TPCA1对BMSCs在C6脑胶质瘤细胞模拟的肿瘤微环境中恶性转化规避的研究
发布时间：2018-08-02 21:11
【摘要】：目的通过构建骨髓间充质干细胞BMSCs(Bone Mesenchymal Stem Cells)与C6脑胶质瘤细胞间接共培养模型,同时用药物TPCA1处理所建共培养细胞模型,探讨规避BMSCs在C6模拟的肿瘤微环境中恶性转化的机制,为BMSCs作为临床肿瘤靶向治疗载体的安全应用提供参考。方法1.实验分组研究共分为两部分:1.1第一部分分组如下:空白对照组单独培养的BMSCs阳性对照组单独培养的C6脑胶质瘤细胞实验组BMSCs与C6脑胶质瘤间接共培养及处理组1.2第二部分分组如下:空白对照组BMSCs单独培养阳性对照组C6脑胶质瘤细胞单独培养实验组BMSCs与C6脑胶质瘤间接共培养,分为三个亚组:第一组:共培养不作处理第二组:共培养加DMSO第三组:共培养加TPCA1处理(300noml/L)2.实验方法2.1细胞间接共培养技术2.2显微镜观察BMSCs形态学变化2.3 CCK8检测TPCA1药物对细胞毒性的大小2.4流式细胞术鉴定BMSCs2.5 Q-PCR检测各组细胞STAT3、NF-κB和C-myc m RNA的表达2.6 Western Blot检测各组细胞P-STAT3、NF-κB和C-myc蛋白的表达2.7免疫荧光检测各组细胞P-STAT3、NF-κB和C-myc蛋白的表达2.8迁移和侵袭实验检测细胞的迁移和侵袭能力实验结果1第一部分实验结果1.1原代提取的BMSCs经流式细胞技术鉴定CD90的阳性表达率99.8%,CD29的阳性表达率为92.5%,CD34和CD45均不表达。1.2显微镜下观察单独培养的BMSCs细胞形态排列规整,集落漩涡状生长;与C6脑胶质瘤细胞间接共培养的BMSCs形态排列明显不规整;C6胶质瘤细胞排列杂乱;TPCA1处理的间接共培养细胞随着药物浓度的增加,细胞的增值速度明显减慢,细胞的形态比未处理的共培养组细胞规整。1.3 CCK8检测随着药物浓度的增加,共培养的BMSCs的增值速度逐渐下降。1.4 Q-PCR检测各组细胞STAT3、NF-κB和C-myc m RNA的表达量随着药物浓度的增加逐渐下降。2第二部分实验结果2.1显微镜下观察,阴性对照组细胞排列相对规整,阳性对照组细胞排列相对杂乱,实验组中TPCA1处理组的细胞的规整性明显比未处理组好。2.2 TPCA1处理组细胞迁移和侵袭能力明显低于未处理组(p0.05)。2.3 TPCA1处理组细胞P-STAT3、NF-κB和C-myc m RNA表达和蛋白的表达明显低于未处理组(p0.05)。结论1 TPCA1可以抑制处于C6脑胶质瘤模拟的肿瘤微环境内的BMSCs的细胞增值,抑制BMSCs STAT3、NF-κB和C-myc m RNA的表达,并具有浓度依赖性,其最佳抑制浓度为200nmol/L。2与C6脑胶质瘤细胞间接共培养的BMSCs生物学特性发生改变,其STAT3和NF-κB存在过度表达与激活,其下游靶基因C-myc表达明显升高。TPCA1处理后的间接共培养BMSCs其各项瘤化指标均受到抑制,说明TPCA1对处于C6脑胶质细胞模拟的瘤肿瘤微环境中的BMSCs的瘤化具有规避作用。
[Abstract]:Objective to establish an indirect co-culture model of bone marrow mesenchymal stem cells (BMSCs (Bone Mesenchymal Stem Cells) and C6 glioma cells, and to explore the mechanism of avoiding the malignant transformation of BMSCs in C6 simulated tumor microenvironment. To provide a reference for the safe application of BMSCs as a clinical tumor targeted therapy vector. Method 1. The experimental group was divided into two parts: the first part of the study was divided into two parts: BMSCs and C6 glioma cells were cultured separately in blank control group, BMSCs positive control group, C6 glioma cell culture group, indirect co-culture and treatment group 1.2, respectively. The second part was divided as follows: BMSCs and C6 glioma were cultured in BMSCs group and C6 glioma indirect co-culture group in blank control group BMSCs alone, positive control group C6 glioma cell culture alone. They were divided into three subgroups: the first group: co culture without treatment the second group: co culture plus DMSO the third group: co culture plus TPCA1 treatment (300noml/L) 2. Methods 2. 1 cell indirect coculture technique 2. 2 microscopically observed the morphological changes of BMSCs. 2. 3 CCK8 detection of cytotoxicity of TPCA1 drugs by 2. 4 flow cytometry the expression of STAT3, NF- 魏 B and C-myc m RNA in cells of each group was detected by BMSCs2.5 Q-PCR. 2. 6 Western Blot detection. Expression of P-STAT3NF-kappa B and C-myc protein in the cells of each group; Immunofluorescence assay for the expression of P-STAT3NF-kappa B and C-myc protein in each group; Detection of migration and invasion ability of cells in the first part of the experiment results 1. 1. 1 Primary generation extraction of P-STAT3NF-kappa B and C-myc protein. The positive expression rate of CD90 was 99.8% by flow cytometry. The positive expression rate of CD29 was 92.5%. The morphological arrangement of BMSCs cells cultured in vitro was observed under microscope without the expression of CD34 and CD45. Colony whirlpool growth, BMSCs morphological arrangement with C6 glioma cells was obviously irregular, and the proliferation rate of C6 glioma cells increased with the increase of drug concentration, and the proliferation rate of C6 glioma cells was decreased with the increase of drug concentration, and the proliferation rate of C6 glioma cells was decreased with the increase of drug concentration. The morphology of cells was higher than that of untreated cocultured cells. 1. 3 CCK8 was detected with the increase of drug concentration. The increment rate of co-cultured BMSCs was decreased gradually. 1.4 Q-PCR was used to detect the expression of STAT3NF-kB and C-myc m RNA in the cells of each group. 2. 2 the second part of the experiment showed that the cells in the negative control group were relatively regular under the microscope. The cells in the positive control group were relatively disorderly. The cell migration and invasion ability of the TPCA1 treated group was lower than that of the untreated group (p0.05) .2.3 TPCA1 group. The expression and protein expression of P-STAT3NF- 魏 B and C-myc m RNA were significantly lower in the TPCA1 treated group than in the untreated group (p0.05). Conclusion 1 TPCA1 can inhibit the proliferation of BMSCs and the expression of BMSCs STAT3NF- 魏 B and C-myc m RNA in the microenvironment of C6 glioma in a concentration-dependent manner. The best inhibitory concentration was that the biological characteristics of BMSCs in indirect co-culture of 200nmol/L.2 and C6 glioma cells were changed, and its STAT3 and NF- 魏 B were overexpressed and activated. The expression of C-myc in the downstream target gene was significantly increased. After treated with TPCA1, all the tumor markers of BMSCs were inhibited, which indicated that TPCA1 could avoid the tumorigenesis of BMSCs in the tumor microenvironment simulated by C6 glia cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3
