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目的筛选人卵巢癌HO8910细胞株中的干细胞并观察其生物学特性。方法以常规培养的HO8910细胞为基础(对照组),采用紫杉醇结合无血清培养基悬浮培养法筛选卵巢癌干细胞(干细胞组)。对筛选出的干细胞分别采用MTT法检测细胞的增殖情况;体外侵袭实验检测细胞侵袭能力;流式细胞术检测CD24、CD44、CD45、CD133、CD117表达及Hoechst33342染色阳性率;Western blot检测ABCG2、Nanog、Oct4及BCRP等干细胞标志基因的蛋白表达,并对卵巢癌干细胞在裸鼠体内的致瘤性进行检测。结果 (1)在紫杉醇结合无血清培养筛选条件下,部分HO8910细胞能够较好的生长,具有干细胞特性。(2)干细胞组各时间点的细胞增殖能力较对照组明显增强。(3)干细胞组穿膜细胞数较对照组明显增多。(4)干细胞组CD24+、CD44+、CD45+的阳性表达率与对照组比较,差异均无统计学意义;CD133+、CD117+的阳性表达率及Hoechst33342染色阳性率与对照组比较,差异均有统计学意义(P<0.05)。(5)干细胞组ABCG2、Nanog、Oct4、BCRP等干细胞标志基因的蛋白表达水平与对照组比较均明显增强。(6)干细胞组细胞在裸鼠体内致瘤性明显高于对照组。结论紫杉醇结合无血清培养基悬浮培养法能够筛选出具有干细胞特征的卵巢癌干细胞;卵巢癌干细胞在体外及体内的生物学特性较普通卵巢癌细胞有很大差异。
Objective To screen the stem cells in human ovarian cancer cell line HO8910 and observe its biological characteristics. Methods Based on routinely cultured HO8910 cells (control group), ovarian cancer stem cells (stem cell group) were screened by paclitaxel and serum-free medium suspension culture. Cell proliferation was detected by MTT assay. Stromal invasiveness assay was used to detect cell invasiveness. Flow cytometry was used to detect the expression of CD24, CD44, CD45, CD133 and CD117 and the positive rate of Hoechst33342 staining. ABCG2 and Nanog , Oct4 and BCRP and other stem cell marker gene protein expression, and ovarian cancer stem cells in nude mice tumorigenicity were detected. Results (1) In the combination of paclitaxel and serum-free culture, some HO8910 cells grew well and had stem cell characteristics. (2) Stem cell group at each time point of cell proliferation than the control group was significantly enhanced. (3) The number of transmembrane cells in stem cell group was significantly higher than that in control group. (4) The positive rates of CD24 +, CD44 + and CD45 + in stem cell group were not significantly different from those in control group. The positive expression rate of CD133 +, CD117 + and the positive rate of Hoechst33342 staining in control group were significantly different P <0.05). (5) The expression of ABCG2, Nanog, Oct4, BCRP and other stem cell marker genes in stem cell group were significantly increased compared with the control group. (6) Stem cell group in nude mice tumorigenicity was significantly higher than the control group. Conclusion Paclitaxel combined with serum-free medium suspension culture method can screened ovarian cancer stem cells with stem cell characteristics; biological characteristics of ovarian cancer stem cells in vitro and in vivo than ordinary ovarian cancer cells are very different.