人脑胶质瘤干细胞SU2耐辐射实验研究

Experimental studies on radioresistance of human glioma stem cells SU2

目的探讨人脑胶质瘤干细胞SU2对辐射的耐受性及可能机制。方法U251细胞常规培养，SU2细胞分别在无血清DMEM／F12培养基、含10％胎牛血清（FBS）的DMEM培养基中培养后得到悬浮干细胞球（SU2）和贴壁分化细胞（SU2-FaS）。U251、SU2、SU2-FBS接受0、2、10、15Gy剂量x线照射后24h流式细胞仪检测侧群细胞（SP）、CD133＋细胞比例；接受0、246、8Gy剂量照射1周后绘制剂量存活曲线，确定放射敏感性参数，即细胞经2GyX线照射后的存活分数（SF2）；Balb／C裸鼠35只分为SU2干细胞移植组、SU2-FBS移植组、空白对照组，前2组又都分为3个亚组（n=5），分别脑内原位接种受照过0、10、20GyX线的1x10s个细胞，5μL SU2干细胞悬液和SU2．FBS细胞悬液，空白对照组注入5乩生理盐水，21d后解剖取脑，HE染色观察致瘤情况并计算肿瘤体积，免疫组织化学染色检测肿瘤巢蛋白（nestin）、毛细血管扩张性共济失调突变（ATM）蛋白的表达。结果SU2干细胞中SP、CD133＋细胞比例随着辐射剂量的提高而增加，SP细胞从5．63％±0．71％增加到22．05％±3．33％；CD133＋细胞由4．60％±0．82％增至17．89％±1．33％，差异有统计学意义体0．051；SU2干细胞的生存曲线位于其他细胞的上方，对辐射的抗拒性（SF2=0．7I±0．03）比SU2．FBS（SF2=0．54±0．04）和U251（SF2=0．57±0．04）要高，差异有统计学意义（氏0．05）。与0GySU2-FBS移植组比较，O、10GySU2干细胞移植组小鼠移植瘤体积较大，差异有统计学意义（P〈0．05），nestin、ATM的阳性表达较强。结论体外培养的胶质瘤干细胞SU2与同类分化细胞SU2-FBS相比具有更强的耐辐射性，这与其高表达ATM有关。

Objective To investigate the radioresistance of human glioma stem cell line SU2. Methods SU2 cells were cultured in DMEM containing 10% fetal bovine serum or serum-free DMEM/F12 to obtain differentiated adherent tumor cells （SU2-FBS） and neurosphere-like tumor stem cells （SU2）, respectively; cells U251 were also chosen. Cells U251, SU2 and SU2-FBS were irradiated by liner accelerator with different radiation dosages （0, 2, 10 and 15 Gy）; and then, the percentages of CD133＋ cells and side population （SP） cells were analyzed with flow cytometry. Survival curve and radiosensitivity parameters were determined one week after 0, 2, 4, 6 and 8 Gy radiation. Irradiated tumor cells were further orthotopicaUy transplanted into 35 Balb/C nude mice, dividing into 7 groups （n=5）： SU2＋0 Gy radiation group, SU2＋10 Gy radiation group, SU2＋20 Gy radiation group, SU2-FBS＋0 Gy radiation group, SU2-FBS＋10 Gy radiation group, SU2-FBS＋20 Gy radiation group and blank control group; SU2 or SU2-FBS was given at lxl06 cells/5 p,L and blank control group was recived 5 mL normal saline.21 d after the transplantation, intracranial tumor formation rate and size of transplantable tumorswere observed by HE staining; the expressions of nestin and ataxia telangiectasia mutated （ATM） protein in transplanted tumors were detected. Results The proportion ofCD133＋ cells and SP cells increased with the radiation dosages in the irradiated cells; SP cells increased from5.63%±0.71% to 22.05%±3.33% （P〈0.05） and CD133＋ cells from 4.60%±0.82% to 17.89%±1.33% （P〈0.05）. These data indicated that the cells which expressed stem cells markers survived preferentially because of their dominant radioresistance. The dose-survival curves of SU2 cells was on top of that of other cells, and the resistance ratio of the SU2 cells （SF2=0.71±0.03） was higher than that of the SU2-FBS cells （SF2=0.54_＋0.04） and U251 cells （SF2=0.57±0.04）, with significant difference （P〈0.05）. As compared with that in the SU2-FBS＋0 Gy radiation group, the tumor sizes in the SU2＋0 Gy radiation group and SU2＋10 Gy radiation group was significantly larger （P〈0.05）, and the nestin and ATM expressions in these two groups were increased. Conclusions The glioma stem cells SU2 have stronger resistant than the SU2-FBS ceils, which might be related to ATMhigh expression.