人卵巢癌HO8910细胞株中干细胞的筛选及生物学特性鉴定

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。

Isolation of cancer stem cells from ovarian cancer cell line HO9810 and identification of their biological characteristics

Objective To isolate cancer stem cells from ovarian cancer cell line HO9810 and identify their biological characteristics.Methods On the bases of conventional culture(group C),the ovarian cancer stem cells were isolated with serum-free culture system and paclitaxel(group P).Changes of cell proliferation and invasion were measured respectively by MTT and transwell chamber cell invasion assay.Expressions of CD24,CD44,CD45,CDl33,CD117 and Hoechst 33342 positive rate were detected by flow cytometry.Protein expressions of ABCG2,Nanog,Oct4 and BCRP were detected by Western blot.The growth ability of cancer stem cells in nude mice was observed.Results(1)The partly HO9810 cell could steadly grow under the serum-free culture system and paclitaxel,and had distinguishing feature of stem cells.(2)The growth rate of ovarian cancer stem cells was significantly strengthened.(3)The invasion cell number in group P was significantly different from that in group C.(4)The positive rates of CD24+,CD44+ and CD45+ in group P were not significantly different from those in group C,which of CD133+,CD117+ and Hoechst 33342 were significantly different between groups of P and C.(5)Compared with group C,the proteins of ABCG2,Nanog,Oct4 and BCRP were significantly enhanced.(6)In vivo test in nude mice showed that the growth ability of cancer stem cells was higher in group P than that in group C.Conclusion Paclitaxel and serum-free culture system can isolate ovarian cancer stem cells which have many features of stem cells.Their biological characteristics in vitro and vivo are significantly different from those of normal cultured ovarian cancer cells.