Effects Of Aldehyde Dehydrogenase2(ALDH2) On PC12Cells Impaired By Mimic Ischemia/Reperfusion

[Objective] To resuscitate and subculture PC12cells, then re-oxygenate PC12cells with two hours (2h) after2h,4h,8h and12h hypoxia respectively in the Three gas incubator. To screen out the time point that with better vigor of the PC12cells and lower expression of ALDH2in the PC12cells after detection. To construct cukaryotic expression vector pEGFP-N1-ALDH2and its mutant vector pEGFP-N1-ALDH2(AAA), then transfect them into PC12cells respectively. To investigate the protection effects of ALDH2on PC12cells which are impaired by mimic ischemia and reperfusion.[Methods]1.Rapid lysis PC12cells (differentiated), and vaccinate them into6holes cell culture plate with1×105cell density, exchange the culture solution after24h. When the degrees of PC12cells fusion reach about90%, subculture them after0.25%trypsinization and centrifugate.2. Model the hypoxia/re-oxygenation PC12cells:â‘ .Fabricate the model of hypoxia/re-oxygenation PC12cells to investigate the best proportion of O2and N2.â‘¡. To detect the vigor with MTT and observe the growth with inverted microscope on the established model of hypoxia/re-oxygenation PC12cells with the hypoxia time of2h,4h,8h and12h.â‘¢.Detect the expression of ALDH2with Western Blot (WB) on each time point of PC12cells, and screen out the time point that the PC12cells with better condition and with lower ALDH2expression for the continuous experiment.3. Construct eukaryotic expression vector pEGFP-N1-ALDH2and its mutant vector pEGFP-N1-ALDH2(AAA), sequencing them by double restriction enzyme digestion.4. Transfect the plasmid (pEGFP-N1), the wild type plasmid (pEGFP-N1-ALDH2), the mutant plasmid (pEGFP-N1-ALDH2(GAAâ†'AAA)) into PC12cells separately with different concentration and cell density that have been designed which uses the Lipofectamine LTX and plus. Determine the cell density and plasmid concentration with the best transfect efficiency which are observed by inverted microscope, validate them with RT-PCR and Western Blot after transfect.5.Two hours re-oxygenation after4h hypoxia for the three groups with the best transfect condition, then count the cells of each group and detect the vigor of each group with MTT. After that, detect the apoptosis of each group by Flow Cytometry after staining each group with Annexin V-FITC/PI.[Results]1. PC12cells are successfully resuscitated and passaged; it can observed that the PC12cells are in good condition, with strong transmission of light, with elipse cell body, with long apophysis beside the two sides by inverted microscope.2.â‘ .The model of hypoxia/re-oxygenation PC12cells is successfully constructed; choose5%CO2+5%02+90%N2as the condition of hypoxia culture.â‘¡.The following can be observed by inverted microscope:the shape of2h hypoxia group PC12cells has no obviously changed, shortened apophysis and partial cell death happened to the4h hypoxia group, the shape of those dead cells became round and floated in the culture solution, the8h and12h hypoxia group appear mass cell death with round cell shape floating in the culture solution, all groups are compared with the normal control group; the cell counting results shows:the quantity has no obviously changed to the2h hypoxia group (p>0.05), significantly decrease of the cell number happened to the4h,8h and12h hypoxia group (p<0.05), especially to the8h and12h hypoxia group(p<0.01), all groups are compared with the normal control group; MTT results shows:the vigor of the2h hypoxia group has no obviously changed (p>0.05), significantly descend of the cell vigor happened to the4h,8h and12h hypoxia group (p<0.05), especially to the8h and12h hypoxia group(p<0.01), all groups are compared with the normal control group;â‘¢. Western Blot results show that the expression of ALDH2in the2h hypoxia group has no obviously difference (p>0.05), significantly decrease of the ALDH2expression happened to the4h,8h and12h hypoxia group (p<0.05), all groups are compared with the normal control group; the expression of ALDH2in the8h and12h hypoxia group also decrease compared to the 4h hypoxia group (p<0.05)3. Eukaryotic expression vector pEGFP-N1-ALDH2and its mutant vector PEGFP-N1-ALDH2(GAAâ†'AAA) which with correct sequencing after double restriction enzyme digestion are successfully constructed.4. The RT-PCR results show that the expression of ALDH2in the PC12cells which transfect with wild type pEGFP-N1-ALDH2is higher than the blank plasmid group and the mutant plasmid group (p<0.05); The WB results are accordance to the RT-PCR results.5. Two hours re-oxygenation after4h hypoxia for the three groups with the best transfect condition, compared to the simple hypoxia group, the survival number of PC12cells is obviously higher in the wild type plasmid group (p<0.05), the blank plasmid group has no obviously difference (p=0.474. p>0.05), the mutant type plasmid group has decreased cells (p<0.05); The amount of cells in the mutant plasmid group is obviously decreased compared to the blank plasmid group (p<0.05); The MTT results are accordance to the cell counting results; The results that detect by Flow Cytometry show that, compared to the simple hypoxia group, the apoptosis ratio is obviously decreased in the wild plasmid group (p<0.05) while it is no significant change in the blank plasmid group and the mutant plasmid group(p>0.05).[Conclusion]1.The condition of constructing the model of hypoxia/re-oxygenation to the differentiated PC12cells is5%CO2+5%O2+90%N2;2.The recombinant plasmid wild type (pEGFP-N1-ALDH2) and its mutant type (pEGFP-N1-ALDH2(AAA)) can transfect into PC12cells through Liposome method;3. The wild type ALDH2plays a protective effect on PC12cells in the process of hypoxia/re-oxygenation injury through which inhibiting the apoptosis and promoting cell survival.