细胞周期素依赖性蛋白激酶调控细胞死亡的研究
发布时间：2019-06-20 00:02
【摘要】： 第一部分 CDK1与细胞凋亡 目的：探讨CDK1在细胞凋亡中的重要作用及其机制研究。 材料和方法：先后构建了表达CDK1的质粒、突变了CDK1主要磷酸化位点(包括Thr161和Tyr15)的质粒和能有效沉默CDK1表达的RNAi，以肝癌细胞Hela为研究对象，以紫外打击为凋亡的主要诱导方式，采用MTT法、流式细胞学分析、流式细胞学分选技术、免疫荧光分析、蛋白质免疫共沉淀技术、Western blot分析等技术分析细胞在遭受外界打击的条件下，CDK1蛋白与凋亡相关蛋白的相互作用关系。 结果： 1)我们成功构建了能够表达CDK1蛋白的真核表达质粒，能够有效沉默CDK1表达的RNAi和突变了Thr161位蛋白和Tyr15位蛋白的表达CDK1质粒；高表达CDK1的细胞在紫外线打击下凋亡率显著增加；沉默CDK1表达的细胞在紫外线打击下凋亡率显著下降；进而发现，当细胞引入突变了Tyr15位蛋白的CDK1质粒时，紫外诱导的G1期特异性细胞凋亡是增加的；当细胞引入突变了Thr161位蛋白的CDK1质粒时，紫外打击导致的细胞的G1期凋亡相对有所减少，免疫共沉淀显示带有Flag尾Tyr15突变体其Thr161位点可以被磷酸化。 2)在低血清培养诱导的细胞G1期阻滞模型中，，当紫外打击后根据不同的时间点我们对细胞进行了分选，与对照组相比，紫外打击组细胞内Phospho-cdc2(Tyr15)的表达是逐渐下降，而同时出现了Phospho-cdc2(Thr161)表达的上升；对照组和紫外打击组内CDK2处于低水平表达，没有出现显著性改变；凋亡相关蛋白检测发现抗凋亡蛋白Bc12的表达上调。 3)对细胞紫外线打击后分别提取胞核蛋白和胞浆蛋白，结果显示紫外打击组胞核中Phospho-cdc2(Tyr15)表达与对照组无明显差异，而胞浆中出现了显著下降。 4)免疫荧光分析显示，细胞在紫外打击后少量的Phospho-cdc2(Tyr15)出现了聚集于凋亡小体处的现象，Phospho-cdc2(Thr161)浓聚在胞浆中。 结论： 1)CDK1表达过多可以促使凋亡的发生，抑制CDK1的表达可以减少细胞的凋亡。 2)通过向细胞引入外源性表达的CDK1相关磷酸化位点突变体后其Thr161蛋白可以被磷酸化，而磷酸化的后果是促发了凋亡。而引入Thr161突变的CDK1并没有显著抑制凋亡的发生。 3)从时间的观点看来，CDK1在G1期如果出现了异常激活则可能直接导致凋亡的发生；从空间的角度理解，CDK1 Thr161磷酸化后若异常停留在胞浆中，和(或)CDK1Tyr15以磷酸化的方式入核或在胞浆中去磷酸化也是凋亡发生的必然条件之一。 第二部分 CDK1与细胞自噬 目的： 探讨CDK1在细胞自噬中的重要作用。 材料和方法： 先后构建了高表达CDK1的质粒和能有效沉默CDK1表达的RNAi，以肝癌细胞Hela为研究对象，通过DHanks液饥饿为主要诱导方式，采用流式细胞学分析、免疫荧光分析、电镜和Western blot分析等技术分析细胞在饥饿所诱导的自噬中，CDK1蛋白在其中的作用。 结果： 当细胞导入高表达CDK1后，我们可以见到相对于对照组，其自噬相关蛋白表达增加，自噬泡更多且更大；相反，当我们沉默CDK1的表达后，细胞自噬的现象明显受到抑制。 结论： CDK1表达过多可以促使细胞自噬的发生，抑制CDK1的表达可以减少细胞的自噬。
[Abstract]:the first part CDK1 vs. The purpose of cell apoptosis: to study the important role of CDK1 in the apoptosis of cells Materials and methods: The expression of the plasmid of CDK1, the plasmid of the main phosphorylation site of CDK1 (including Thr161 and Tyr15) and the RNAi which can effectively silence the expression of CDK1 were constructed. The methods of induction, MTT, flow cytometry, flow cytometry, immunofluorescence, protein-immunoprecipitation and Western blot were used to analyze the CDK1 protein and apoptosis. related egg Results:1) We successfully constructed the eukaryotic expression plasmid which can express the CDK1 protein, can effectively silence the RNAi of the CDK1 expression and the expression of the expression CDK1 of the Tyr15-bit protein and the high expression CD. The apoptosis rate of the cells of K1 cells increased significantly under the ultraviolet ray; the apoptosis rate of the cells expressed by the silence CDK1 was significantly decreased under the ultraviolet ray; furthermore, when the cells were introduced into the CDK1 plasmid with the mutation of the Tyr15-bit protein, the apoptosis of the UV-induced G1 phase-specific cells was increased; and when the cells were introduced