宫颈及宫颈癌间质成纤维细胞的分离、鉴定与衰老检测研究

背景与目的：宫颈癌作为最常见的妇科恶性肿瘤之一，近年来发病呈年轻化趋势。目前认为人类乳头瘤病毒(human papillomavirus，HPV)的感染，特别是高危型HPV感染是其发生的主要因素。已有研究证实，在肿瘤发生的微环境中，间质成纤维细胞衰老可能促进上皮性肿瘤的发生，但宫颈癌的发生是否也伴有间质成纤维细胞的衰老鲜见报道。本研究拟对正常宫颈和宫颈癌间质成纤维细胞进行特异性研究，以揭示慢性炎症诱导宫颈癌发生的机制，旨在为宫颈癌的诊治提供新的理论依据。方法：分离纯化正常宫颈成纤维细胞(normal fibroblasts，NFs)和宫颈癌相关成纤维细胞(cancer-associated fibroblasts，CAFs)，通过ELISA法检测细胞因子白介素-6(interleukin-6，IL-6)和血管内皮生长因子(vascular endothelial growth factor，VEGF)的分泌水平；并用衰老相关β-半乳糖甘酶染色(senescence-associated β-galactosidase，SA-β-gal)、细胞计数和蛋白质印记法(Western blot)分别鉴定细胞衰老、绘制细胞生长曲线和检测NFs与CAFs中p16蛋白表达。结果：CAFs分泌的细胞因子IL-6和VEGF比NFs高2倍以上(P<0.05)；CAFs中SA-β-gal的活性比NFs高；而CAFs中p16水平的表达水平比NFs中高。结论：间质成纤维细胞衰老可能与宫颈癌的发生和HPV密切相关，研究细胞因子介导的宫颈癌间质衰老可能有助于宫颈癌的预防、诊断和治疗。

Isolation,identification, and senescence examination of cervical cancer and normal cervical fibroblasts

Background and purpose: Cervial cancer is most common gynecological malignancy. In recent years, the number of the young patients has obviously risen. It is now believed that almost all cases of cervical cancer are caused by exposure to a high-risk human papilloma virus (HPV). It has demonstrated that epithelial cancer can induce the senescence of normal stromal fibroblasts and senescent fibroblasts can promote tumor growth. This study by comparing the secretion of interleukin-6(IL-6) and vascular endothelial growth factor(VEGF) helped us to understand the mechanism of cervical cancer, and providesd more adequate theoretical and experimental basis for clinical early diagnosis and treatment of the disease. This study characterized normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) to understand the mechanism how the chronic inflammation induced cervical cancer, and to provide novel theoretical basis for clinical diagnosis and treatment of the disease. Methods: NFs and CAFs were isolated and purified from normal and cervical cancer tissues. The expression levels of IL-6 and VEGF in cell culture medium were measured by ELISA and cellular senescence was detected by senescence-associated beta galactosidase (SA-β-gal) staining. The senescent marker p16 was analyzed by Western blot. Results: Our data showed that the expressions of both IL-6 and VEGF were higher in CAFs than in NFs (P<0.05), and that the blue staining of SA-β-gal and the expression of p16 and VEGF were stronger in CAFs than in NFs. Conclusion: Cervical epithelial cancer may be resulted from the senescence of stromal fibroblasts and HPV infection. Studying on cytokine-mediated stromal senescence may have a potential value for prevention, diagnosis, and treatment of cervical cancer.