| Hypertrophic scar is the main attention of plastic and reconstructive surgery, being the result of the wound healing. Scar formation is a process in the charge of multi-factors, material mechanism is not clear. And the effect of manifold treatment method is not successful. Micro-surrounding research display that the process of angiogenesis plays an important role in wound healing. Recent investigation proved that angiogenesis have tightly related with the formation of hypertrophic scar. Following the researching for the tumor's development, more powerful angiogenesis inhibitor are discovered and applied. Thus, new attempted chances are offered for studying the effect on angiogenesis in the different diseases including the hypertrophic scar formation.Recently, cytotherapy and gene therapy are hotspot in medicine and life science field. It brings a new hope to therapy many diseases. A key question is to select seed cells and transgene vehicles. Endothelium cells have a highly self-renew ability and demonstrate exetensive diferentiation. They are considered as an ideal seed cells and rrangene vehicles.Gene transfer approaches are used both viral vector including retrovirus-mediated gene transfer, adenovirus-mediated gene transfer and non-viral vector including calcium ion sedimentation, electroporation. Replication-deficient adenoviral vectors (AdVec), which can transfer gene with high efficiency both in vivo and in vitro. infects nearly all type of cells with the advantage of ready infection of both cycling and noncycling cells, low toxicity and easy delivery. Human umbilical vein endothelial cells as vehicles have been report using AdVec; However, studying the feasibility of HUVEC with AdVec containing delivered-gene is far from complete and many important issues are awaiting answers, including infection efficiency, infection time, the optimal AdVec infection tilter, the change of antigenic phenotype of HUVEC infected with AdVec in pre- or post-infected time, the differentiation potential of HUVEC after infected with AdVec, the change of cell cycle of HUVEC using AdVec in pre- or post-infected time. These need systemic discussing.Therefore, this study aims to investigate the properties of HUVEC infected with AdVec systemically, using HUVEC derived from umbilical vein which will include the following five parts:Part one: Objective To get the full length of human METH1 cDNA and express it in mammalian cell stably. Methods METH1 was amplified by RT-PCR, and cloned into pCDNA3.0 after confirmed by sequence analysis. HepG2 cells were transfected by Lipofectamine?reagent and then selected in medium with G418. Theexpression level of METH1 was detected by RT-PCR and Western blot. Results METH1 with expected length was effectively amplified, and matched the published sequence encoding mature peptide[GI:5725505] completely as shown by sequence analysis. Eukaryotic vector expressing METH1 was got by gene cloning, and cells expressing METHI stably were got by selection with G418 3 weeks after transfection. RT-PCR and Western blot showed high level expression of METHI. Conclusion Full length of human METHI gene is cloned successfully and expressed in HepG2 stably, and this makes a basis for the study of effects of METHI on hypertrophic scar angiogenesis.Part two: Objective To explore the growth inhibitory effects of pCDNA3.0 vector expression of METHI gene on human umbilical vein endothelial cells(HUVEC) in vitro. To investigate the HUVEC apoptosis by using flow cytometry. Methods To construct eukaryotic vector pCDNA3.0 expressing METHI and transfect the vector to HepG2 cells by Lipofectamine?000 reagent. HUVEC were expanded in vitro., MTT method was used to examine the effects of HepG2/ METHI and HepG2 supernatant on HUVEC proliferation. The ratio of apoptosis to HepG2/METHl group was increased. Results Eukaryotic vector pCDNA3.0 expressing METHI was successfully constructed, and HepG2 cells expressing METHI stably were got after transfection. The result of MTT showed that the group of HepG2/METHl inhibited the pr...
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