【作者】 靳晓亮；

【导师】 杨波；

【作者基本信息】 郑州大学 ， 神经外科， 2009， 硕士

【摘要】 背景与目的:脑胶质瘤是颅内最常见的肿瘤,虽然在脑胶质瘤的分子、基因方面的研究已经取得了许多进步,但患者的预后仍不满意,目前手术切除仍是脑肿瘤治疗的首选方法,但根治性切除后复发率高是影响其长期生存率的主要因素。而国内外均有文献报道显示MSCs具有抑瘤特性,本实验组在之前的研究中已经观察到hMSCs可以抑制肿瘤细胞的生长。另随着C6大鼠胶质瘤细胞系的成熟发展,研究人员认为C6胶质瘤细胞的相关性研究对于人脑胶质瘤的基础研究具有参考和指导价值。本实验在建立人羊膜间充质干细胞(human mesenchymal stem cells hMSCs)及人脐带间充质细胞(human umbilicalcord stem cells hUSCs)的体外培养和鉴定方法的基础上,探讨hMSCs及hUSCs对于C6胶质瘤细胞不同共培养方法培养后可能出现的结果。在本研究中,我们将通过在体外实验中分别进行hMSCs与hUSCs和C6共培养,观察hMSCs与hUSCs和C6的相互作用,进一步明确hMSCs与hUSCs对C6生长的影响的不同,以期为进一步的研究提供参考和方向的选择。材料与方法:无菌条件下取正常足月剖腹产胎儿的羊膜和脐带采用组织块培养法及胰酶消化法分离并于含10%胎牛血清(fetal bovine serum,FBS)的MEM/F12培养基进行培养。取P3代hMSCs及hUSCs采用免疫组织化学与流式细胞仪对其间充质干细胞表面标志CD29、CD44、HLA-ABC及CD29、CD44进行鉴定和其细胞周期判定。取P3代的hMSCs及hUSCs,配制成浓度为1.0×10~6个/ml的直接共培养工作液与1.0×10~6个稳定传代3代以上C6单细胞悬液分别采用直接共培养和间接共培养的方法进行共培养,共分5组,羊膜直接共培养组(A组),羊膜间接共培养组(B组),脐带直接共培养组(C组),脐带间接共培养组(D组),空白对照组(E组),后光镜下观察细胞生长情况,收集C6进行流式细胞测定和电镜超微结构观察。结果:用酶消化法分离羊膜中的MSCs,可在体外进行培养,进而通过贴壁分离法不断得到纯化。用酶消化法及组织块培养法分离脐带中的MSCs,可在体外进行培养,进而通过贴壁分离法不断得到纯化。hMSCs及hUSCs免疫细胞化学染色显示CD44和CD29均为阳性反应:hMSCs及hUSCs的细胞周期分析具有干细胞的典型周期特性,流式细胞仪检测传代hMSCs阴性对照9.39%,CD29阳性细胞比率为82.53%,CD44阳性细胞比率为90.86%,HLA-ABC阳性细胞比率为89.55%。流式细胞仪检测传代hUSCs阴性对照7.61%,CD29阳性细胞比率为70.44%,CD44阳性细胞比率为75.50%.透射电镜观察见C6细胞不同程度出现细胞间连接消失,观察像核分裂相减少,细胞体积缩小,细胞器空泡样变及髓样变改变,甚者出现细胞核固缩,出现典型细胞凋亡形态,对照组E组细胞仍呈旺盛生长状态。C6细胞中bcl-2随时间延长阳性表达率呈下降趋势,不同时间比较差异具有显著性(P＜0.01);C、D两组较A、B两组阳性表达率降低趋势更为明显,且两两比较差异具有显著性(P＜0.01);B、D两组较A、C两组阳性表达率降低趋势更为明显(P＜0.01)。C6细胞随时间延长细胞平均凋亡率呈上升趋势,不同时间比较差异具有显著性(P＜0.01);C、D两组较A、B两组阳性表达率上升趋势更为明显,且两两比较差异具有显著性(P＜0.01);B、D两组较A、C两组阳性表达率上升趋势更为明显(P＜0.01)。结论:1.Bcl-2在C6中呈高表达。2.hMSCs与hUSCs能够降低C6克隆团细胞之间的黏附作用。3.hMSCs及hUSCs与C6共培养后C6增殖能力减弱。4.hMSCs及hUSCs与C6共培养后C6中bcl-2的表达随时间延长呈降低趋势。5.hMSCs及hUSCs与C6共培养后C6细胞凋亡率随着时间延长呈上升趋势。6.采用间接共培养方法共培养后C6细胞bcl-2的表达降低趋势更为明显。7.hUSCs与C6共培养后C6细胞凋亡率表达上升趋势更为明显。更多还原

