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目的:自人卵巢癌细胞系SK-OV-3中分离干/祖细胞并进行鉴定。方法:采用无血清球形体形成法从SKOV-3中分离培养卵巢癌干/祖细胞;采用实时定量PCR和蛋折质印迹法测定球形体细胞干/祖细胞相关标志ABCG2、Oct-4、Nanog基因和蛋白的表达;流式细胞仪检测其耐药性;双层软琼脂检测其克隆形成能力;NOD/SCID小鼠检测其体内致瘤性。结果:球形体细胞表达干/祖细胞相关标志Oct-4、ABCG2、Nanog;对顺铂高耐药;在双层软琼脂上克隆形成率达(13.67±1.48)%;1 000个球形体形成细胞就能在NOD/SCID鼠中成瘤。结论:采用无血清培养基中球形体形成法从SKOV-3细胞系中可以分离出具有干/祖特性的卵巢癌细胞,可为今后研究卵巢癌的发生、发展、复发及其化疗药物筛选提供简便实用的体外模型。
Purpose: To isolate and identify stem / progenitor cells from human ovarian cancer cell line SK-OV-3. Methods: The ovarian cancer stem / progenitor cells were isolated and cultured from human ovarian cancer cell line SKOV-3 by serum-free spheroid formation method. ABCG2, Oct-4 and Nanog were detected by real-time PCR and Western blotting The expression of genes and proteins were detected by flow cytometry. The drug resistance was detected by flow cytometry. The ability of colony formation was detected by double soft agar. NOD / SCID mice were used to detect the tumorigenicity in vivo. Results: Spheroid somatic cells expressed the markers of stem / progenitor cells (Oct-4, ABCG2, Nanog), and were highly resistant to cisplatin. The rate of clonal formation on double soft agar was (13.67 ± 1.48)% Cells can become tumorigenic in NOD / SCID mice. CONCLUSION: Ovarian cancer cells with stem / ancestral characteristics can be isolated from SKOV-3 cell line by serum-free culture of spheroid formation, which can be used to study the occurrence, development, recurrence and chemotherapeutic drug screening of ovarian cancer Simple and practical in vitro model.