异时相Cyclin B1的表达与细胞凋亡的关系及其机制的探讨

发布时间：2018-06-07 01:41

本文选题：Cyclin + B1　；参考：《华中科技大学》2009年博士论文

【摘要】： 第一部分：四环素可控表达Cyclin B1的载体构建和单克隆细胞株的筛选目的：筛选四环素诱导细胞周期素B1(Cyclin B1)可控表达的293单克隆细胞株(Tetracycline-Regulated Expression 293 cells，T-REx~(TM)-293)。方法：用多聚酶链反应(polymerase chain reactions，PCR)的方法从人胎肝cDNA文库中调出带有酶切位点的Cyclin B1基因全长，在37℃经过BamH1和X-bal1双酶切过夜后，与经过同样酶切后的pcDNA4／TO／myc-HisB载体在16度连接过夜，转化BL21细菌感受态，然后涂在带有氨苄抗性的LB培养板上，37度培养过夜，挑细菌克隆，摇菌，测序鉴定。接着大提测序正确的质粒，用脂质体Lipofectamine~(TM)2000转染T-REx~(TM)-293细胞，用含有杀稻瘟菌素(Blasticidin，5μg／ml)和腐草霉素(Zeocin，200μg／ml)双药物的DMEM培养两周后，将细胞传96孔板，等单个细胞扩增出单细胞克隆。最后，用Western blot和流式细胞仪检测单克隆细胞株在加入四环素诱导后目的基因的表达情况。结果：构建的pcDNA4／TO／myc-HisB-Cyclin B1载体，经英骏公司测序鉴定序列正确。筛选出的单克隆细胞株，在未加四环素时没有外源性的Cyclin B1表达，在加入四环素后，3小时就有表达，随时间的增加，Cyclin B1表达量也增加，48小时最多。结论：筛选出的T-REx~(TM)-293单克隆细胞株能经四环素可控表达Cyclin B1。第二部分：G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡目的：利用四环素诱导Cyclin B1可控表达的293单克隆细胞株来证明G_0／G_1期异时相Cyclin B1的表达促进细胞凋亡方法：挑选出一个最优的Thymidine的浓度，其能最大程度的阻滞细胞于G_0／G_1期。接着，将细胞大量阻滞在G_0／G_1期，然后在阻滞好的细胞中加入微量的四环素诱导Cyclin B1表达48小时，最后用Annexin V-PI双标法和API法检测细胞凋亡的量和凋亡的周期特异性，同时用Western blot检测细胞周期素依赖蛋白激酶1(Cdk1)的活性情况。结果：用Thymidine处理细胞后，绝大部分细胞都同步化在G_0／G_1期。在四环素诱导Cyclin B1表达48小时后，诱导组比非诱导组的Cyclin B1蛋白总量明显增加，细胞凋亡明显增加。此时，活性的Cdk1(Thr~(161)磷酸化)蛋白量较未诱导组也明显增加。结论：G_0／G_1期异时相Cyclin B1的表达能促进细胞凋亡。第三部分：Cyclin B1导致的G_0／G_1期特异性细胞凋亡机制的探索目的：探索在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了激活Cdk1外，Cyclin B1还可能和哪些蛋白相互作用，或者Cyclin B1／Cdk1能通过磷酸化哪些蛋白质起作用。方法：我们筛选好的单克隆细胞株在加入四环素后能表达带有Myc和His标签的外源性Cyclin B1。将未诱导的和用四环素诱导48小时的细胞裂解后，分别加入10μgMyc的单克隆抗体，用免疫沉淀(immune precipitation，IP)的方法将可能和Cyclin B1结合的蛋白沉淀下来，接着将蛋白混合物进行SDS PAGE电泳，用考马斯亮蓝染色法和银染法找出未诱导组和诱导组之间的差异蛋白，然后用质谱(mass spectrometry，MASS)分析出差异蛋白的名称。结果：用质谱鉴定出的蛋白有细胞周期素依赖蛋白激酶一(Cyclin-dependentkinase 1，，Cdk1)，细胞周期素依赖蛋白激酶二(Cyclin-dependent kinase 2，Cdk2)，有丝分裂阻滞缺失样蛋白一(Mitotic arrest deficient-like protein 1，MAD1-like 1，MAD1L1)和热休克蛋白八(heat shock 70kDa protein 8 isoform 1)。结论：在由Cyclin B1导致的G_0／G_1期特异的细胞凋亡中，除了磷酸化Thr~(161)残基激活Cdk1后导致细胞凋亡外，Cyclin B1(Cyclin B1／Cdk1)与Cdk2、MAD1L1和Hsp70的结合都有可能参与其中。
[Abstract]:Part I: Construction of vector for tetracycline controlled expression of Cyclin B1 and screening of monoclonal cell lines
Objective: to screen the 293 monoclonal cell lines (Tetracycline-Regulated Expression 293 cells, T-REx~ (TM) -293) that can be controlled by tetracycline induced cyclin B1 (Cyclin B1).
Methods: using the polymerase chain reaction (polymerase chain reactions, PCR), the whole length of the Cyclin B1 gene with the enzyme cut site was transferred from the cDNA Library of human fetal liver. After the night of BamH1 and X-bal1 double enzyme at 37 C, the pcDNA4 / TO / myc-HisB carrier after the same enzyme was connected for the night at 16 degrees, then the bacterial receptive state was transformed. After being coated on the LB culture plate with ampicillin resistance, 37 degrees were cultured for the night, bacterial cloning, bacteria shaking, sequencing identification were carried out. Then the correct plasmid was sequenced and T-REx~ (TM) -293 cells were transfected with liposome Lipofectamine~ (TM) 2000, and the two drugs were cultured with rice blast (Blasticidin, 5 u g / ml) and humomycin (Zeocin, 200 mu g / ml). After two weeks, the cells were passed through 96 orifice plates and single cells were amplified by single cell clone. Finally, the expression of the target gene was detected by Western blot and flow cytometry after the induction of tetracycline.
