细胞周期素依赖性蛋白激酶调控细胞死亡的研究
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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。
PartⅠ
CDK1 and Apoptosis
Object:Investigate the role of the CDK1 in apoptosis.Materials and Methods:We constructed expressed CDK1 vector,CDK1 siRNA,expressed CDK1-p15-mut vector and expressed CDK1-p161-mut vector.Livercancer cell line Hela for this study,transfecting the Hela cell line withthese plasmids and RNAi,analysising the effect of CDK1 on the cell cycle andcell apoptosis after ultraviolet strike by MTT reduction assay,flow cytometry,immunoprecipitation,immunofluorescence,Western blot analysis andpostsorting immunoprecipitation and western analysis.
Result:
1) We succesfully constructed the high expressed CDK1 vector,high expressedCDK1-p15-mut vector,high expressed CDK1-p161-mut vector and CDK1 siRNA.Transfecting these plasmids in cells,we could find that high expressedCDK1 and CDK1-p15-mut would enhance the ratio of apoptosis after UVradiation;high expressed CDK1-p161-mut and RNAi CDK1 would decrease theratio of apoptosis after UV radiation;the result of IP present that thedeficient of CDK1 cannot be phosphorylated at Tyr 15 but can at Thr161.
2) Hela cell delay in G1 phase under the culture of low serum DMEM.AfterUVradiation,wepostsorted the G1 phase cells to do Western Blot.We findthe expression of Phospho-cdc2 (Tyr15) was in a low level,and theexpression of Phospho-cdc2 (Thr161) was high.There is no significientchange in the expression of CDK2.The expression of apoptotic-relatedprotein of Bc12 was upregulation.
3) In apoptotic cells,the expression of Phospho-cdc2 (Tyr15) had no significiant change in nucleus,but downregulation in cytoplasm comparewith control group.
4) The result of IF appeared that a small bit of Phospho-cdc2 (Tyr15) puttogether in apoptosis body.A great deal of Phospho-cdc2 (Thr161)gathered in cytoplasm.
Conclusion:
1) CDK1 acts an important role in cell apoptosis.
2) The phosphorylation of cdc2 (Thr161) is the centre key in G1 apoptosis.
3) The expression of CDK1 in different time and space result in thedifferent function.
PartsⅡ
CDK1 and Autophagy
Object:Investigate the role of the CDK1 in Autophagy.Materials and Methods:We constructed high expressed CDK1 vector and CDK1 siRNA.Transfecting the Hela cell line with the plasmid and RNAi,analysising theeffect of CDK1 on autophagy after starvation by flow cytometry,immunofluorescence,electronmicroscope and Western blot analysis.
Result:With the high expression of CDK1,cells after starvation would producemuch more autophagic protein to against deficiency of energy and DNA damagesubsequent.
Conclusion:CDK1 act an important role in cell autophagy.
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。
PartⅠ
CDK1 and Apoptosis
Object:Investigate the role of the CDK1 in apoptosis.Materials and Methods:We constructed expressed CDK1 vector,CDK1 siRNA,expressed CDK1-p15-mut vector and expressed CDK1-p161-mut vector.Livercancer cell line Hela for this study,transfecting the Hela cell line withthese plasmids and RNAi,analysising the effect of CDK1 on the cell cycle andcell apoptosis after ultraviolet strike by MTT reduction assay,flow cytometry,immunoprecipitation,immunofluorescence,Western blot analysis andpostsorting immunoprecipitation and western analysis.
Result:
1) We succesfully constructed the high expressed CDK1 vector,high expressedCDK1-p15-mut vector,high expressed CDK1-p161-mut vector and CDK1 siRNA.Transfecting these plasmids in cells,we could find that high expressedCDK1 and CDK1-p15-mut would enhance the ratio of apoptosis after UVradiation;high expressed CDK1-p161-mut and RNAi CDK1 would decrease theratio of apoptosis after UV radiation;the result of IP present that thedeficient of CDK1 cannot be phosphorylated at Tyr 15 but can at Thr161.
2) Hela cell delay in G1 phase under the culture of low serum DMEM.AfterUVradiation,wepostsorted the G1 phase cells to do Western Blot.We findthe expression of Phospho-cdc2 (Tyr15) was in a low level,and theexpression of Phospho-cdc2 (Thr161) was high.There is no significientchange in the expression of CDK2.The expression of apoptotic-relatedprotein of Bc12 was upregulation.
3) In apoptotic cells,the expression of Phospho-cdc2 (Tyr15) had no significiant change in nucleus,but downregulation in cytoplasm comparewith control group.
4) The result of IF appeared that a small bit of Phospho-cdc2 (Tyr15) puttogether in apoptosis body.A great deal of Phospho-cdc2 (Thr161)gathered in cytoplasm.
Conclusion:
1) CDK1 acts an important role in cell apoptosis.
2) The phosphorylation of cdc2 (Thr161) is the centre key in G1 apoptosis.
3) The expression of CDK1 in different time and space result in thedifferent function.
PartsⅡ
CDK1 and Autophagy
Object:Investigate the role of the CDK1 in Autophagy.Materials and Methods:We constructed high expressed CDK1 vector and CDK1 siRNA.Transfecting the Hela cell line with the plasmid and RNAi,analysising theeffect of CDK1 on autophagy after starvation by flow cytometry,immunofluorescence,electronmicroscope and Western blot analysis.
Result:With the high expression of CDK1,cells after starvation would producemuch more autophagic protein to against deficiency of energy and DNA damagesubsequent.
Conclusion:CDK1 act an important role in cell autophagy.
引文
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