| Angiogenesis plays an important role in tissue replacement and healing and requires the coordinated action of a variety of cells and growth factor receptors.Induction of angiogenesis by using biochemical stimulation has been plagued by the complexities of obtaining a mature vascular morphology and function, i.e., vessels that are well perfused, yet not hyperpermeable. Indeed, induction of angiogenesis only with VEGF induces a vascular response that is unphysiologically permeable. To redress this limitation, bioengineering approaches to deliver multiple growth factors to provide both angiogenic and maturation signals have been explored.As an alternative to local administration of multiple growth factors, the approach of delivering HIF-1??ODD, the transcription factor that was removed the oxygen-sensitive degradation domain(ODD) can regulate a number of proangiogenic signals under normoxic conditions has been presented. Results from transgenic animals, morphometric analyses support that a mature angiogenic response may be obtainable through gene therapy with a form of HIF-1 that is stable under normoxic conditions. So we plan to campare the HIF-1??ODD gene with VEGF165 gene for angiogenesis and vessels mature.The use of direct viral vector injection in the ischemic tissue has its associated disadvantages. The direct viral vector injection may initiate host inflammatory and immune responses which may interfere with the repair process. Similarly, as compared with the ex vivo cell based gene delivery approach, direct viral vector gene delivery may be less efficient in initiating a therapeutically significant angiogenic response. Moreover, the injected viral vectors may poorly transfect host tissue and hence the delivered vector may be lost quickly. In order to overcome these undesired complications, using cells as the gene vector represents a rational therapeutic measure for treatment of ischemic diseases. We choose fibroblast cells as the gene vector because fibroblast cells are easy to culture and demonstrate a low rate of immunity-assisted rejection. Some studies found that heterogeneic fibroblast cells could survive in host for 2 to 8 weeks. For many ischemic diseases, for example, chronic wound, myocardial ischemia and tissue engineering, two weeks'therapy was enough. Moreover, these xenogenous cells could be rejected by host at last, so there was no danger of tumor formation. So we plan to choose the fibroblast cells as the gene vector.The pedicle skin flap transplantation is one major tissue repairing method, however, the pedicle can be devided at last three weeks later. The autotransplanted fat was use to repair the facial deformity and hump the breast, however, the survival of the autotransplanted fat was low. If the revascularization can be improved after operation, the period before the pedicle being divided will be shorten and the survival of the autotransplanted fat will be enhanced. We plan to choose the rat pedicle flap and the fat autotransplantation as the animal model.Our goal in this work was to explore transplantation of HIF-1??ODD gene transfected NIH3T3 cells for rat pedicle flap and fat autotransplantation. Through observing the living period of NIH3T3 cells after being transplanted to rat, we plan to explore the affect of the xenogeneic fibroblast cells as the gene vector. Through observing the period before the pedicle being divided, the survival of the autotransplanted fat, the capillary density and the matue vessels percent, we plan to explore the affect of HIF-1??ODD gene for matue neovascularization.Experiment 1 Lenti/HIF-1??ODD/EGFP constructionObjective: To construct Lenti/HIF-1??ODD/EGFP. Methods: Designed a pair of primers, added the SmaI cleavage site and the NheI cleavage site before and behind HIF-1??ODD gene, PCR for pCEP4/HIF-1??ODD, ran electrophoresis for PCR products, retrieved the glue, SmaI and NheI cleaved the products, ran electrophoresis and retrieved the glue. SmaI and NheI cleaved the pRRLsin/LacZ alpha(+), retrieved the larger fragment, connected the products of two steps and got the pRRLsin/HIF-1??ODD/EGFP. Transduced pLP1?pLP2?pLP/VSVG and pRRLsin/HIF-1??ODD/EGFP into 293T cells and cellected the supernatant. Result: The results of the PCR, SmaI and NheI cleavage and sequencing were correct. Conclusions: Lenti/HIF-1??ODD/EGFP construction was achieved. Experiment 2 Lenti/VEGF165/EGFP constructionObjective: To construct Lenti/ VEGF165/EGFP. Methods: Designed a pair of primers, added the SmaI cleavage site and the NheI cleavage site before and behind VEGF165 