蛋白激酶C抑制剂TAM对人脑胶质瘤细胞放射敏感性的影响及其机制研究

发布时间：2018-04-16 14:41

本文选题：TAM + 人脑胶质瘤细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：目的探讨蛋白激酶C（PKC）抑制剂他莫昔芬（tamoxifen，TAM）联合X射线对人脑胶质瘤A172和U251细胞生长、细胞周期、凋亡和放射敏感性的影响及其机制，构建人脑胶质瘤裸鼠原位瘤，在体内验证TAM的放射增敏作用。方法（一）体外实验1、MTT法检测不同TAM浓度、不同处理时间脑胶质瘤A172、U251细胞的增殖变化；2、划痕实验观察脑胶质瘤A172、U251细胞的迁移能力；3、克隆形成实验检测TAM对脑胶质瘤A172、U251细胞的放射敏感性的影响；4、流式细胞仪测定TAM联合X射线对脑胶质瘤A172、U251细胞的周期分布和细胞凋亡的影响；5、western-blot法检测细胞内蛋白表达水平的变化。（二）体内实验：构建裸鼠脑胶质瘤原位模型，将成瘤的裸鼠随机分为对照组、放疗组、TAM组、放疗+TAM组，每组5只。MRI评估原位瘤裸鼠肿瘤体积大小，处死裸鼠取瘤组织，免疫组化检测瘤组织中与凋亡相关的蛋白Bad的变化。结果（一）体外实验：1、TAM可显著抑制人脑胶质瘤A172、U251细胞的增殖，当TAM浓度为10μmol/L时，A172细胞的存活率为95.1%，U251细胞的存活率为93.9%，且与对照组相比无统计学意义，因此后续实验研究选择10μmol/L药物浓度作为实验浓度。2、细胞划痕实验显示TAM联合X射线可显著抑制人脑胶质瘤A172、U251细胞的迁移能力。3、TAM联合X射线照射作用脑胶质瘤细胞时，细胞克隆存活率随照射剂量的增加而减少（p＜0.01），，多靶单击模型拟合剂量存活曲线，A172细胞单纯照射组的平均致死剂量（D0）和准阈剂量（Dq）分别2.46Gy和2.97Gy，照射+TAM组的D0和Dq分别为1.97Gy和2.80Gy，A172细胞的放射增敏比（SER）为1.25；U251细胞单纯照射组的D0和Dq分别为2.60Gy和0.91Gy，照射+TAM组的D0和Dq分别为1.78Gy和1.34Gy，U251细胞的放射增敏比（SER）为1.46。4、TAM联合X射线使人脑胶质瘤A172、U251细胞发生G2/M期阻滞且不可逆转。5、TAM联合X射线可显著增加人脑胶质瘤A172、U251细胞的凋亡率（p＜0.01）。6、western-blot结果显示，TAM联合X射线作用脑胶质瘤A172、U251细胞能显著抑制PKCι和Cdk7的活性，周期蛋白cyclinB1的表达减少，而凋亡蛋白Bad的表达明显增加，caspase3的活性增加，PARP的活性降低。（二）体内试验：1、构建原位脑胶质瘤A172细胞的荷瘤鼠模型，MRI检查结果显示照射+TAM组肿瘤体积比其他实验组肿瘤体积明显小，免疫组化显示照射+TAM组肿瘤组织Bad表达增高。结论（1）TAM能明显抑制人脑胶质瘤A172和U251细胞的增殖，呈浓度和作用时间依赖性；（2）TAM联合X射线能显著抑制人脑胶质瘤A172和U251细胞的迁移能力，TAM能增加人脑胶质瘤A172和U251细胞的放射敏感性（；3）TAM联合X射线可显著增加脑胶质瘤A172和U251细胞的G2/M期阻滞，促进脑胶质瘤细胞的凋亡；（4）TAM能够通过抑制PKCι的活性增加脑胶质瘤A172和U251细胞的辐射敏感性。同时TAM能够通过抑制Cdk7的活性进而降低G2/M期相关蛋白cyclinB1的表达，导致G2/M期的阻滞增加，凋亡蛋白Bad的升高，增加caspase3活性并降低PARP的活性，增强X射线诱导的凋亡作用。（5）TAM联合X射线能有效抑制肿瘤生长。
[Abstract]:Objective to investigate the protein kinase C (PKC) inhibitor tamoxifen (tamoxifen, TAM) and X ray on the growth of human glioma A172 cells and U251 cell cycle, apoptosis and radiosensitivity of tumor and its mechanism, construction of human glioma in nude mice, sensitized in vivo validation of TAM radiation.
