【作者】 何乐亚；

【导师】 龚建平；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【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^TM2000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：G₀／G₁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G₀／G₁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G₀／G₁期。接着，将细胞大量阻滞在G₀／G₁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G₀／G₁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(Thr¹⁶¹磷酸化)蛋白量较未诱导组也明显增加。结论：G₀／G₁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G₀／G₁期特异性细胞凋亡机制的探索目的：探索在由Cyclin B1导致的G₀／G₁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G₀／G₁期特异的细胞凋亡中，除了磷酸化Thr¹⁶¹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。更多还原

【Abstract】 Objective:A monoclone cell line was needed for expressing Cyclin B1 induced byTetracycline.Methods:The full sequence of Cyclin B1 was got from fetal liver cDNA library byPCR.After both cutted by BamH1 and X-ball at 37℃overnight,the product of Cyclin B1and empty vector pcDNA4/TO/myc-HisB were ligased at 16℃overnight by T4 DNAligase.The constructed pcDNA4/TO/myc-HisB-Cyclin B1 was sequenced,and the rightone was used to transfect T-REx^TM-293 cells using Lipofectamine^TM2000.The cells werecultured in DMEM containing Blasticidin （5μg/ml）and Zeocin （200μg/ml）for two weeks.Then they were seeded into 96 well plates.So,different clones were got,which amplifyingfrom a single cell.After cyclin B1 induced for different time by Tetracycline,cells wereharvested,and Cyclin B 1 expression was tested by Western blot and flow cytometry.Results:The pcDNA4/TO/myc-HisB-Cyclin B1 vector was successfully constructed,and the sequence was right.The monoclone cells could express Cyclin B 1-myc-his as earlyas 3 hours after Tetracycline added to DMEM,and the highest amount of CyclinB 1-myc-his was expressed 48 hours after Tetracycline added.Conclusions:The monoclone T-REx^TM-293 cell line could express Cyclin B1 inducedby Tetracycline under control. Objective:‘Unscheduled expression of Cyclin B1 in G₀/G₁ phase increasing cellapoptosis’was wanted to be confirmed by using a monoclone cell line,which couldexpress Cyclin B1 induced by Tetracycline.Methods:Cells were caused to arrest in G₀/G₁ phase by Thymidine firstly.ThenTetracycline was added to induce Cyclin B1 expression for 48 hours,and thecontrol group was added no Tetracycline.At last,the cell apoptosis was detected byAnnexin V-PI and API methods.The activity of Cdk1 was also confirmed byWestern blot at the same time.Results:Most cells were arrested in G₀/G₁ phase cause of Thymidine.CyclinB1 protein and cell apoptosis increased after Cyclin B1 expression for 48 hoursinduced by Tetracycline.And the activity of Cdk1 (Thr¹⁶¹phosphorylation)was alsoincreased.Conclusions:Unscheduled expression of Cyclin B1 in G₀/G₁ phase couldincrease cell apoptosis. Objective:Study the proteins doing functions in specific apoptosis of G₀/G₁phase cause of Cyclin B1,which can interact with Cyclin B1 or be phosphorylatedby Cyclin B1/Cdk1 complex.Methods:The monoclone cell line could express Cyclin B1 with a Myc and Histag after Tetracycline added.Cells uninduced or induced by Tetracycline for 48hwere both lysed,then 10pg Myc antibody was added to each.IP （immuneprecipitation）was used for detect proteins which might interact with Cyclin B1,thenthey were run in SDS-PAGE.The proteins were confirmed by mass spectrometry,after different bands cutted from SDS-PAGE by coomassie blue staining and silverstaining.Results:The four proteins confirmed by MASS were Cdk1 （Cyclin-dependentkinase 1）,Cdk2 （Cyclin-dependent kinase 2）,MAD1L1 （Mitotic arrest deficient-likeprotein 1,MAD1-like 1）and heat shock 70kDa protein 8 isoform 1.Conclusions:Cyclin B1 （or Cyclin B1/Cdk1）could interact with Cdk2,MAD1L1and Hsp70,which might had functions in specific apoptosis of G₀/G₁ phase cause ofCyclin B1,besides the activation of Cdk1.更多还原