gene, PCR for pcDNA3.1(-)/VEGF165, ran electrophoresis for PCR products, retrieved the glue, SmaI and NheI cleaved the products, ran electrophoresis and retrieved the glue. SmaI and NheI cleaved the pRRLsin/LacZ alpha(+), retrieved the larger fragment, connected the products of two steps and got the pRRLsin/ VEGF165/EGFP. Transduced pLP1?pLP2?pLP/VSVG and pRRLsin/VEGF165/EGFP into 293T cells and cellected the supernatant. Result: The results of the PCR, SmaI and NheI cleavage and sequencing were correct. Conclusions: Lenti/ VEGF165/EGFP construction was achieved.Experiment 3 Recombinated lentivirus transfect NIH3T3 cellsObjective: To get HIF-1??ODD gene, VEGF165 gene and EGFP gene stable transfected NIH3T3 cells respectively. Methods: Lenti/HIF- 1??ODD/EGFP?Lenti/VEGF165/EGF and Lenti/EGFP transfected NIH3T3 cells respectively. The transfected cells were selected by flow cell sorter and were identified by real time PCR, Western Blot and ELISA. Result: The results of real time PCR, Western Blot and ELISA showed that the Lenti/HIF- 1??ODD/EGFP transfected NIH3T3 cells expressed mouse VEGF?bFGF and Ang-1 protein highly and Lenti/VEGF165/EGFP transfected NIH3T3 cells expressed human VEGF165 highly. Conclusions: Lenti/HIF-1??ODD/EGFP stable transfected NIH3T3 cells, Lenti/VEGF165/EGFP stable transfected NIH3T3 cells and Lenti/ EGFP stable transfected NIH3T3 cells were gained. Experiment 4 Observe survival time of NIH3T3 cells in rat bodyObjective: To explore the survival time of NIH3T3 cells in rat body. Methods: Lenti/EGFP transfected NIH3T3 cells and CM-DiI labeled NIH3T3 cells were transplanted into subcutaneous fascial layer of rats respectively. After 1, 2, 3 and 4 weeks, the green fluorescent Lenti/EGFP transfected NIH3T3 cells and red fluorescent CM-DiI labeled NIH3T3 cells were observed by fluorescence microscopy respectively. Result: From 1 to 3 weeks, the number of green fluorescent cells and red fluorescent cells was similar, however, the number decreased after the 4th week. Conclusions: NIH3T3 cells can survive in rat body for at last 3 weeks.Experiment 5 NIH3T3 cells transfected by HIF-1??ODD gene for rat flapObjective: To shorten the period before the rat flap pedicle being divided by transplanting NIH3T3 cells transfected by HIF-1??ODD gene into subcutaneous fascial layer of the flap. Methods: Thirty SD rats were divided into 3 groups. Group A received Lenti/HIF-1??ODD/EGFP transfected NIH3T3 cells, group B received Lenti/VEGF165/EGFP transfected NIH3T3 cells, group C received Lenti/EGFP transfected NIH3T3 cells. After 4 days, the pedicles of were divided. The percent of the flap survival area was evaluated. Immunofluorescence assay was used to detect the CD31 and SMA. The level of circulating human VEGF-165, mouse VEGF, mouse IGF-1 and mouse bFGF were quantified using according ELISE kit. Result: The percent of the flap survival area of the group A and B were 100%, however, it was about 40% in group C. The density of vessels of group A was 3 fold higher than which of group C. The density of vessels of group B was 4 fold higher than which of group C.The percent of the mature vessels of group A was 53%, group B 10% and group C 39%. Conclusions: Transplantation of NIH3T3 cells transfected by HIF-1??ODD gene into subcutaneous fascial layer of the flap could shorten the period before the rat flap pedicle being divided.Experiment 6 NIH3T3 cells transfected by HIF-1??ODD gene for rat fat autotransplantationObjective: To improve the survival of the rat autotransplanted fat by adding NIH3T3 cells transfected by HIF-1??ODD gene into fat granules. Methods: Eighteen SD rats were divided into 3 groups. Group A received Lenti/HIF-1??ODD/EGFP transfected NIH3T3 cells, group B received Lenti/VEGF165/EGFP transfected NIH3T3 cells, group C received Lenti/EGFP transfected NIH3T3 cells. The cells were added into the fat granules which were autotransplanted into dorsal subcutaneous stratum respectively. After 6 months, the percent of the fat survival was evaluated. HE staining and immunofluorescence assay was used to detect vessels. Result: The percent of the fat survival of the group A was 99.3%,B 76.7% and C 20.0%. The density of vessels of group A was 5 fold higher than that of group C. The density of vessels of group B was silimar with that of group A. Conclusions: Adding of NIH3T3 cells transfected by HIF-1??ODD gene into fat granules could improve the survival of the rat autotransplanted fat and was better than adding of NIH3T3 cells transfected by VEGF165 gene. |
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