Both Gene TGF-β1and BFGF Modified BMSCs In Vitro Experiment

Objective：To construct the recombinant human basic fibroblast growth factor(bFGF)eukaryotic expression vector and human transforming growth factor-β1(TGF-β1)eukaryotic expression vector. They were transfected into the bone marrowmesenchymal stem cells(BMSCs) of Sprague Dawley(SD)rats by Lipofectamine2000reagent. And to investigate in vitro effects of the recombinant vector on theproliferation of the BMSCs. In order to explore the feasibility of both two genesmodified BMSCs，and know whether they had synergetic effect on proliferation ofBMSCs.To further study the combination with gene engineering and periodontaltissue engineering on therapy in alveolar bone reconstruction.Method：The bFGF and TGF-β1cDNA encoding the full-length human bFGF gene andhuman TGF-β1gene were amplificated from the expression plasmidpDONR223-bFGF、pDONR223-TGF-β1by PCR, using primers containing HindⅢand XhoⅠ restriction enzyme.Then the cDNA digested with HindⅢ andXhoⅠ, andsubcloned into the same site of the plasmid pcDNA3.1(+) vector.The recombinantswere named pcDNA3.1(+)-bFGF and pcDNA3.1(+)-TGF-β1, and were identified byenzyme digestion analysis and sequencing.The two eukaryotic expression vectorswere transfected into BMSCs of SD rats by Lipofectamine2000.The expression ofbFGF and TGF-β1were analyzed by using Western blot and RT-PCR.And using MTTcolorimetric assay analyzed the proliferation of the BMSCs.The OD value of MTT ofeach group were calculated by T test.Result：（1）Eukaryotic expression vector pcDNA3.1(+)-bFGF and pcDNA3.1(+)-TGF-β1were digested with restriction enzyme, and agarose gel electrophoresisshowed there were fragments of468bp(human bFGF)、1173bp(human TGF-β1) and5428bp(pcDNA3.1(+)).Comparing with modified gene,0.43%of the DNA sequencemutationed (447th: G�'T;467th: A�'G;) and no amino acid has changed in bFGF.The DNA sequence of TGF-β1was identical to the report in GenBank and didnot revealed any mutation.Both bFGF gene and TGF-β1gene expressed inpcDNA3.1(+)-bFGF+pcDNA3.1(+)-TGF-β1group.(2) Twenty-four hours after transfection,the results of RT-PCR and Western blotdemonstrated that the recombinant vector pcDNA3.1(+)-bFGF andpcDNA3.1(+)-TGF-β1were expressed in BMSCs, and the relatived molecular massof the bFGF protein was about17×103, while the relatived molecular mass of theTGF-β1protein expressed was about43×103. The results suggested that these twoprotein had good biological activity in BMSCs of SD mouse.Twenty-four hours after transfection,comparison of clinical data among thegroups: The OD value of MTT in pcDNA3.1(+)-bFGF group、pcDNA3.1(+)-TGF-β1group、pcDNA3.1(+)-bFGF+pcDNA3.1(+)-TGF-β1group、pcDNA3.1(+) group were significant higher than control group(P＜0.01);pcDNA3.1(+)-bFGF group、 TGF-β1group、 pcDNA3.1(+)-bFGF+pcDNA3.1(+)-TGF-β1group were significant higher than pcDNA3.1(+) group(P＜0.01);pcDNA3.1(+)-bFGF group was significant higher than pcDNA3.1(+)-bFGF+pcDNA3.1(+)-TGF-β1group(P＜0.01);TGF-β1group and pcDNA3.1(+)-bFGF+pcDNA3.1(+)-TGF-β1group were no significant statistical significance.Conclusion：（1）The eukaryotic expression vector pcDNA3.1(+)-bFGF and pcDNA3.1(+)-TGF-β1have been successfully constructed.（2）The eukaryotic expression vector pcDNA3.1(+)-bFGF and pcDNA3.1(+)-TGF-β1can be expressed in BMSCs of SD mouse.Both bFGF gene and TGF-β1genecould modify BMSCs together.（3）The gene bFGF gene and TGF-β1gene modified BMSCs individually canstimulate proliferation of the BMSCs cultured in vitro. Both bFGF gene and TGF-β1gene modified BMSCs could stimulate its proliferation,but they hadn’t synergeticeffect.