TPCA1對BMSCs在C6腦膠質(zhì)瘤細胞模擬的腫瘤微環(huán)境中惡性轉(zhuǎn)化規(guī)避的研究
發(fā)布時間：2018-08-02 21:11
【摘要】：目的通過構(gòu)建骨髓間充質(zhì)干細胞BMSCs(Bone Mesenchymal Stem Cells)與C6腦膠質(zhì)瘤細胞間接共培養(yǎng)模型,同時用藥物TPCA1處理所建共培養(yǎng)細胞模型,探討規(guī)避BMSCs在C6模擬的腫瘤微環(huán)境中惡性轉(zhuǎn)化的機制,為BMSCs作為臨床腫瘤靶向治療載體的安全應用提供參考。方法1.實驗分組研究共分為兩部分:1.1第一部分分組如下:空白對照組單獨培養(yǎng)的BMSCs陽性對照組單獨培養(yǎng)的C6腦膠質(zhì)瘤細胞實驗組BMSCs與C6腦膠質(zhì)瘤間接共培養(yǎng)及處理組1.2第二部分分組如下:空白對照組BMSCs單獨培養(yǎng)陽性對照組C6腦膠質(zhì)瘤細胞單獨培養(yǎng)實驗組BMSCs與C6腦膠質(zhì)瘤間接共培養(yǎng),分為三個亞組:第一組:共培養(yǎng)不作處理第二組:共培養(yǎng)加DMSO第三組:共培養(yǎng)加TPCA1處理(300noml/L)2.實驗方法2.1細胞間接共培養(yǎng)技術(shù)2.2顯微鏡觀察BMSCs形態(tài)學變化2.3 CCK8檢測TPCA1藥物對細胞毒性的大小2.4流式細胞術(shù)鑒定BMSCs2.5 Q-PCR檢測各組細胞STAT3、NF-κB和C-myc m RNA的表達2.6 Western Blot檢測各組細胞P-STAT3、NF-κB和C-myc蛋白的表達2.7免疫熒光檢測各組細胞P-STAT3、NF-κB和C-myc蛋白的表達2.8遷移和侵襲實驗檢測細胞的遷移和侵襲能力實驗結(jié)果1第一部分實驗結(jié)果1.1原代提取的BMSCs經(jīng)流式細胞技術(shù)鑒定CD90的陽性表達率99.8%,CD29的陽性表達率為92.5%,CD34和CD45均不表達。1.2顯微鏡下觀察單獨培養(yǎng)的BMSCs細胞形態(tài)排列規(guī)整,集落漩渦狀生長;與C6腦膠質(zhì)瘤細胞間接共培養(yǎng)的BMSCs形態(tài)排列明顯不規(guī)整;C6膠質(zhì)瘤細胞排列雜亂;TPCA1處理的間接共培養(yǎng)細胞隨著藥物濃度的增加,細胞的增值速度明顯減慢,細胞的形態(tài)比未處理的共培養(yǎng)組細胞規(guī)整。1.3 CCK8檢測隨著藥物濃度的增加,共培養(yǎng)的BMSCs的增值速度逐漸下降。1.4 Q-PCR檢測各組細胞STAT3、NF-κB和C-myc m RNA的表達量隨著藥物濃度的增加逐漸下降。2第二部分實驗結(jié)果2.1顯微鏡下觀察,陰性對照組細胞排列相對規(guī)整,陽性對照組細胞排列相對雜亂,實驗組中TPCA1處理組的細胞的規(guī)整性明顯比未處理組好。2.2 TPCA1處理組細胞遷移和侵襲能力明顯低于未處理組(p0.05)。2.3 TPCA1處理組細胞P-STAT3、NF-κB和C-myc m RNA表達和蛋白的表達明顯低于未處理組(p0.05)。結(jié)論1 TPCA1可以抑制處于C6腦膠質(zhì)瘤模擬的腫瘤微環(huán)境內(nèi)的BMSCs的細胞增值,抑制BMSCs STAT3、NF-κB和C-myc m RNA的表達,并具有濃度依賴性,其最佳抑制濃度為200nmol/L。2與C6腦膠質(zhì)瘤細胞間接共培養(yǎng)的BMSCs生物學特性發(fā)生改變,其STAT3和NF-κB存在過度表達與激活,其下游靶基因C-myc表達明顯升高。TPCA1處理后的間接共培養(yǎng)BMSCs其各項瘤化指標均受到抑制,說明TPCA1對處于C6腦膠質(zhì)細胞模擬的瘤腫瘤微環(huán)境中的BMSCs的瘤化具有規(guī)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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-3
本文編號：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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-3
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