into the process, When the expression of the CDK1 plasmid of the Thr161-bit protein was changed, the apoptosis of the G1 phase of the cells caused by the ultraviolet-attack was relatively reduced, and the immunoprecipitation showed that the Flag tail Tyr1 5. The Thr161 site of the mutant can be phosphorylated.2) In the cell G1 phase block model induced by low serum culture, the cells were sorted according to different time points after UV-attack, and compared with the control group, the expression of Phospho-cdc2 (Tyr15) in the ultraviolet-striking group was gradually decreased, while Ph. Phospho-cdc2 (Thr161) expression increased; in the control group and in the ultraviolet-striking group, the CDK2 was expressed at a low level and no significant change was observed; and The expression of anti-apoptotic protein Bc12 was found to be up-regulated by the detection of apoptosis-related protein. R15) The expression of r15 showed no significant difference with the control group, and there was a significant decrease in the cytoplasm.4) Immunofluorescence analysis showed that the small amount of Phospho-cdc2 (Tyr15) in the cells appeared to be clustered in the apoptotic body after the ultraviolet. the present at the office Phospho-cdc2 (Thr161) is concentrated in the cytoplasm. 1) The expression of CDK1 can induce apoptosis, and the expression of CDK1 can reduce the apoptosis of the cells. The Thr161 protein of the DK1-related phosphorylation site mutant can be phosphorylated And the effect of phosphorylation is to promote apoptosis. CDK1, which is introduced to the Thr161 mutation, does not significantly inhibit the occurrence of apoptosis.3) From the point of view of time, CDK1 is abnormal in G1 phase. Activation may lead to the occurrence of apoptosis directly; from the point of view of the space, after the phosphorylation of CDK1 Thr161, in that slurry, and (or) CDK1Ty r15 to enter the nucleus in a phosphorylated manner or to dephosphorylation in the cytoplasm It is also the occurrence of apoptosis One of the necessary conditions for CDK1 and autophagy: to study the important role of CDK1 in autophagy. Materials and methods: high expression of CDK1 has been constructed. The plasmid and the RNAi which can effectively silence the expression of the CDK1, the liver cancer cell Hela is the research object, is the main In this paper, the role of CDK1 protein in the autophagy induced by starvation was analyzed by flow cytometry, immunofluorescent analysis, electron microscope and Western blot analysis. to be fine After the introduction of high-expression CDK1, we can see the increase of autophagy-related protein expression and autophagy relative to the control group.
