| ObjectiveThis study aims to to investigate the effects of Survivin over expressionon MS1cell survivin mRNA and protein and cell apoptosis.Methods1.The study used the mouse MS1cell;2.According to the genetic sequence of GenBank provide, to constructthe Survivin gene contained plasmid vector;3.Cultured MS1cells，then transfected into cells by LipofectamineTM2000,and designed blank control group、Lipofectamine TM2000transfection group、negative control group、、interference group（pcDNA3.1-Survivin-V5-IRES-EGFP transfected into cells；4.Expression level of survivin mRNA was assayed by real-time PCRtechnology； Expression level of survivin protein was assayed byWestern bloting.5.The apoptosis and cycle of cells were assayed by flow cytomerry。Results1. After the sequence identification, it was verified that the size anddirection of objective genn segment inserting vector; `2.Liposome–mediated transfection of mouse MS1cell,greenfluorescence was observed under in fluorescence microscopy;3.The result of real time PCR indicated that expression of survivinmRNA of MS1cells was very low。Experimental group increased byapproximately124times than control group。4.Western bloting results showed that expression of survivingproteinum of MS1cells was very low,and the expression ofexperimental group increased significantly。5.The FCM results showed that，the transfection rate was20%。The apoptotic rate of experimental group was （6.04±0.12）％,lower than blank control group（6.69±0.34）％,LipofectamineTM2000transfection group （6.30±0.23）％、negative control group（6.53±0.46）％, experimental group compara with control group hadsignificant difference。Conclusion1.Construct the survivin espression vector of pcDNA3.1-Survivin--V5-IRES-EGFP successfully；2.Survivin expression level was increased by transfecting Survivingene into MS1, meanwhile, the cells apoptosis was reduced.Survivin may reduce the cells apoptosis rate of MS1,the outcomeof the experiment plays a good theoretical for the subsequent animalexperiments. |
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