人羊膜及脐带间充质干细胞与C6胶质瘤细胞分别共培养的实验研究

发布时间：2018-03-23 07:06

本文选题：人羊膜间充质干细胞　切入点：人脐带间充质干细胞　出处：《郑州大学》2009年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background and purpose:
Glioma is the most common intracranial tumors, although the molecules in glioma, genetic research has made many progresses, but the prognosis is still not satisfied with the current preferred method for surgical resection is the treatment of brain tumors, but the high recurrence rate after radical resection is the main factors affecting the long-term survival rate. But the domestic and foreign literatures have showed that MSCs has anti-tumor properties, in a previous study of the experimental group has been observed in the hMSCs can inhibit the growth of tumor cells. The other with the mature development of glioma cell line C6 rats, the researchers think the correlation of C6 glioma cells has reference value for based on the human brain glioma.
In this experiment, the establishment of human amniotic mesenchymal stem cells (human mesenchymal stem cells hMSCs) and human umbilical cord mesenchymal cells (human umbilicalcord stem cells hUSCs) based method for in vitro culture and identification of hMSCs and hUSCs, different in C6 glioma cells were cultured after culture method may occur as a result of. In this study, we will through respectively hMSCs and hUSCs and C6 in vitro co culture, the interaction effect of hMSCs and hUSCs and C6, hMSCs and hUSCs to further clarify the different effects on the growth of C6, to provide the reference and direction for further research.
Materials and methods: under aseptic conditions of normal full-term fetus amniotic membrane and umbilical cord culture method and enzymatic digestion and with 10% fetal bovine serum by tissue (fetal bovine serum, FBS) of the MEM/F12 medium. P3 hMSCs and hUSCs by immunohistochemistry and flow cytometry in mesenchymal stem cell surface markers CD29, CD44, HLA-ABC and CD29, were identified and the cell cycle of CD44. HMSCs and hUSCs determine P3, prepared into a concentration of 1 * 10~6 /ml direct co culture liquid and 1 * 10~6 passage 3 generation to C6 single cell suspension respectively. The direct co culture method and indirect co culture were co cultured, were divided into 5 groups, amniotic membrane in direct co culture group (group A), amniotic membrane in indirect co culture group (group B), umbilical cord direct co culture group (group C), umbilical cord indirect co culture group (D group) and control group (E group), light microscope The growth of the cells was observed, and C6 was collected for flow cytometry and ultrastructural observation of electron microscope.
Results: the digestive enzymes separation of amniotic MSCs can be cultured in vitro, then purified by adherent separation method has been used. Enzyme digestion and tissue culture method to separate the umbilical cord in MSCs can be cultured in vitro, and then through the wall of separation has been purified.HMSCs and hUSCs immunocytochemistry staining showed that CD44 and CD29 were positive reaction: the cell cycle of hMSCs and hUSCs analysis of the characteristics of a typical cycle with stem cell, serum hMSCs negative control 9.39% flow cytometry, CD29 positive cells ratio was 82.53%, CD44 positive cell rate was 90.86%, the rate of HLA-ABC positive cells of 89.55%. were detected by flow cytometry hUSCs negative control 7.61%, CD29 positive cells ratio was 70.44%, the rate of CD44 positive cells was 75.50%.
Transmission electron microscopy showed different degrees of C6 cell cell-cell junction disappeared, as observed mitoses are reduced, cell shrinkage, cell vacuolar degeneration and myeloid changed, what appeared karyopyknosis, typical apoptotic morphology, the control group E cells was still vigorous growth.
Bcl-2 C6 cells with time. The positive expression rate decreased, different time difference was statistically significant (P < 0.01); C, D two group compared with the A, two group of B positive expression rate decreased more obviously, and the 22 difference was statistically significant (P < 0.01); B, D two compared with group A, two group of C positive expression rate decreased more obviously (P < 0.01).
C6 cells with the prolonging of average cell apoptosis rate increased, the different time difference was statistically significant (P < 0.01); C, D two group compared with the A, B two group, the positive expression rate of upward trend is more obvious, and the 22 difference was statistically significant (P < 0.01); B, D two groups compared with A group, C two positive expression rate trend is more significant (P < 0.01).
Conclusion:
1.Bcl-2 is highly expressed in C6.
2.hMSCs and hUSCs can reduce the adhesion between C6 cloned cells.
The proliferation ability of C6 was weakened after co culture of 3.hMSCs and hUSCs with C6.
After co culture of 4.hMSCs and hUSCs and C6, the expression of Bcl-2 in C6 decreased with time.
After co culture of 5.hMSCs and hUSCs and C6, the apoptosis rate of C6 cells increased with time.
6. the decrease trend of bcl-2 expression in C6 cells was more obvious after co culture.
The increase of apoptosis rate in C6 cells was more obvious after co culture of 7.hUSCs and C6.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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