CAJ | 학술논문

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-FBS).U251、SU2、SU2-FBS接受0、2、10、15Gy剂量X线照射后24 h流式细胞仪检测侧群细胞(SP)、CD133+细胞比例;接受0、2、4、6、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、20 GyX线的1×106个细胞/5 μL SU2干细胞悬液和SU2-FBS细胞悬液,空白对照组注入5μL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％; CD 133+细胞由4.60％±0.82％增至17.89％±1.33％,差异有统计学意义(P＜0.05);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=0.71±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(P＜0.05).与0GySU2-FBS移植组比较,0、10 Gy SU2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.
목적 탐토인뇌효질류간세포SU2대복사적내수성급가능궤제. 방법 U251세포상규배양,SU2세포분별재무혈청DMEM/F12배양기、함10％태우혈청(FBS)적DMEM배양기중배양후득도현부간세포구(SU2)화첩벽분화세포(SU2-FBS).U251、SU2、SU2-FBS접수0、2、10、15Gy제량X선조사후24 h류식세포의검측측군세포(SP)、CD133+세포비례;접수0、2、4、6、8Gy제량조사1주후회제제량존활곡선,학정방사민감성삼수,즉세포경2GyX선조사후적존활분수(SF2);Balb/C라서35지분위SU2간세포이식조、SU2-FBS이식조、공백대조조,전2조우도분위3개아조(n=5),분별뇌내원위접충수조과0、10、20 GyX선적1×106개세포/5 μL SU2간세포현액화SU2-FBS세포현액,공백대조조주입5μL생리염수,21d후해부취뇌,HE염색관찰치류정황병계산종류체적,면역조직화학염색검측종류소단백(nestin)、모세혈관확장성공제실조돌변(ATM)단백적표체. 결과 SU2간세포중SP、CD133+세포비례수착복사제량적제고이증가,SP세포종5.63％±0.71％증가도22.05％±3.33％; CD 133+세포유4.60％±0.82％증지17.89％±1.33％,차이유통계학의의(P＜0.05);SU2간세포적생존곡선위우기타세포적상방,대복사적항거성(SF2=0.71±0.03)비SU2-FBS (SF2=0.54±0.04)화U251 (SF2=0.57±0.04)요고,차이유통계학의의(P＜0.05).여0GySU2-FBS이식조비교,0、10 Gy SU2간세포이식조소서이식류체적교대,차이유통계학의의(P＜0.05),nestin、ATM적양성표체교강. 결론 체외배양적효질류간세포SU2여동류분화세포SU2-FBS상비구유경강적내복사성,저여기고표체ATM유관.
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 orthotopically 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 1×106 cells/5 μL and blank control group was recived 5 mL normal saline.21 d after the transplantation,intracranial tumor formation rate and size of transplantable tumors were observed by HE staining; the expressions ofnestin 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 cells,which might be related to ATMhigh expression.