Over-expression Of BCL-2in PC12Cell And The Effect On Apoptosis Caused Byalcohol

Objective: We build the nuclear expresses vector of pcDNA3.1(-)-bcl-2with BCL-2cDNA coloned frommouse，then transfect it to the PC12cell and get the PC12cell with over-experssion BCL-2at last, whichare used for studying the role of BCL-2protein in alcohol-induced apopotosis in the PC12cell.Methods: We obtian the total RNA from mouse spleen and colone the cDNA which reverse transcriptedfrom the total RNA, obtain the BCL-2DNA by RT-PCR and the1%DNA gel electrophoresis. We link theBCL-2to the pMD-19T Vector and extract the restructured DNA after blue-white selecting, cloning, trans-forming. We deal with the DNA of pcDNA3.1(-) and pMD19-T-bcl-2which have been tested by PCR andDNA sequencing using the restrict enzymes of Hind Ⅲ and EcoRⅠat the same time, then we link theneeded fragments with T4DNA ligase after1%DNA gel electrophoresis and test the accuracy of DNA ofpcDNA3.1(-)-bcl-2with the same restrict enzymes after transformation,cloning and extracting DNA fromplasmid.After all of these have been done,we transfect the pcDNA3.1(-) vector and nuclear expressesvector into the PC12cell stably, selected by G418,we can extract the total RNA from three kinds of PC12cell, that is,the normal, the one with pcDNA3.1(-) vector and the one with pcDNA3.1(-)-bcl-2vector, andwe exmain the effect of transfection not only on the nucleic acid level using RT-PCR,but also on theprotein level by the expression of BCL-2protein. The MTT assay was used to measure the activity of threekinds of different cell proliferation after using the different alocohol concentration.Results: The total RNA obtained from mouse spleen is pure and the result of RT-PCR reveals that we getBCL-2. It is vetified cleatly that the whole gene sequence of BCL-2is right and the recombinant plasmidpMD19-T-bcl-2is built successfully by the results of PCR and bacteria liquid sequencing. We link the two needed fragments with T4DNA ligase enzyme after two restrict enzymes effecting on the DNA ofpMD19-T-bcl-2and pcDNA3.1(-), then get the nuclear expresses vector of pcDNA3.1(-)-bcl-2. We extractpure RNA from three kinds of PC12cell, and measure the expression of BCL-2mRNA and protein, at lastthe result shows that we construct the PC12cell with BCL-2over-expression successfully. The result ofMTT assay suggests that there is a higher survival ratio in the transfected pcDNA3.1(-)-bcl-2PC12whenthe the concentration of alcohol is100mmol/L and200mmol/L, compared to the normal PC12cell (P<0.01, P<0.05). But when the concentration reach at400mmol/L,there is not statistical significancebetween two kinds of cells anymore(P>0.05). BCL-2protect the PC12cell from apoptosis induced byalcohol with the concentration of100~200mmol/L.Conclusions:1We construct nuclear expresses vector pcDNA3.1(-)-bcl-2successfully, BCL-2mRNAand protein are expressed in a high level in PC12cell after pcDNA3.1(-)-bcl-2has been transfected intothe PC12cell.2The PC12cell with over-expression BCL-2can resist the apoptosis induced by alcohol atcertain range of concentration.