新型CDK7抑制剂对胶质瘤细胞生物学功能的影响

发布时间：2018-03-24 06:35

本文选题：胶质瘤　切入点：增殖　出处：《南京医科大学》2017年硕士论文

【摘要】：目的胶质瘤呈浸润性生长,具有高度侵袭性,恶性胶质瘤患者病死率高,并且缺乏有效的治疗手段。新型CDK7抑制剂THZ1含有一个丙烯酰胺结构,能共价结合到传统激酶区域外的半胱氨酸残基上,是首个被报道的共价不可逆CDK抑制剂。THZ1在小细胞肺癌、三阴性乳腺癌、神经母细胞瘤、T细胞急淋和其他造血系统肿瘤中显示了较好疗效,其能够抑制多种癌基因的转录,阻滞细胞周期进展,抑制增殖,诱发凋亡。本研究探讨THZ1对人胶质瘤细胞系U87及U251的增殖、凋亡、侵袭能力的影响及细胞周期的阻滞作用,进一步探索THZ1的功能及其抗胶质瘤的可能机制。方法将U87及U251细胞分成空白组、对照组及实验组,对于U87细胞,实验组分别采用终浓度为 25nmol/L、50 nmol/L、100 nmol/L、200 nmol/L、400 nmol/L的THZ1进行处理,对于U251细胞实验组分别采用终浓度为5 nmol/L、10 nmol/L、20 nmol/L、40 nmol/L、80 nmol/L的THZ1进行处理,空白组为加入等量的DMEM完全培养基,溶剂对照组(DMSO组)为含THZ1工作液中等量的DMS0的DMEM完全培养基。实验组加入不同浓度的THZ1处理后,以CCK-8法及平板克隆形成试验分析细胞增殖;Transwell侵袭试验观测各组侵袭能力的变化;流式细胞仪检测各组细胞周期分布及凋亡情况;Western blot实验检测凋亡相关蛋白Caspase-3的表达量。结果 1.THZ1抑制U87及U251的增殖,72小时药物IC50,U87细胞为83.1nmol/L、U251细胞为13.7nmol/L。平板克隆形成试验结果显示对照组克隆形成率为(55.26±8.81)%,5nmol/L处理组的克隆形成率为(19.34±3.41)%,与对照组相比差异有统计学意义(t=6.586,P0.01),10nmol/L处理组未见明显克隆形成,结果显示低剂量THZ1即可明显抑制U251细胞的单克隆形成率。2.胶质瘤细胞U87具有较强的侵袭能力,通过体外transwell侵袭实验发现,对照组细胞数为134±13个,50nmol/L处理组细胞数为72±7个,与对照组相比差异有统计学意义(t=7.141,P0.01),给予50nmol/L THZ1处理24小时后,其侵袭能力明显减弱。所以,THZ1能明显的降低胶质瘤细胞的侵袭性。3.THZ1促进U251细胞凋亡,Caspase-3表达增加。对照组凋亡率为(8.3±2.5)%,THZ1 处理 10nmol/L 组凋亡率为(15.6±3.7)%,20nmol/L 组凋亡率为(39.7±6.0)%,凋亡率随着THZ1浓度的升高而增加。实验组与对照组比较,差异有统计学意义(t分别为2.832和8.367,P0.05)。Western blot结果显示,凋亡相关蛋白Caspase-3表达随着THZ1浓度升高而明显增加。4.THZ1 阻滞U87及U251细胞周期进展,G2期细胞显著增加。流式细胞仪检测结果显示使用浓度为25、50、100nmol/L的THZ1处理U87细胞24h后,G2期随着药物浓度的增加,细胞比例逐渐增加,(分别为31.26± 1.97、34.09±2.09、41.22±2.40)与对照组(17.95±2.83)比较差异有统计学意义(P0.01)。同样,使用浓度为5、10、20nmol/L的THZ1处理U251细胞24h后,G2期随着药物浓度的增加,细胞比例逐渐增加,(分别为9.27±2.01、41.35±0.73、56.92±0.69)与对照组(16.17±1.99)比较差异有统计学意义(P㩳0.01)。结论1.THZ1能够抑制胶质瘤细胞U87及U251的增殖,促进凋亡,抑制侵袭,并阻滞细胞周期进展。2.新型CDK抑制剂THZ1有望成为神经胶质瘤的靶向药物
[Abstract]:Objective glioma infiltrative growth, highly invasive malignant glioma patients, mortality rate is high, and the lack of effective treatment. A novel CDK7 inhibitor THZ1 has a structure of acrylamide, covalently bound to cysteine residues to traditional kinase outside the region, is the first reported covalent irreversible CDK inhibitor.THZ1 in small cell lung cancer, three breast cancer, neuroblastoma, T cell acute lymphocytic leukemia and other hematopoietic tumors showed better curative effect, it can inhibit transcription of oncogenes, progress, cell cycle arrest and inhibit proliferation and induced apoptosis. This study investigated the effect of THZ1 on human glioma cell lines U87 and U251 the proliferation, apoptosis, invasion and inhibition effect of cell cycle, can further explore the mechanism of THZ1 function and anti glioma. Methods U87 and U251 cells were divided into blank group, control group and For the experimental group, U87 cells, experimental group respectively with the final concentration of 25nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 400 nmol/L THZ1 treatment for U251 cells in the experimental group were used at the concentration of 5 nmol/L, 10 nmol/L, 20 nmol/L, 40 nmol/L, 80 nmol/L THZ1 were treated as blank group adding the same amount of DMEM complete medium, solvent control group (group DMSO) an THZ1 containing working fluid in DMS0 DMEM complete medium. Different concentration in experimental group after THZ1 treatment, CCK-8 assay and colony formation test and analysis of changes of Transwell cell proliferation; invasion capability test observed the distribution of cells; each cycle and apoptosis was detected by flow cytometry; the expression of Western blot assay of apoptosis related protein Caspase-3. Results 1.THZ1 inhibited the proliferation of U251 and U87, 72 hours of drug IC50, U87 cell 83.1nmol/L, U251 cell 13.7nmol/L. colony formation test showed that the control group clone formation rate was (55.26 + 8.81)%, the treatment group 5nmol/L clone formation rate was (19.34 + 3.41)%, compared with the control group the difference was statistically significant (t=6.586, P0.01), 10nmol/L treatment group had no obvious colony formation, results showed that the monoclonal low dose of THZ1 can be inhibit U251 cell formation rate of glioma cells.2. U87 has strong ability of invasion in vitro by Transwell invasion assay showed that the cells in the control group were 134 + 13, in 50nmol/L treated cells was 72 + 7, compared with the control group the difference was statistically significant (t=7.141, P0.01), 50nmol/L THZ1 24 hours later, the invasion ability decreased. Therefore, THZ1 can significantly reduce the invasiveness of glioma cells.3.THZ1 promote U251 cell apoptosis, increase the expression of Caspase-3. The apoptosis rate of the control group (8.3 + 2.5)%, THZ1 The apoptosis rate of 10nmol/L group was (15.6 + 3.7)%, the apoptosis rate of 20nmol/L group was (39.7 + 6)%, the apoptosis rate increased with the increase of THZ1 concentration. Compared with the control group, the difference was statistically significant (t = 2.832 and 8.367, P0.05).Western blot results showed that the apoptosis related protein Caspase-3 the expression of THZ1 with the increased concentration of.4.THZ1 U87 increased significantly in U251 and block the cell cycle, a significant increase in G2 phase cells. Flow cytometry results showed that the concentration of THZ1 in U87 cells treated with 24h 25,50100nmol/L, G2 phase with the increase of drug concentration, the proportion of cells increased gradually, respectively (31.26 + 1.97,34.09 + 2.09,41.22 + 2.40) and control group (17.95 + 2.83) the difference was statistically significant (P0.01). Similarly, the use of concentration of THZ1 in U251 cells treated with 24h 5,10,20nmol/L, G2 phase with the increase of drug concentration, the proportion of cells increased gradually ( Were 9.27 + 2.01,41.35 + 0.73,56.92 + 0.69) and control group (16.17 + 1.99) and there was a statistically significant difference (P? 0.01). Conclusion: 1.THZ1 can inhibit U87 and U251 glioma cell proliferation, promote apoptosis, inhibit the invasion, and block the cell cycle progression of.2. CDK THZ1 is expected to become a new inhibitor of glioma the targeted drug

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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