【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
本文编号：2502742
[Abstract]:the first part CDK1 vs. The purpose of cell apoptosis: to study the important role of CDK1 in the apoptosis of cells Materials and methods: The expression of the plasmid of CDK1, the plasmid of the main phosphorylation site of CDK1 (including Thr161 and Tyr15) and the RNAi which can effectively silence the expression of CDK1 were constructed. The methods of induction, MTT, flow cytometry, flow cytometry, immunofluorescence, protein-immunoprecipitation and Western blot were used to analyze the CDK1 protein and apoptosis. related egg Results:1) We successfully constructed the eukaryotic expression plasmid which can express the CDK1 protein, can effectively silence the RNAi of the CDK1 expression and the expression of the expression CDK1 of the Tyr15-bit protein and the high expression CD. The apoptosis rate of the cells of K1 cells increased significantly under the ultraviolet ray; the apoptosis rate of the cells expressed by the silence CDK1 was significantly decreased under the ultraviolet ray; furthermore, when the cells were introduced into the CDK1 plasmid with the mutation of the Tyr15-bit protein, the apoptosis of the UV-induced G1 phase-specific cells was increased; and when the cells were introduced into the process, When the expression of the CDK1 plasmid of the Thr161-bit protein was changed, the apoptosis of the G1 phase of the cells caused by the ultraviolet-attack was relatively reduced, and the immunoprecipitation showed that the Flag tail Tyr1 5. The Thr161 site of the mutant can be phosphorylated.2) In the cell G1 phase block model induced by low serum culture, the cells were sorted according to different time points after UV-attack, and compared with the control group, the expression of Phospho-cdc2 (Tyr15) in the ultraviolet-striking group was gradually decreased, while Ph. Phospho-cdc2 (Thr161) expression increased; in the control group and in the ultraviolet-striking group, the CDK2 was expressed at a low level and no significant change was observed; and The expression of anti-apoptotic protein Bc12 was found to be up-regulated by the detection of apoptosis-related protein. R15) The expression of r15 showed no significant difference with the control group, and there was a significant decrease in the cytoplasm.4) Immunofluorescence analysis showed that the small amount of Phospho-cdc2 (Tyr15) in the cells appeared to be clustered in the apoptotic body after the ultraviolet. the present at the office Phospho-cdc2 (Thr161) is concentrated in the cytoplasm. 1) The expression of CDK1 can induce apoptosis, and the expression of CDK1 can reduce the apoptosis of the cells. The Thr161 protein of the DK1-related phosphorylation site mutant can be phosphorylated And the effect of phosphorylation is to promote apoptosis. CDK1, which is introduced to the Thr161 mutation, does not significantly inhibit the occurrence of apoptosis.3) From the point of view of time, CDK1 is abnormal in G1 phase. Activation may lead to the occurrence of apoptosis directly; from the point of view of the space, after the phosphorylation of CDK1 Thr161, in that slurry, and (or) CDK1Ty r15 to enter the nucleus in a phosphorylated manner or to dephosphorylation in the cytoplasm It is also the occurrence of apoptosis One of the necessary conditions for CDK1 and autophagy: to study the important role of CDK1 in autophagy. Materials and methods: high expression of CDK1 has been constructed. The plasmid and the RNAi which can effectively silence the expression of the CDK1, the liver cancer cell Hela is the research object, is the main In this paper, the role of CDK1 protein in the autophagy induced by starvation was analyzed by flow cytometry, immunofluorescent analysis, electron microscope and Western blot analysis. to be fine After the introduction of high-expression CDK1, we can see the increase of autophagy-related protein expression and autophagy relative to the control group.
【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
【引证文献】
相关期刊论文 前2条
1 李瑞;郑亚莉;周学兵;王炜;;Cdk1在HepG2细胞的表达及活性对肝癌细胞凋亡的影响[J];宁夏医科大学学报;2011年06期
2 徐纪伟;孙丹华;;大鼠脊髓半切损伤后细胞周期蛋白激酶1的表达增强[J];细胞与分子免疫学杂志;2013年10期
相关硕士学位论文 前1条
1 李瑞;CDK1在HepG2细胞的表达及活性对肝癌细胞凋亡的影响[D];宁夏医科大学;2011年
本文编号：2502742
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