缺氧诱导胶质瘤细胞肿瘤干样细胞形成体外初步研究

Hypoxia induces cancer stem-like cells from glioma cells

目的通过体外研究初步探讨缺氧可以诱导胶质瘤细胞逆分化形成肿瘤干样细胞。方法1采用CD133+细胞免疫磁珠分选法分离胶质瘤细胞得到CD133-细胞群。2缺氧处理CD133-细胞48 h后行免疫荧光染色鉴定肿瘤干细胞样标记蛋白(SOX2,OCT-4,KLF-4,Nanog,CD133)表达情况,并分别缺氧处理0、3、6、9、12、24 h及0、12、24、48、72 h行qRT-PCR及Western blot检测。流式细胞术检测胶质瘤细胞缺氧培养后CD133+细胞比例变化,检测时间为缺氧培养1、3、5、7 d。3缺氧处理胶质瘤细胞1、3、5、7 d后流式分析细胞周期及凋亡改变。结果 1荧光检测结果表明,肿瘤干细胞样标记蛋白在CD133-胶质瘤细胞缺氧处理48 h后高表达;qRT-PCR及Western blot检测结果表明,缺氧处理胶质瘤细胞可以明显上调肿瘤干细胞样蛋白表达(P<0.05),其中qRT-PCR显示SOX-2,OCT-4及Nanog最高表达在缺氧后9 h,KLF-4及CD133在缺氧后12 h表达最高。Western blot蛋白检测显示OCT-4最高表达在缺氧后12 h,CD133表达最高在缺氧24 h后,KLF-4及Nanog最高表达则在缺氧48 h后,SOX-2在缺氧72 h后表达量最高。另外随着缺氧时间的延长,CD133+细胞数比例逐渐升高(P<0.05)。2细胞周期结果提示缺氧处理肿瘤细胞后,G0/G1期延长,G2/M+S缩短(P<0.05);凋亡结果提示常氧培养组更易凋亡(P<0.05)。结论缺氧可以诱导CD133-胶质瘤细胞肿瘤干样细胞形成。

Objective To explore whether cancer stem-like cells （CSCs） can be induced under hypoxia from glioma cells in vitro. Methods Glioma cell lines U87 and GL261 and primary glioma cells cultured from pathologically confirmed glioma patients were cultured in DMEM/F12 ＋ 10% FBS, and magnetic-activated cell sorting （MACS） was used to sort CD133^- cells. The markers of CSCs, CD133, SOX-2, OCT-4, KLF-4 and Nanog, were detected by immunofluorescence staining after the CD133^- cells exposure to hypoxia for 48 h. qRT-PCR and Western blotting were used to detect the expression of above markers at RNA and protein levels under hypoxia in 0, 3, 6, 9, 12 and 24 h or 0, 12, 24, 48 and 72 h respectively. Flow cytometry was used to detect the proportion of CD133 ＋ cells, cell cycle and cell apoptosis in 1, 3, 5 and 7 d after hypoxia. Results Immunofluorescence staining demonstrated the above CSCs markers were highly expressed in 48 h after hypoxia. Both qRT-PCR and Western blotting showed obviously increase in the expression of markers （P 〈 0.05 ）. Specifically, qRT-PCR proved that SOX-2, OCT-4 and Nanog reached the highest expression in 9 h after hypoxia, and KLF-d and CD133 in 12 h. Western blotting displayed the highest expression of CD133 was seen in 12 h, that of KLF-4 and Nanog in 48 h, and that of SOX2 in 72 h. With the elapse of hypoxic treatment, the proportion of CD133^＋ was also increased greatly （P 〈 0.05 ）. What＇ s more, hypoxia treatment induced CD133- cells arrested in G0/G1 phase, decreased the cells at G2/M ＋ S phase （P 〈0.05）, and resulted in lower apoptotic rate than the cells under normoxia （P 〈 0.05 ）. Gonelusion Hypoxia induces the formation of CSCs from glioma cells.