CAJ | 학술논문

目的对悬浮培养的脑胶质母细胞瘤干细胞球中的标记滞留细胞进行表型研究.方法通过荧光标记滞留分析区分胶质母细胞瘤干细胞球中的标记滞留细胞及标记阴性细胞;通过体外克隆分析比较上述两类细胞的自我更新能力;以Western blot检测分化前后分化标记物的表达,评估其体外分化能力;给予放、化疗处理后检测细胞增殖,评估其放、化疗敏感性;通过免疫缺陷鼠颅内种植,评估其成瘤能力.结果胶质瘤干细胞球细胞经初始DiI荧光标记示踪2周后,可区分慢增殖的DiI滞留细胞,其比例＜10％及快增殖的DiI阴性细胞.DiI滞留细胞经多次传代克隆分析,其克隆成球率保持恒定,而DiI阴性细胞呈显著下降(P＜0.05).DiI滞留细胞分化后分化标记物GFAP,PDGFRα以及βⅢ-tubulin蛋白表达相对于分化前显著上调(P＜0.05).给予10 Gy剂量照射或500μm/l替莫唑胺处理72 h后,DiI滞留细胞增殖相对于处理前无显著改变,而DiI阴性细胞增殖显著下降(P＜0.05).仅约100个DiI滞留细胞在最长追踪观察6个月时在免疫缺陷鼠颅内已可成瘤,而相应数量的DiI阴性细胞未能成瘤.结论胶质母细胞瘤干细胞球中呈现慢增殖特性的标记滞留细胞相对于标记阴性细富集了肿瘤干细胞.
목적 대현부배양적뇌효질모세포류간세포구중적표기체류세포진행표형연구.방법 통과형광표기체류분석구분효질모세포류간세포구중적표기체류세포급표기음성세포;통과체외극륭분석비교상술량류세포적자아경신능력;이Western blot검측분화전후분화표기물적표체,평고기체외분화능력;급여방、화료처리후검측세포증식,평고기방、화료민감성;통과면역결함서로내충식,평고기성류능력.결과 효질류간세포구세포경초시DiI형광표기시종2주후,가구분만증식적DiI체류세포,기비례＜10％급쾌증식적DiI음성세포.DiI체류세포경다차전대극륭분석,기극륭성구솔보지항정,이DiI음성세포정현저하강(P＜0.05).DiI체류세포분화후분화표기물GFAP,PDGFRα이급βⅢ-tubulin단백표체상대우분화전현저상조(P＜0.05).급여10 Gy제량조사혹500μm/l체막서알처리72 h후,DiI체류세포증식상대우처리전무현저개변,이DiI음성세포증식현저하강(P＜0.05).부약100개DiI체류세포재최장추종관찰6개월시재면역결함서로내이가성류,이상응수량적DiI음성세포미능성류.결론 효질모세포류간세포구중정현만증식특성적표기체류세포상대우표기음성세부집료종류간세포.
Objective To conduct the phenotypic study for the label-retaining cells in suspension cultured brain glioblastoma stem cell spheres.Methods Label-retaining assay was used to distinguish the label-retaining cells from label negative cells in the stem cell spheres of glioblastoma.In vitro clonal analysis was used to compare the self-renewal capacity of the above two types of cells.Western blot was used to detect the expression of differentiation markers before and after differentiation,and their in vitro differentiation capacity was assessed.After giving the radiotherapy or chemotherapy treatment,cell proliferation was detected,and their radiotherapy and chemotherapy sensitivities were assessed.Implantation of the tumor cells into the brain of immunocompromised mice was used to assess their tumorigenic ability.Results Two weeks after initial DiI fluorescently labeled tracer,the glioblastoma stem cell sphere cells distinguished the slow proliferative DiI retaining cells,their ratio was ＜ 10％ with the fast proliferative DiI negative cells.After several passages clonal analysis of the DiI retaining cells,the ball forming rate of clone remained constant,while the DiI negative cells were decreased significantly (P ＜0.05).After the DiI retaining cells were differentiated,the protein expressions of the differentiation markers GFAP,PDGFRα,and βⅢ-tubulin were significantly upregulated compared with those before differentiation (P ＜ 0.05).After giving an irradiation dose of 10 Gy or being treated with 500 μm/L temozolomide for 72 h,the proliferation of DiI retaining cells did not have any changes compare with that before treatment,while the proliferation of DiI negative cells was decreased significantly (P ＜0.05).Only about 100 DiI retaining cells could form tumors in the immunodeficient mice brains at the longest follow-up observation of 6 months,whereas a corresponding number of DiI negative cells failed to form tumors.Conclusion Relative to the label negative cells,showing the label-retaining cells with slow proliferation properties in the glioblastoma stem cell spheres enriched tumor stem cells.