Methods (1) in vitro experiment 1, detection of different concentrations of TAM MTT method, different treatment time of A172 brain glioma, proliferation of U251 cells; 2, scratches experimental observation of brain glioma A172, the migration ability of U251 cells; 3, clone forming test of TAM on brain glioma A172, the radiosensitizing effect of U251 cells the 4, TAM and X; X-ray determination of glioma A172 cells by flow cytometry, cell cycle distribution and apoptosis effect of U251 cells; 5, the protein expression of Western-blot was detected by changes in the level.
(two) in vivo: construct nude mice brain glioma orthotopic model of nude mice were randomly divided into the tumor control group, radiotherapy group, TAM group, +TAM radiotherapy group, 5 rats in each group of.MRI to assess the in situ tumor size in nude mice, mice were killed and the tumor tissue changes, immunohistochemistry and apoptosis detection in tumor tissue the protein of Bad.
Results (1) in vitro experiment: 1 TAM inhibited glioma A172 cell proliferation of U251, when the TAM concentration is 10 mol/L, the survival rate of A172 cells was 95.1%, the survival rate of U251 cells was 93.9%, and compared with the control group had no statistical significance, so the follow-up experiment research 10 mol/L concentration as the experimental concentration of.2, cell scratch test showed that TAM combined with X ray can significantly inhibit human glioma A172, U251 cell migration ability of.3, TAM and X ray irradiation of glioma cells, cell survival rate with the dose increase and decrease (P < 0.01), multi target click the model fitting dose survival curve, the mean lethal dose of A172 cells irradiated group (D0) and the quasi threshold dose (Dq) were 2.46Gy and 2.97Gy, +TAM D0 and Dq irradiation group were 1.97Gy and 2.80Gy, A172 cells radiosensitization ratio (SER) is 1.25; U251 cells alone The irradiation group D0 and Dq were 2.60Gy and 0.91Gy, +TAM D0 and Dq irradiation group were 1.78Gy and 1.34Gy, U251 cells radiosensitization ratio (SER) of 1.46.4, TAM combined with X ray to human glioma A172, U251 cells G2/M phase arrest and irreversible.5, TAM combined with X ray significantly the increase of human glioma A172, the apoptosis rate of U251 cells (P < 0.01).6, Western-blot results showed that TAM combined with X ray effect of A172 glioma cells, U251 can significantly inhibit PKC L and the activity of Cdk7, reduce the expression of cyclin cyclinB1, and apoptosis protein Bad expression was significantly increased, the increase of Caspase3 activity, decreased the activity of PARP. (two) in vivo experiment: 1 mice model of orthotopic A172 glioma cells, MRI examination results showed that the irradiation group +TAM the tumor volume was significantly smaller than the other experimental group tumor volume, immunohistochemistry showed that irradiation of tumor tissue in +TAM group Bad Up to the rise.
Conclusion (1) TAM can inhibit glioma cell proliferation of A172 and U251, in a concentration and time dependent effect; (2) the migration ability of TAM combined with X radiation can significantly inhibit human glioma A172 and U251 cells, TAM can increase the radiation sensitivity of human glioma cells (A172 and U251 3; sensibility TAM) combined with X ray can significantly increase the glioma A172 and U251 cells G2/M phase arrest and apoptosis of brain glioma cells; (4) TAM can increase the radiation sensitivity of A172 glioma and U251 cells by inhibiting PKC iota activity. TAM can also by inhibiting the activity of Cdk7 and decreased the expression of G2/M the related protein cyclinB1, resulting in a delay in G2/M phase increased, increased apoptosis protein Bad, increase the activity of Caspase3 and decreased PARP activity, enhanced apoptosis induced by X - ray. (5) TAM combined with X ray can effectively inhibit tumor growth.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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