The Effect Of Proliferation And Differentiation After Transfected PEGFP-N1/Igf1Plasmid On Rat Dental Follicle Cells

Objective: The Construction of gene Igf1and Egfp fusion expressionvector pEGFP-N1/Igf1. The pEGFP-N1/Igf1was transfected on SD ratdental follicle cells with the mediate of liposome to analysis the earlyproliferation and differentiation effect of the gene and provides a referencefor the use of Igf1gene in the repair and regeneration of periodontal tissueengineering.Methods:1. The primers were designed according to the SD rat Igf1gene sequence in the GeneBank. Target bands were acquired by Nested PCRand bonding it to pMD-19T vector and then the cloning vectorpMD-19T/Igf1was constructed.2. Digestion of pMD-19T/Igf1andeukaryotic expression vector pEGFP-N1by the restriction endonucleaseEcoR Ⅰ and Xho Ⅰ. Digested Igf1fragments and pEGFP-N1fragmentswere connected with the ligase Solution Ⅰ. After that, product wastransformed to E. coli DH5α. PCR restriction enzyme digestion andsequencing identification was done to make sure the product was right.3. The primary SD rat dental follicle cells were cultured after that. Using thefourth generation of cells, the cell source was identificated. Experimentalgroups:①blank group②pEGFP-N1/Igf1group③pEGFP-N1+liposome group④pEGFP-N1/Igf1+liposome.4. The observation ofgreen fluorescence: The expression of green fluorescent was observed every24hours after transfection, by the inverted fluorescence microscope，continuously for96hours.5. Cell viability test (MTT method):96-wellplates seeded cells. The cells were detected every24hours after transfection,continuously for8days.6. ALP quantitative detection (colorimetricmethod):24-well plates seeded cells. The cell culture medium was collectedevery24hours after transfection, to detect the quantity of ALP,continuously for6days.7. Type Ⅰ collagen Col1α1and Col1α2geneexpression analysis (real-time PCR method):6-well plates seeded cells. Thecells were collected after being transfected for48hours, taking the blankgroup as reference to calculate the relative gene expression levels of othergroups.Results:1. Identified by PCR, restriction enzyme digestion, sequencingplasmid pEGFP-N1/Igf1was successfully constructed.2. Greenfluorescence observation: The amount of expression of green fluorescentwas strongest after transfection for48hours.3. Viability of cells:Theoverall trend: After transfection for48to96hours, cell viability of group③and④which used the liposomes were below group①and②(P <0.05). After transfection for96hours（group①<②）: The difference wasstatistically significant (P<0.05). The remaining time points all werestatistically significant (P>0.05). After transfection for48hours （group③<④）：The difference was statistically significant (P<0.05). The remainingtime points all were statistically significant (P>0.05).4. The quantity of theALP which in the cell culture medium: the overall trend: group①and④were closer to. Group②was always higher than other groups within96hours and achieve the best peak at the72hours. Group③was alwayslower than the other groups within96h. After transfection for24and96hours: group②>④>①>③. The differences were statistically significant(P<0.05). After transfection for48and72hours: group②>①>④>③. Thedifference between group①and④was not statistically significant(P>0.05). The remaining groups all were statistically significant (P<0.05).5.The expression of Col1α1and Col1α2gene (after transfection for48hours):The relative expression amount of Col1α1: group④>②>①>③and therelative expression amount of Col1α2: group④>②>①>③.Conclusion:1. Eukaryotic expression vector pEGFP-N1/Igf1plasmidwhich fusion the Egfp to the C-terminal of Igf1gene was successfullyconstructed.2. Liposome Lipofectamine2000mediated transfectionstrongest expression of target proteins aftter transfected for48hours.3. Theliposome Lipofectamine2000is toxic to the SD rat dental follicle cells.Plasmid pEGFP-N1/Igf1plays a role in the proliferation of SD rat dental follicle cells. Naked plasmid gets the max-effects of cell proliferation aftertransfected for48hours while the Liposome-mediated transfection wasafter transfected for96hours.4. pEGFP-N1/Igf1plasmid can enhanceextracellular ALP activity of SD rat dental follicle cells and has a certaineffect in osteogenic differentiation. The cytotoxic effect of the liposomesreduces extracellular ALP activity while the liposome grouppEGFP-N1/Igf1plasmid also can promote strong effects of ALP expressionat48hours and72hours.5. pEGFP-N1/Igf1plasmid can promote theexpression of Col1α1and Col1α2in the SD rat dental follicle cells aftertransfected for48hours. It has a certain effect in osteogenic differentiationand the effect is related to time we need to continue.