Effect Of Sense And Antisense VEGF165 CDNA Transfection On The Growth Of Hypertrophic Scar-derived Fibroblasts In Vitro

[Objective] We subcloned human VEGF165 cDNA into the pcDNA3.1(+/-) eukaryotic expression vector, which named sense group and antisense group. Including empty vector group and control group,VEGF sense,and VEGF antisense were transferred into human hyperplastic scar fibroblasts in vitro. Expression of VEGF mRNA was detected by reverse transcription-PCR (RT-PCR);VEGF protein expression was detected by using enzyme-linked immunosorbent assay(ELISA).The growth of dermal fibroblasts was measure by MTT.All the measures were used to explore the effect on the growth of hypertrophic scar-derived fibroblasts by sense and antisense VEGF165 gene.[Methods] Human dermal fibroblasts were isolated from hypertrophic scar by serum supplemented media method, and immunocytochemical staining was used to characterize the cell lineage.Those eukaryoticexpression plasmids of pcDNA3.1(+)/hVEGF165,pcDNA3.1(-)/hVEGF165 targeting on VEGF were constructed and transfected into human dermal fibroblasts plus pcDNA3.1(+),which named sense group,antisense group and empty vector group respectively,and a positive clone was selected by G418.Control group was not subjected to transfection. Expression of VEGF mRNA was detected by reverse transcription-PCR (RT-PCR); VEGF protein expression was detected by using enzyme-linked immunosorbent assay(ELISA). The growth of dermal fibroblasts was measure by MTT.[Results] (1)Human dermal fibroblasts were isolated from hypertrophic scar by serum supplemented media method, CK7 and V9 immunohistochemical staining was used to characterize the cell lineage. The purity of HHSFb reached over 90%.(2)Those eukaryotic expression plasmids of pcDNA3.1(+)/hVEGF165,pcDNA3.1(-) /hVEGF165 targeting on VEGF were constructed successfully. (3)200μg/mL was the Optimum screening concentration for HHSFb cells,50μg/mL G418 was the amplification concentration.(4)The sense VEGF,antisense VEGF and empty vector pcDNA3.1(+) could be transfected successfully into HHSFb cells mediated with liposome formulation,and the positive clone of the three groups was selected by G418.(5)Expression of VEGF mRNA and VEGF protein were increased obviously in sense group,but reduced obviously in antisense group, as compared with those in empty vector group and control group.(6)The proliferation of the four groups in vitro on the whole were the same.[Conclusion] (1)We constructed successfully sense and antisense VEGF165 eukaryotic expression vector and transferred it into HHSFb cells in vitro mediated by liposome formulation to obtain stable expression cell lines.(2)It was confirmed that VEGF165 antisense could effectively inhibit the expression of endogenous VEGF protein when it was transfected into cells,meanwhile the sense VEGF165 could up-regulation the expression of VEGF.But there were no difference in the morphologic and the growth rate of HHSFb cells.(3)Antisense VEGF gene therapy applied to restrain the scar tissue hyperplasia study is required to conduct further in vivo.