本文编号：2160739
[Abstract]:Objective to establish an indirect co-culture model of bone marrow mesenchymal stem cells (BMSCs (Bone Mesenchymal Stem Cells) and C6 glioma cells, and to explore the mechanism of avoiding the malignant transformation of BMSCs in C6 simulated tumor microenvironment. To provide a reference for the safe application of BMSCs as a clinical tumor targeted therapy vector. Method 1. The experimental group was divided into two parts: the first part of the study was divided into two parts: BMSCs and C6 glioma cells were cultured separately in blank control group, BMSCs positive control group, C6 glioma cell culture group, indirect co-culture and treatment group 1.2, respectively. The second part was divided as follows: BMSCs and C6 glioma were cultured in BMSCs group and C6 glioma indirect co-culture group in blank control group BMSCs alone, positive control group C6 glioma cell culture alone. They were divided into three subgroups: the first group: co culture without treatment the second group: co culture plus DMSO the third group: co culture plus TPCA1 treatment (300noml/L) 2. Methods 2. 1 cell indirect coculture technique 2. 2 microscopically observed the morphological changes of BMSCs. 2. 3 CCK8 detection of cytotoxicity of TPCA1 drugs by 2. 4 flow cytometry the expression of STAT3, NF- 魏 B and C-myc m RNA in cells of each group was detected by BMSCs2.5 Q-PCR. 2. 6 Western Blot detection. Expression of P-STAT3NF-kappa B and C-myc protein in the cells of each group; Immunofluorescence assay for the expression of P-STAT3NF-kappa B and C-myc protein in each group; Detection of migration and invasion ability of cells in the first part of the experiment results 1. 1. 1 Primary generation extraction of P-STAT3NF-kappa B and C-myc protein. The positive expression rate of CD90 was 99.8% by flow cytometry. The positive expression rate of CD29 was 92.5%. The morphological arrangement of BMSCs cells cultured in vitro was observed under microscope without the expression of CD34 and CD45. Colony whirlpool growth, BMSCs morphological arrangement with C6 glioma cells was obviously irregular, and the proliferation rate of C6 glioma cells increased with the increase of drug concentration, and the proliferation rate of C6 glioma cells was decreased with the increase of drug concentration, and the proliferation rate of C6 glioma cells was decreased with the increase of drug concentration. The morphology of cells was higher than that of untreated cocultured cells. 1. 3 CCK8 was detected with the increase of drug concentration. The increment rate of co-cultured BMSCs was decreased gradually. 1.4 Q-PCR was used to detect the expression of STAT3NF-kB and C-myc m RNA in the cells of each group. 2. 2 the second part of the experiment showed that the cells in the negative control group were relatively regular under the microscope. The cells in the positive control group were relatively disorderly. The cell migration and invasion ability of the TPCA1 treated group was lower than that of the untreated group (p0.05) .2.3 TPCA1 group. The expression and protein expression of P-STAT3NF- 魏 B and C-myc m RNA were significantly lower in the TPCA1 treated group than in the untreated group (p0.05). Conclusion 1 TPCA1 can inhibit the proliferation of BMSCs and the expression of BMSCs STAT3NF- 魏 B and C-myc m RNA in the microenvironment of C6 glioma in a concentration-dependent manner. The best inhibitory concentration was that the biological characteristics of BMSCs in indirect co-culture of 200nmol/L.2 and C6 glioma cells were changed, and its STAT3 and NF- 魏 B were overexpressed and activated. The expression of C-myc in the downstream target gene was significantly increased. After treated with TPCA1, all the tumor markers of BMSCs were inhibited, which indicated that TPCA1 could avoid the tumorigenesis of BMSCs in the tumor microenvironment simulated by C6 glia cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R73-3
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