【摘要】 目的 构建含HGF基因真核表达载体pEGFP－N1-HGF，观察HGF基因在人脐静脉内皮细胞中表达。为HGF用于血管重建的基因治疗提供实验依据。方法 由重组质粒PBSKSHGF中扩增出HGF基因,将其克隆到含增强型绿色荧光蛋白的真核表达载体EGFP－N1中,应用脂质体将重组质粒EGFP－N1-HGF转染到人脐静脉细胞株ECV304中,Ｇ418筛选获得稳定表达克隆,采用荧光显微镜观察、RT-PCR、免疫细胞化学方法鉴定。结果 经G418筛选后可形成抗性细胞克隆；荧光显微镜下观察到有绿色荧光;RT-PCR能扩增出HGFmRNA预期的699bp片段；免疫细胞化学证实转染HGF基因的细胞有HGF蛋白的表达。结论 重组质粒EGFP－N1-HGF能够在细胞株ECV304转录、表达。
Construction of EGFP labeled recombinant vector containing HGF and its expression in vein endothelial cells
CHANG Bing-mei,LI Mei-ning,XIE Jun,YANG Tao,TIAN Xin-rui,CHENG Niu-liang,NIU Bo.
Biochemistry & Molecular Biology Department of Shanxi Medical University,Taiyuan 030001,China
【Abstract】 Objective To investigate hepatocyte growth factor （HGF） gene transfection and expression in human umbilical vein endothelial cells（ ECV304） with liposome in vitro.Methods The recombinant plasmid was constructed and transfected into human umbilical vein endothelial cells with liposome：GeneSHUTTLE-20, and positive clones were selected by G418. The transfection and the expression of HGF in ECV304 were tested by RT-PCR and immunohistochemistry.Results After G418 selection, the cell clones were obtained successfully. Under a fluorescent microscope, the expression of enhanced green fluorescent protein（EGFP） was seen in cells transfected with vector contain EGFP gene. RT-PCR and immunohistochemistry proved that there was transcription of HGF gene in transfection cells and there was expression of HGF in these cells.Conclusion The recombinant of EGFP-N1-HGF is an effective expression vector. The recombinant of EGFP- HGF transcript and express in the transfected cells.
【Key words】 hepatocyte growth factor （HGF）;endothelial cells; EGFP
Hepatocyte growth factor （HGF） was first identified in 1984［1,2］ and purified as a potent mitogen of primary cultured hepatocytes［3,4］. Molecular cloning revealed that it is a heterodimeric molecule composed of a 69-kDa chain and a 34-kDaβ -chain. HGF is an important paracrine mediator of epithelial-mesenchymal cell interactions. It is secreted by mesenchymal cells and affects epithelial cell proliferation, motility and morphology. HGF is a multifunctional protein expressed in a variety of cell types and tissues. Exogenous HGF was cardioprotective, through its anti-apoptotic effect on cardiomyocytes.When endogenous HGF was neutralized with a specific antibody, the numbers of myocyte cell deaths increased markedly, the infarct area expanded, and the mortality increased to 50%, as compared with a control group in which there was no mortality［5］.Because HGF is a member of the endothelium-specific growth factors, we hypothesized that HGF may play a role in cardiovascular disease. In our study, recombination in bacteria, an EGFP labeled recombinant eukaryotic vector containing HGF was constructed quickly and efficiently expressed in human umbilical vein endothelial cells. It maybe provid a strategy of angiogenesis in cardiovascular disease.
1 Materials and methods
1.1 Plasmids, bacterial strains, cell lines and major reagents The plasmid PBSKSHGF contain in HGF coding sequence , plasmid pEGFP-N1（containing EGFP gene）; DH5α strains, human umbilical vein endothelial cells（ ECV304） preserved in our laboratory. Liposome geneSHUTTLE-20 were purchased from Quantumbiotechnologys; PCR Amplification Kit，DNA Ligation Kit，RNA-PCR Kit（AMV）were purchased from TaKaRa Biotechnology. Antibodies raised against peptides derived from human HGF antibody were purchased from Sant Cruz Biotechnology,immunohistochemical SABC kit antwere purchased from HuaMei Biotechnology.
1.2 Cell culture Human umbilical vein endothelial cells（ ECV304） were obtained from the second hospital, Shanxi Medical University and cultured in RPMI1640 medium with 10% fetal calf serum.
1.3 Construction of EGFP labled recombinant vector containing HGF The DNA encoding the HGF was generated as a fragment using PCR. The template was PBSKSHGF that had been constructed in our laboratory. The interesting gene obtained by PCR then inserted into EGFP-N1 vector, a kind of EGFP labeled vector. Then the EGFP-HGF plasmid vector is transformed into competent E.coli DH5α.Then plasmids were extracted from the bacteria and analysised by restriction endonucleases and DNA sequencing.
1.4 Cell culture and transfection ECV304 cells were maintained in RPMI1640 medium supplemented with 10% fetal calf serum. Well growing cells were harvested, inoculated in 35 mm dishes on 1x105 cells/ml's concentration, let to get 50%-60% confluence. Liposome-DAN mixture was made up, 800μl serum-free medium was added to liposome-DNA mixture. And the mixture was homogenized, then poured into 35 mm dishes, and negative and vacant control were set up. 2 ml complete medium was added to the dishes until they had been incubated for twenty-four hours. When the cells fully grew, the cells were regenerated by 1 to 3. Then the medium was replaced with G418 culture medium for selective culture when the cells got 80% confluence. Fourteen days later, all the cells in the control dishes died and maintenance does of G418 was used to continue culture. The resistant clones were formed after twenty days, then the 3 resistant clones were transferred to 24-well culture plates one by one, two of them were filled with vacant vetor transfection ECV304 cells. When the cells had fully grown under 37℃,5% CO2 condition, they were disgeted by pancreatin, then transferred to 50 ml culture bottle for amplification.