【Abstract】 Background and Objective:Glioma is the most common intracranial tumor,although the elements in glioma, the study of a lot of progress has been made,but the prognosis of the patients are still not satisfied.At present surgical resection is still the preferred method of treatment, but the radical resection of the high relapse rate after the impact of its long-term survival rate is the main factor.Recently,we can fine some reports about the MSCs can depress the growing of the tumor.Our team have proved that MSCs can depress the growing of the tumor stem cells during the previous experiments.Because the C6 golima cells can be get easily and stably,so the basic research about C6 golima cells can enrich the knowledge of golima.This experiment in the establishment of hMSCs and hUSCs in vitro culture and identification methods on the basis of hMSCs and hUSCs to observe the probably different result which can be get by different method of co-culture between the MSCs and C6 golima cells.In this study,we will carry out in vitro seperatly with the method of co-culture between the MSCs and C6,initially found that the deferent interaction between the MSCs and C6,further defined in the choice ang reference of the next research. Materials and Methods:Under sterile conditions for the normal full-term fetus amniotic caesarean section and umlilicalcord,refine hMSCs and hUSCs from them, culture in containing 10%fetal bovine serum(fetal bovine serum,FBS) DMEM/F12 the culture medium with the method of explant tissue and digestion.P3 hMSCs and hUSCs from the immunohistochemistry and flow cytometry analysis:CD29,CD44 positive expression;flow cytometry test cycle with stem cell characteristics;P3 generation hMSCs and hUSCs co-culture with C6 directly and indirectly,there will be 5 groups,Direct train between hMSCs and C6(Group A),Indirect train between hMSCs and C6(Group B),Direct train between hUSCs and C6(Group C),Indirect train between hUSCs and C6(Group D),Blank control of C6(Group E),collected a total of cultured C6 for flow cytometry determination and electron-microscopic analysis.Results:Digestive enzymes separation of amniotic MSCs,can be cultivated in vitro and then through the wall of separation has been continuously purification.Digestive enzymes and explant tissues separation of umbilicalcord MSCs,can be cultivated in vitro and then through the wall of separation has been continuously purification.Immunocytochemical staining showed that CD44 and CD29of hMSCs and hUSCs are positive;the cell cycle analysis of hMSCs and hUSCs with stem cell characteristics of a typical cycle,FCM passage hMSCs negative control 9.39%,CD29 positive cells ratio was 82.53%,CD44 positive cells ratio was 90.86%,HLA-ABC positive cells ratio was 89.55 percent,hUSCs negative control7.61%,CD29 positive cells ratio was 70.44%,CD44 positive cells ratio was 75.50%.Under the electron-microscopic,we can find that all the four groups show the jionts tail off,the volume tail off,cellural organ denaturation,even the apoptotic body can be find,Group E grow up vigorously.Group A、B、C、D show that the bcl-2 postive rate of C6 reduce with the time run out,and the Group C、D will be more remarkable tha Group A、B;Group A、Cwill be more remarkable tha Group A、B;Group A、B、C、D show that the bcl-2 postive rate of C6 reduce with the time run out,and the Group C、D will be more remarkable tha Group A、B;Group A、Cwill be more remarkable tha Group A、B.Conclusions:1.Bcl-2 in C6 was in high expression.2.hMSCs and hUSCs can reduce C6-cell cloning between the role of adhesion.3.C6 proliferation weakened in co-culture with hMSCs and hUSCs.4.Bcl-2 in C6 after the co-culture will be lower with timing flow.5.The apoptosis rate of C6 after the co-culture will be upper with timing flow.6.Indirect co-culture group of C6 depressed more extremly.7.hUSCs depress the growing of C6 more extremly.更多还原