The Effect On Proliferationafter Transfection Of PEGFP-N1/Igf1and PEGFP-N1/Bmp2Plasmid To SD Rat Dental Follicle Cells

Objective: The culture and identification of SD rat dental follicle cellsin vitro, the construction of pEGFP-N1/Igf1and pEGFP-N1/Bmp2eukaryon express vectors, and use liposome to transfect them on rat dentalfollicle cells, then test their early effect on cell proliferation, to provide areference for further application of them in periodontal tissue engineering.Methods:1.Culture and identify SD rat dental follicle cells in vitro,choosed the fourth generation of cells to do the latter experiment.2.Designed the primers of SD rats’ Igf1and Bmp2genes, and the two geneswere amplificated by PCR technique, then constructed the vectors ofpMD-19T/Igf1and pMD-19T/Bmp2.3.Digested pMD-19T/Igf1and rpEGFP-N1，pMD-19T/Bmp2and pEGFP-N1, thenconnected Igf1，Bmp2fragments and pEGFP-N1fragments, transformed the products to E. coliDH5a and do the identifications.4.Divided the fourth generationofcells intofive groups:①blank group②liposome group③pEGFP-N1/Igf1+liposome④pEGFP-N1/Bmp2+liposome⑤pEGFP-N1/Igf1+pEGFP-N1/ Bmp2+liposome.5.Observed the green fluorescence: did the observationfromthe second dayafter transfection, last for4days, and once a day.6. Cellviability test through MTT: detected the cells once a day after transfection,and last for4days.7.Used Bradford to detect the protein concentration in thecells: The Bradford working fluid was prepared, then measured the cells’absorbance by spectropholometry, and calculated the protein concentration.Results:1.SD Rats’ DFCs were successfully purified and cultured invitro, among them the viability of the fourth generation cell is better.2. ThepEGFP-N1/Igf1and pEGFP-N1/Bmp2eukaryon express vectors weresuccessfully constructed.3.Observation of Green fluorescence: Theexpressed amount of green fluorescent was strongest at48hours aftertransfection.4.Viability ofcells:④＞⑤＞③＞①＞②at the first,third andforth days after transfection, the differences between group④and⑤haveno sense in statistics（p＞0.05）, but the differences between the othersgroups have（p＜0.05）.⑤＞④＞③＞①＞②at the second day aftertransfection, the differences between group④and⑤have no sense instatistic（sp＞0.05）, the others’hav（ep＜0.05）.5. The proteinconcentrationin cells: Results are consistent with MTT, the protein concentrations aresignificantly higher in group③,④and⑤than those in the other threegroups, the differences between group④and⑤have no sense in statistics（p＞0.05）. Besides group②the protein concentrations beganto rise fromthe second day, increased significantly on the third day, then went down on the fourth day.Conclusions:1.The pEGFP-N1/Igf1and pEGFP-N1/Bmp2eukaryonexpress vectors were successfully constructed.2. The expressed amount ofgreen fluorescent was strongest at48hours after transfection throughLiposome Lipofectamine2000.3. Liposome Lipofectamine2000was toxicto DFCs of SD rats.4.Plasmids pEGFP-N1/Igf1and pEGFP-N1/Bmp2canenhance the proliferation of DFCs of SD rats, But it doesn’t makesignificant differences when both of them are applicated,compared to usesingle pEGFP-N1/Bmp2plasmid, so further research is needed.