CAJ | 학술논문

目的探索一种简单、高效、稳定、可靠、经济的胶质瘤干细胞分离和纯化方法. 方法取对数生长期U87细胞,应用无血清干细胞培养基,置含1％O2的缺氧培养箱中培养,10d后传代培养并离心收集第一代肿瘤球(G1),连续传代培养获得第5代肿瘤球(G5).免疫组化染色检测G5肿瘤球中巢蛋白(nestin)、CD133和缺氧诱导因子2α (HIF2α)的表达;Western blotting检测G1～G5肿瘤球中HIF2α蛋白的表达;免疫细胞化学染色检测G5分化细胞中胶质纤维酸性蛋白(GFAP)、O1及βⅢ微管蛋白(βⅢ-Tublin)的表达.6只裸鼠按随机数字表法均分为实验组和对照组(每组3只),于左侧背部皮下分别注射105个/5 μL G5、U87细胞,25 d后处死裸鼠,取出肿瘤,免疫组织化学染色检测肿瘤中nestin的表达. 结果 G5肿瘤球中nestin、CD133、HIF2α阳性细胞率分别为91％±5％、95％±4％和98％±2％; Western blotting检测显示G1～G5肿瘤球中HIF2α蛋白的表达逐渐增加;G5细胞能分化为表达βⅢ-Tublin、GFAP、O1的子细胞;6只裸鼠均长出肿瘤,实验组的肿瘤体积较大,且肿瘤组织nestin阳性细胞率(47％±11％)明显高于对照组(5％±2％). 结论使用功能性分离和纯化方法获得的G5肿瘤球细胞,表达胶质瘤干细胞的特异性标记物、具有三系分化能力和较强的体内成瘤能力,符合胶质瘤干细胞的特征.
목적 탐색일충간단、고효、은정、가고、경제적효질류간세포분리화순화방법. 방법 취대수생장기U87세포,응용무혈청간세포배양기,치함1％O2적결양배양상중배양,10d후전대배양병리심수집제일대종류구(G1),련속전대배양획득제5대종류구(G5).면역조화염색검측G5종류구중소단백(nestin)、CD133화결양유도인자2α (HIF2α)적표체;Western blotting검측G1～G5종류구중HIF2α단백적표체;면역세포화학염색검측G5분화세포중효질섬유산성단백(GFAP)、O1급βⅢ미관단백(βⅢ-Tublin)적표체.6지라서안수궤수자표법균분위실험조화대조조(매조3지),우좌측배부피하분별주사105개/5 μL G5、U87세포,25 d후처사라서,취출종류,면역조직화학염색검측종류중nestin적표체. 결과 G5종류구중nestin、CD133、HIF2α양성세포솔분별위91％±5％、95％±4％화98％±2％; Western blotting검측현시G1～G5종류구중HIF2α단백적표체축점증가;G5세포능분화위표체βⅢ-Tublin、GFAP、O1적자세포;6지라서균장출종류,실험조적종류체적교대,차종류조직nestin양성세포솔(47％±11％)명현고우대조조(5％±2％). 결론 사용공능성분리화순화방법획득적G5종류구세포,표체효질류간세포적특이성표기물、구유삼계분화능력화교강적체내성류능력,부합효질류간세포적특정.
Objective To explore a new and simple approach of functional isolation and purification of glioma stem cells.Methods U87 cells at the logarithmic phase were cultured at serum-free stem cell medium under hypoxia (1％O2); 10 d after that,the first generation (G1) of tumorspheres was collected by centrifugation,and continuous passage culture was performed till the fifth generation (G5) of t umorspheres was harvested; the expressions of CD 133,nestin and hypoxia inducible factor 2 alpha (HIF2α) in G5 tumorspheres were detected by immumohistochernical staining; Western blotting was employed to detect the HIF2α protein expression in G1-G5 tumorspheres; immunocytochemical staining was used to detect the expressions of glial fibrillary acidic protein (GFAP),O1 and βⅢ-Tublin in G5 differentiated cells.Six nude mice were randomly divided into experimental group and control group (n=3); they were,respectively,received left dorsal subcutaneous injection of 105 cells/5 μL G5 and U87 cells; 25 d after that,the nude mice were sacrificed and the tumors were removed; immunohistochemistry was used to detect the nestin expression in the tumors.Results The percentages of nestin,CD133 and HIF2α positive cells were 91％ ±5％,95％ ±4％ and 98％ ±2％,respectively,in the G5 turnorspheres; Western blotting indicated that the HIF2α protein expression from G1 to G5 cells was gradually increased; G5 cells can differentiate into daughter cells expressed βⅢ-Tublin,GFAP and O1.Six nude mice could grow tumors,and the tumor sizes and numbers ofnestin positive cells in the experimental were obviously larger than those in control group (nestin positive cell rate was 47％±11％ and 5％±2％,respectively).Conclusions G5 cells can express glioma stem cell -specific markers (CD133,nestin and HIF2α),and enjoy three-line differentiation capacity and strong in vivo tumorigenicity,which share all properties ofglioma stem cells.Approach in this study is a simple,reliable and effective approach to isolate and purify glioma stem cells.