瘤变组织及小儿包皮组织原代培养出HPV16阳性及HPV16阴性的成纤维细胞。间接免疫荧光染色发现X光照射后,HPV16阳性细胞中53BP1、BRCA1、NBS1、RPA32灶点多于HPV16阴性细胞(P<0.05),E7感染细胞中53BP1、RPA32、NBS1灶点少于pLV感染细胞(P<0.05),均为6h(2Gy)或4h(5Gy)达峰。结论利用原代培养CIN成纤维细胞成功建立了研究DNA损伤中E7作用的模型,在宫颈癌前病变发展过程中,HPV E7能够抑制DNA双链断裂修复应答反应。
Objective To explore the role of HPV E7 in DNA damage response by primary culture of HPV-positive CINⅢ fibroblasts and HPV-negative pediatric foreskin fibroblasts. Methods Clinicopathologically confirmed HPV16 positive CIN Ⅲ fresh cervical tissue and HPV16-negative pediatric foreskin tissue were digested with collagenase A, HPV16-fibroblasts were infected with lentivirus E7 or pLV, , 2Gy or 5Gy) were exposed to HPV16-negative and HPV16-positive cells and cells infected by pLV or E7 lentivirus for 0-8h, respectively. DNA double-strand breaks were induced in the cells. Indirect immunofluorescence assay was used to detect the DNA double-strand breaks associated protein 53BP1, BRCA1 , NBS1, RPA32 response characteristics. Results The HPV16 positive and HPV16 negative fibroblasts were successfully cultured from cervical intraepithelial neoplasia and pediatric foreskin. Indirect immunofluorescence staining showed that there were more foci of 53BP1, BRCA1, NBS1 and RPA32 in HPV16 positive cells than those of HPV16 negative cells (P <0.05) after X-ray irradiation. The number of 53BP1, RPA32 and NBS1 in E7 infected cells was less than pLV infected cells (P <0.05), both peaked at 6h (2Gy) or 4h (5Gy). Conclusion The model of E7 in DNA damage was successfully established by primary culture of CIN fibroblasts. HPV E7 can inhibit DNA double-strand break repair response in the development of cervical precancerous lesions.