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目的:通过对比并改良常见的原代(CAFs)细胞培养方法,建立一种成功率较高的宫颈癌相关成纤维细胞(CAFs)原代培养的方法。方法:取确诊为宫颈癌患者的活检或手术标本,通过对消化时间、重悬液量及培养基血清浓度进行改良原代细胞培养,观察细胞生长状况,并比较其与传统组织块贴壁法和酶消化法的细胞生长率。运用时间差酶消化及反复贴壁法进行分离纯化后,用免疫荧光的方法对宫颈癌相关成纤维细胞的纯度进行鉴定。结果:改良培养法消化时间为30min,血清浓度为20%时成功率最高(66.67%)。反复运用时间差酶消化及反复贴壁法可以得出纯度较高的宫颈癌相关成纤维细胞。结论:本研究成功建立了一种成功率较高的宫颈癌相关成纤维细胞改良培养方法,为后期探讨CAFs在肿瘤形成、进展中的作用打好实验基础。
OBJECTIVE: To establish a method of primary culture of cervical cancer-associated fibroblasts (CAFs) with high success rate by comparing and improving common primary (CAFs) cell culture methods. Methods: The biopsies or surgical specimens of patients diagnosed as cervical cancer were collected. The primary cell culture was performed by digestion time, resuspension volume and serum concentration of the culture medium. The cell growth was observed and compared with traditional tissue block adhesion And cell growth rate of enzymatic digestion. Using time difference enzyme digestion and repeated adherent method for isolation and purification, immunofluorescence method to identify the purity of cervical cancer-related fibroblasts. Results: The modified culture method digestion time was 30min, the highest success rate (66.67%) when serum concentration was 20%. Repeated use of time and poor enzyme digestion and repeated adherent method can be drawn higher purity of cervical cancer-related fibroblasts. Conclusion: This study successfully established a high success rate of cervical cancer-related fibroblasts improved culture methods for the later study of CAFs in tumor formation and progress in lay the foundation for the experiment.