Results: the constructed pcDNA4 / TO / myc-HisB-Cyclin B1 carrier was correct by the sequencing and identification sequence of the company. The screened monoclonal cell line was not expressed as a exogenous Cyclin B1 at the time of tetracycline. After adding tetracycline, it was expressed in 3 hours. As time increased, the expression of Cyclin B1 increased and the maximum of 48 hours was increased.
Conclusion: the screened T-REx~ (TM) -293 monoclonal cell line can express Cyclin B1. by tetracycline.
The second part: the expression of G_0 / G_1 phase Cyclin B1 promotes cell apoptosis.
Aim: to demonstrate the expression of Cyclin / B1 in G_0 / G_1 phase by promoting the apoptosis of 293 monoclonal cell lines induced by tetracycline in Cyclin B1.
Methods: the optimum concentration of Thymidine was selected, and the cells could block the cells at the G_0 / G_1 stage to the maximum extent. Then, the cells were blocked in G_0 / G_1 period, and then the Cyclin B1 expression was induced by the addition of trace tetracycline in the blocked cells for 48 hours. Finally, the amount of apoptosis and the apoptosis were detected by the Annexin V-PI double standard method and API method. Cyclical specificity of the cell cycle was detected, and the activity of cyclin dependent kinase 1 (Cdk1) was detected by Western blot.
Results: after the cells were treated with Thymidine, most of the cells were synchronized in the G_0 / G_1 phase. After 48 hours of Cyclin B1 expression induced by tetracycline, the total amount of Cyclin B1 protein in the induction group was obviously increased and the apoptosis increased obviously. At this time, the activity of Cdk1 (Thr~ (161) phosphorylation) protein was also increased significantly than that of the uninduced group.
Conclusion: the expression of G_0 / G_1 phase Cyclin B1 can promote cell apoptosis.
The third part: the mechanism of G_0 / G_1 phase specific apoptosis induced by Cyclin B1.
Objective: To explore the interaction between Cyclin B1 and what proteins in G_0 / G_1 specific apoptosis induced by Cyclin B1, in addition to activating Cdk1, or that Cyclin B1 / Cdk1 can act by which proteins can be phosphorylated.
Methods: after adding tetracycline, our screened monoclonal cell lines could express the exogenous Cyclin B1. with Myc and His tags, which were uninduced and induced by tetracycline for 48 hours of cell lysis, adding 10 mu gMyc monoclonal antibodies, and using immune precipitation, IP to combine with Cyclin B1. The protein was precipitated and the protein mixture was followed by SDS PAGE electrophoresis. The difference protein between the uninduced and the induced groups was found by coloring and silver staining. Then the name of the differential protein was analyzed by mass spectrometry (mass spectrometry, MASS).
Results: the proteins identified by mass spectrometry were cyclin dependent protein kinase 1 (Cyclin-dependentkinase 1, Cdk1), cyclin dependent protein kinase two (Cyclin-dependent kinase 2, Cdk2), and mitotic block deletion like protein 1 (Mitotic arrest deficient-like protein 1, MAD1-like 1, MAD1L1) and heat shock protein eight (heat) (heat) Shock 70kDa protein 8 isoform 1).
Conclusion: in G_0 / G_1 specific apoptosis induced by Cyclin B1, the combination of Cyclin B1 (Cyclin B1 / Cdk1) with Cdk2, Cyclin and G_1 may participate in the apoptosis of cells except the phosphorylated Thr~ (161) residue activates Cdk1.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

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