1.5 RT-PCR analysis RT-PCR was performed using the RT-PCR kit, according to the manufacturer's instructions. To synthesize complementary DNA （cDNA）, 4 μg of total RNA and 750 ng of random primers were used. Oligonucleotides （0.5 μmol/L） used for amplification were: FP（5′CATACCCGGGATGGTTGTAAATGGGATT3′）RP（5′ACATCTGCAGCTTCAGCTA-TGACTGT 3）′,PCR reactions were performed in 25 μl, using 1.5 mmol/L MgCl2, 200 μmol/L of each dNTP, and 1 μl of cDNA reaction. PCR cycles were as follows: 95°C for 5 minutes for denaturation step, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 1 minute. then 10μl of RT-PCR product was checked with 1% agarose gel electrophoresis.
1.6 Immunohistochemistry Expression of HGF detected by immunohistochemical SABC technique in ECV304 cells. Cells were incubated on cover slip which placed in 6-well culture plates . Then cells fixed at 4 °C for 5 minutes by cold methyl aldehyde. Cove slips were incubated for 1 hour in blocking solution, phosphate-buffered saline （PBS）, pH 7.4, supplemented with 10% calf serum albumin and 1% goat serum. After two washes in PBS, Cove slips were incubated for 4 hours at 4°C with rabbit anti-HGF antibody . Controls using nonimmune IgGs were also performed . Horseradish peroxidase-linked secondary antibody incubation was performed for 1 hour at room temperature. Signals were visualized with diaminobenzidine and analyzed by light microscopy.
2.1 Identification of recombinant PGFP-HGF The products of recombinant PGFP-HGF digested with PstI and BamHⅠwere 4679bp fragment and 2221bp fragment（Lane 2 in fig.1）,while digested with KpnI and BamHⅠwere 2200 bp fragment and 4700bp fragment（Lane 3 in fig.1） .The recombinant plasmid were reconstructed ,and the results of restriction endonucleases analysis were shown in fig.1.
2.2 Transfection of ECV304 cells with plasmid DNA and G418 selection of resistant cell strain ECV304 cells transfected with plasmid DNA by liposome were screened with G418 up to 14th day. The negative control cells were all dead. There were cell clones formed in other dishes. Then maintaining dose of G418 was used to 17th day when cell degeneration and necrosis all disappeared and the resistant cells formed positive clone and gradually grew up. The expression of EGFP located in the plasma and nucleus under the inverted fluorescent microscopy. （Fig.2）
2.3 RT-PCR analysis of monoclonal cell strain after being selected by G418 The RNA of the monoclonal cells screened by G418 was extracted using Trizol. A 699 bp strap was amplified in the EGFP-HGF transfected cells by RT-PCR, but there was no special strip amplified in the vacant carrier EGFP transfected cells and the negative control cells （Fig.3）. The result showed that there was no HGF mRNA expression in the vacant carrier EGFP transfected cells, but the mRNA expression in the EGFP-HGF trasfected cells. It could be considered that the EGFP-HGF has transfected ECV304 cell successfully.
2.4 Immunohistochemical results Immunohistochemical results demonstrated that HGF antigen staining occurred predominantly within inflammatory and epithelium cell plasm. （fig.4）
HGF is well known as a mesenchyme-derived pleiotropic（polyphenic） factor that regulates cell growth, cell motility, and morphogenesis of various types of cells and is thus considered a humoral mediator of epithelial-mesenchymal interactions. Nobuaki Nakano previously reported that HGF has a unique characteristic to stimulate only endothelial cell growth but not vascular smooth muscle cells （VSMC） growth［6,7］. Moreover, the presence of specific receptor of HGF, C-MET, has been detected in vascular tissues［8］. However, there are no reports as to how HGF is regulated in vascular tissues. Because HGF is a member of the endothelium-specific growth factors, we hypothesized that HGF may play a role in cardiovascular disease. In this study, ECV304 cell clones were obtained successfully. Under a fluorescent microscope, the expression of enhanced green fluorescent protein（EGFP） was seen in cells transfected with vector contain EGFP gene. RT-PCR and immunohistochemistry proved that there was transcription of HGF gene in transfection cells and there was expression of HGF in these cells. Therefore the recombinant of pEGFP-N1-HGF is an effective expression vector. HGF is thus a potent agent for strategies designed to promote therapeutic angiogenesis［9］.
In the research , a novel reporting gene enhanced green fluorescent proteins （EGFP）was labeled. The major advantage of EGFP to the biologist is their ability to exhibit intrinsic fluorescence ; for most other fluorescent proteins a cofactor is required. This property is endowed on EGFP thanks to three amino acids that cyclise （Ser65-Tyr66-Gly67） and then undergo an oxidation step during a complex maturation process［10］. The fluorophore is deeply buried within the hydrophobic core of EGFP that is, itself, made up of β-sheet with a central а-helix containing the fluorophore. EGFP-N1 is vector Fusions to the N terminus of EGFP retain the fluorescent properties of native protein allowing the localization of fusion protein in vivo, with no intervening in -frame stop codons. The recomebinant EGFP vector can be transfected into mammalian cells using any standard transfection method. As required ,stable transformants can be selected using G418. EGFP-N1 encodes a variant of wild type GFP which has been optimized for brighter and high expression in mammalian cell. Therefore the EGFP labeled recombinant eukaryotic vector containing HGF was constructed quickly and efficiently expressed in human umbilical vein endothelial cells. It maybe provide a strategy of detect the target proteins.
（The figs on attaches the page 1）
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作者单位： 030001 山西太原，山西医科大学生物化学与分子生物学教研室