| Partâ… CDK1 and ApoptosisObject: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. Partsâ…¡CDK1 and AutophagyObject: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. |
