| Obective To clone and construct the eukaryotic expression plasmid for human epidermal growth factor (hEGF) gene and human vascular endothelial growth factor 121 (hVEGF121) gene with signal peptide . To transfect the hEGF gene plasmid into adult epidermal keratinocytes and transfect the hVEGF121 gene plasmid into adult dermal fibroblasts. And to investigate the expression and secretion of hEGF gene in transfected keratinocytes and the expression and secretion of hVEGF121 gene and the biological activities of hVEGF121 protein in transfected fibroblast. Methods Section â… : After two pairs of primers were designed and synthesized,the cDNA fragment of human epidermal growth factor (EGF) gene and signal peptide (SP) gene were amplified from total RNAs extracted from human renal tissue of a 3-month-old fetus.The amplified cDNA fragments were cloned into pGEM-T vector.The expression plasmids were verified by double endonuclease digestion and DNA sequence analysis.Then plasmid pGEM-T-SP was digested with Nhe â… and EcoR â… ,pGEM-T-EGF was prepared by double digestion with EcoR â… and Hind â…¢, plasmids pcDNA3.1(+) was digested with Nhe â… and Hind â…¢.Further,EGF cDNA and SP cDNA fragments were ligased with T4 ligase between the Nhe â… and Hind â…¢ restriction sites in the liner plasmid pcDNA3.1(+) to form the plasmid pcDNA3.1(+)-hEGF. The plasmid pcDNA3.1(+)-hEGF,digested with Nhe â… and Hind â…¢,was found to contain the hEGF cDNA sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis analysis.And the coding sequence of hEGF cDNA in the pcDNA3.1(+)-hEGF was confirmed by DNA sequence analysis . Section â…¡:Primary adult epidermal keratinocytes isolated from human skin by two-step combined dissociation were cultured in defined keratinocyte-SFM (DKSFM) and were indentified by morphology,immunohistochemistry and transmissive electromicroscope.The eukaryotic expression plasmids pcDNA3.1(+)-hEGF were transfected into keratinocytes in EGF-free medium mediated by Lipefectin2000.As cultured 48 hours,the transcription and expression of hEGF gene in transfected keratinocytes was investigated by RT-PCR and agarose gel electrophoresis analysis,the biological activety of hEGF protein in supernatants secreted by keratinocytes which transfected hEGF gene was investigated by radioactive immunoassay and Hacat cells MTT assay. Sestion â…¢:Total RNAs were extracted from human liver tissue of a 3-month-old fetus and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with the amplified products cloned intopGEM-T vector.DNA sequence analysis and agarose gel electrophoresis analysis were performed before the amplified cDNA-fragments were cloned into the eukaryotic expression plasmid pcDNA3.1(+).The recombinant of plasmid pcDNA3.1(+)-hVEGF121,digested with Hind â…¢ and BamH â… ,was found to contain the hVEGF121 cDNA sequence by RT-PCR and agarose gel electrophoresis analysis.And the coding sequence of hVEGF121 cDNA in the plasmid was confirmed by DNA sequence analysis again. Section â…£:The adult dermal fibroblasts were isolated , cultured in DMEM with 15% fetal bovine serum (FBS) and were indentified by morphology and immunohistochemistry.The plasmid pcDNA3.1(+)-hVEGF121 was transfected into fibroblasts in VEGF-free medium mediated by Lipefectin2000.Cultured 48 hours,the transcription and expression of hVEGF121 gene in transfected fibroblasts was investigated by RT-PCR and agarose gel electrophoresis analysis,the hVEGF121 protein level in supernatant of transfected fibroblasts culture was determined by enzyme linked immunosorbent assay (ELISA).The biological activities of hVEGF121 protein was tested by observing the growth rate of the human umbilical vein endothelial cells (HUVEC) after being stimulated with the said supernatant,and by performing the Mile's assay in Kun-ming rats. ResultsSetion I:After RT-PCR using two pairs of primers,two bands (90 bp and 180 bp) were obtained and identified respectively.The bands were confirmed as signal peptide cDNA fragment (with the full length of SP being 66 bp) and EGF cNDA fragment (with the full length of EGF being 159 bp) by DNA sequence analysis after cloned into pGEM-T vector.Further,the SP and EGF cDNA fragments were ligased and inserted into plasmid pcDNA3.1(+).The bands of 230 bp and 5.4 kb were identified from digested plasmid pcDNA3.1(+)-SP-EGF.And the correct nucleotide sequence for the full length of SP-EGF cNDA fragment was confirmed by DNA sequence analysis from the extracted and purified plasmid pcDNA3.1(+) -SP-EGF. Section â…¡:Adult epidermal keratinocytes are all capable of be isolated and cultured in vitro steadily and rapidly.The plasmid pcDNA3.1(+)-SP-EGF were successfully introduced into cultured keratinocytes.These transgenic cells were able to secrete hEGF protein to certain extent,with biological activities to enhance the growth rate of Hacat cells in vitro. Section â…¢:After RT-PCR using a pair of primers,two bands were identified.The band (460 bp) shorter in length was confirmed as hVEGF121 (with the full length of hVEGF121 being 444 bp) by DNA sequence analysis after inserted into pGEM-T vector.Then the hVEGF121 cDNA fragment was ligased and cloned into plasmidpcDNA3.1(+). The bands of 460 bp and 5.4 kb were identified by double digestion.And the corroct nucleotide sequence was confirmed by DNA sequence analysis. Section â…£:Adult dermal fibroblasts were successfully isolated and cultured in vitro steadily and rapidly.With Lipefectin2000, the hVEGF121 gene in plasmid pcDNA3.1(+)-hVEGF121 was successfully transfected into cultured fibroblasts in vitro.These transfected cells were able to secrete hVEGF121 protein to certain extent,with biological activities to enhance the growth of HUVEC in vitro and improve vascular permeability. Conclusion 1. The eukaryotic expression plasmids for hEGF gene and hVEGF121 gene are successfully constructed. 2. Adult epidermal keratinocytes and dermal fibroblasts are all capable of be isolated and cultured in vitro steadily and rapidly. 3. Human keratinocytes modified with exogenetic hEGF cDNA can express and secrete active hEGF. 4. Dermal fibroblasts in vitro transfected with exogenetic hVEGF121 cDNA can express and secrete active hVEGF121. KEY WORDS skin,keratinocyte,fibroblast,epidermal growth factor (EGF),vascular endothelial growth factor (VEGF)...
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