创伤刺激大鼠C6胶质瘤肿瘤干细胞后对其致瘤性的影响

The Influence of Trauma on the Tumorigenicity of Rat C6 Glioma Tumor Stem Cells in vivo

目的观察创伤增强大鼠C6胶质瘤细胞中侧群(C6-SP)细胞致瘤性的作用,并探讨其作用机制。方法用流式细胞法分选出大鼠C6-SP细胞,通过MTT实验、克隆形成实验和流式细胞术检测细胞周期观察其生物学特性;免疫荧光法和流式细胞术检测CD133表达;原位种植评估其致瘤性。建立大鼠C6-SP细胞模型10 d后,对大鼠脑部行原位致伤和异位致伤,设立空白对照组,计算各组肿瘤体积,免疫组化检测Ki67;建立大鼠CM-DiI染色C6-SP细胞模型10 d后,对大鼠脑部行原位和异位致伤,设立空白对照组,行脑组织冰冻切片观察各组CM-DiI染色C6-SP细胞有无迁移。结果 C6-SP细胞在无血清培养中可形成肿瘤球,C6-SP细胞倍增时间较普通C6细胞短,单细胞克隆形成率C6-SP细胞(77.0%)高于普通C6细胞(16.4%);C6-SP中49.7%±5.2%的细胞处于G0/G1期,普通C6细胞处于G0/G1期的占35.2%±4.3%;C6-SP细胞CD133表达呈阳性;C6-SP细胞(500/10μL)原位移植2周后100%成瘤。原位致伤组肿瘤体积、Ki67表达水平大于异位致伤组和空白对照组(P<0.05),异位致伤组和空白对照组之间差异无统计学意义(P>0.05),原位致伤组和异位致伤组致伤部位脑组织可见红染肿瘤细胞,空白对照组未见红染肿瘤细胞。结论 C6-SP细胞是大鼠胶质瘤肿瘤干细胞;肿瘤生长部位的创伤会增强C6-SP细胞的致瘤性,同时,创伤还引起C6-SP细胞在脑内迁移。

Objective To investigate the influence of trauma on the tumorigenicity of rat glial tumor stem cells（C6-side population cell,C6-SP） in vivo.Methods Rat glial tumor stem cells C6-SP were isolated by flowcytometry.The biological behavior of C6-SP cells were observed by means of MTT experiment,single cell cloning,and cell cycle study with FCM CD133 expression was measured by immunofluorescence and FCM..The tumorigenicity of C6-SP cells in vivo was evaluated by in situ tumor growth after intracranial implantation.The rat model was established by intracranial implantation of C6-SP cells.10 days later,the rats in experimental groups were subjected to orthotopic or ectopic trauma.24 days later,brain specimen was retrieved,gross tumor volume was measured,and Ki67 was evaluated by immunochemistry.The migration of stem cell was studied by the method to observe the relocation of C6-SP cells.Results Clustrus tumor growth was seen when C6-SP cell was cultured in serum-free medium.The doubling time of C6-SP cell was shorter than ordinary C6 cell.Single stem cell cloning efficiency of C6-SP cell was 77% whereas that of ordinary C6 cell was 16.4%.Among cloned C6-SP cell and ordinary C6 cell,49.7%±5.2% and 35.2%±4.3% were at G0/G1 phase respectively.CD133 expression of C6-SP cells was positively shown by immunofluorescence.Tumorigenesis was 100% 2 weeks after in situ implantation of C6-SP cells.Gross tumor volume and Ki67 expression of orthotopic trauma group were larger and higher than those of ectopic trauma group or blank control group（P0.05） whereas difference was insignificant in the later two groups（P0.05）.Red stained cells relocation was seen in both traumatized groups and absent in controls.ConclusionC6-SP cells are the tumor stem cells（TSCs） for rat glioma.Trauma at the lesional site could increase tumorigenicity of the C6-SP cells.Moreover,trauma could induce migration of C6-SP cells in brain.