本文選題：Cyclin + B1��； 參考：《華中科技大學(xué)》2009年博士論文

【摘要】： 第一部分：四環(huán)素可控表達(dá)Cyclin B1的載體構(gòu)建和單克隆細(xì)胞株的篩選 目的：篩選四環(huán)素誘導(dǎo)細(xì)胞周期素B1(Cyclin B1)可控表達(dá)的293單克隆細(xì)胞株(Tetracycline-Regulated Expression 293 cells，T-REx~(TM)-293)。 方法：用多聚酶鏈反應(yīng)(polymerase chain reactions，PCR)的方法從人胎肝cDNA文庫(kù)中調(diào)出帶有酶切位點(diǎn)的Cyclin B1基因全長(zhǎng)，在37℃經(jīng)過BamH1和X-bal1雙酶切過夜后，與經(jīng)過同樣酶切后的pcDNA4／TO／myc-HisB載體在16度連接過夜，轉(zhuǎn)化BL21細(xì)菌感受態(tài)，然后涂在帶有氨芐抗性的LB培養(yǎng)板上，37度培養(yǎng)過夜，挑細(xì)菌克隆，搖菌，測(cè)序鑒定。接著大提測(cè)序正確的質(zhì)粒，用脂質(zhì)體Lipofectamine~(TM)2000轉(zhuǎn)染T-REx~(TM)-293細(xì)胞，用含有殺稻瘟菌素(Blasticidin，5μg／ml)和腐草霉素(Zeocin，200μg／ml)雙藥物的DMEM培養(yǎng)兩周后，將細(xì)胞傳96孔板，等單個(gè)細(xì)胞擴(kuò)增出單細(xì)胞克隆。最后，用Western blot和流式細(xì)胞儀檢測(cè)單克隆細(xì)胞株在加入四環(huán)素誘導(dǎo)后目的基因的表達(dá)情況。 結(jié)果：構(gòu)建的pcDNA4／TO／myc-HisB-Cyclin B1載體，經(jīng)英駿公司測(cè)序鑒定序列正確。篩選出的單克隆細(xì)胞株，在未加四環(huán)素時(shí)沒有外源性的Cyclin B1表達(dá)，在加入四環(huán)素后，3小時(shí)就有表達(dá)，隨時(shí)間的增加，Cyclin B1表達(dá)量也增加，48小時(shí)最多。 結(jié)論：篩選出的T-REx~(TM)-293單克隆細(xì)胞株能經(jīng)四環(huán)素可控表達(dá)Cyclin B1。 第二部分：G_0／G_1期異時(shí)相Cyclin B1的表達(dá)促進(jìn)細(xì)胞凋亡 目的：利用四環(huán)素誘導(dǎo)Cyclin B1可控表達(dá)的293單克隆細(xì)胞株來證明G_0／G_1期異時(shí)相Cyclin B1的表達(dá)促進(jìn)細(xì)胞凋亡 方法：挑選出一個(gè)最優(yōu)的Thymidine的濃度，其能最大程度的阻滯細(xì)胞于G_0／G_1期。接著，將細(xì)胞大量阻滯在G_0／G_1期，然后在阻滯好的細(xì)胞中加入微量的四環(huán)素誘導(dǎo)Cyclin B1表達(dá)48小時(shí)，最后用Annexin V-PI雙標(biāo)法和API法檢測(cè)細(xì)胞凋亡的量和凋亡的周期特異性，同時(shí)用Western blot檢測(cè)細(xì)胞周期素依賴蛋白激酶1(Cdk1)的活性情況。 結(jié)果：用Thymidine處理細(xì)胞后，絕大部分細(xì)胞都同步化在G_0／G_1期。在四環(huán)素誘導(dǎo)Cyclin B1表達(dá)48小時(shí)后，誘導(dǎo)組比非誘導(dǎo)組的Cyclin B1蛋白總量明顯增加，細(xì)胞凋亡明顯增加。此時(shí)，活性的Cdk1(Thr~(161)磷酸化)蛋白量較未誘導(dǎo)組也明顯增加。 結(jié)論：G_0／G_1期異時(shí)相Cyclin B1的表達(dá)能促進(jìn)細(xì)胞凋亡。 第三部分：Cyclin B1導(dǎo)致的G_0／G_1期特異性細(xì)胞凋亡機(jī)制的探索 目的：探索在由Cyclin B1導(dǎo)致的G_0／G_1期特異的細(xì)胞凋亡中，除了激活Cdk1外，Cyclin B1還可能和哪些蛋白相互作用，或者Cyclin B1／Cdk1能通過磷酸化哪些蛋白質(zhì)起作用。 方法：我們篩選好的單克隆細(xì)胞株在加入四環(huán)素后能表達(dá)帶有Myc和His標(biāo)簽的外源性Cyclin B1。將未誘導(dǎo)的和用四環(huán)素誘導(dǎo)48小時(shí)的細(xì)胞裂解后，分別加入10μgMyc的單克隆抗體，用免疫沉淀(immune precipitation，IP)的方法將可能和Cyclin B1結(jié)合的蛋白沉淀下來，接著將蛋白混合物進(jìn)行SDS PAGE電泳，用考馬斯亮藍(lán)染色法和銀染法找出未誘導(dǎo)組和誘導(dǎo)組之間的差異蛋白，然后用質(zhì)譜(mass spectrometry，MASS)分析出差異蛋白的名稱。 結(jié)果：用質(zhì)譜鑒定出的蛋白有細(xì)胞周期素依賴蛋白激酶一(Cyclin-dependentkinase 1，，Cdk1)，細(xì)胞周期素依賴蛋白激酶二(Cyclin-dependent kinase 2，Cdk2)，有絲分裂阻滯缺失樣蛋白一(Mitotic arrest deficient-like protein 1，MAD1-like 1，MAD1L1)和熱休克蛋白八(heat shock 70kDa protein 8 isoform 1)。 結(jié)論：在由Cyclin B1導(dǎo)致的G_0／G_1期特異的細(xì)胞凋亡中，除了磷酸化Thr~(161)殘基激活Cdk1后導(dǎo)致細(xì)胞凋亡外，Cyclin B1(Cyclin B1／Cdk1)與Cdk2、MAD1L1和Hsp70的結(jié)合都有可能參與其中。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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