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Objective: To investigate the feasibility of establishing a murine new vessels model with Lentiviral vector (LVs) mediated human vascular endothelial growth factor-165 (pcDNA3.1/ VEGF165) gene., Methods: Lentivirus plasmid carrying a human VEGF165 was constructed and was used to transfect mouse's NIH/3T3 cells. The NIH/3T3 cells with high secretion of VEGF were injected into the skeletal muscle of mouse to establish a mouse new vessels model by implantation of vascular endothelium growth factor (VEGF) gene. The external secretion of VEGF was measured with ELISA. Histological examination was carried out after injection. The expression of CD31 was assessed with immunohistochemical method., Results: The lenti-VEGF165-EGFP was correctly constructed and confirmed by restriction endonuclease analysis, polymerase chain reaction and DNA sequencing analysis. Lentivirus plasmid carrying a human VEGF165 was constructed. lenti-VEGF165-EGFP was used to transfect mouse's NIH/ 3T3 cells, and human VEGF165 gene was assessed. NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model. The external secretion of VEGF in peripheral blood was measured with ELISA. The legs became swollen in experimental group 14 d after injection. It found the cells expression of CD31 44 d after injection, and histological analysis showed the swollen tissue was composed of small new vessels., Conclusions: NIH/3T3 cells mediated VEGF gene implantation can produce stable and effective mouse new vessels model.

Objective: To approach the effect of the donor antigenic specificity CD4+CD25+ regulatory T cell (Treg) on cellular immune tolerance function in rat composite tissue allotransplantation (CTA)., Methods: Use the method of immunomagnetic beads to separate CD4+CD25+ Treg, (1 x 10(6))CD4+CD25+ Treg was transfused to rat CTA model. Collected peripheral blood 30 days after operation, and used nylon wool column to separate B cell and T cell. With the stimulation of IgM, detected B cell proliferation and the level of IgG and IgA in serum. Observed the effect of CD4+CD25+ Treg on B cell and T cell function and the survival of allotransplants, and analyzed the data by statistics., Results: The purity of separated CD4+CD25+ Treg was 95.6%. The CPM of T cell of normal control group, topical intervention group, systemic intervention group and non-intervention group were (2436 +/- 358), (2273 +/- 136), (2338 +/- 228) and (3749 +/- 245). The CPM of B cells of normal control group, topical intervention group, systemic intervention group and non-intervention group were (2418 +/- 348), (2252 +/- 127), (2315 +/- 218) and (3720 +/- 224), there was a significant difference in these groups (P < 0.01). The serum level of IgG and IgA of topical intervention group and systemic intervention group were (12.56 +/- 1.30), (2.38 +/- 0.21), (13.48 +/- 1.23) and (2.86 +/- 0.24) g/L, and of normal control group was (12.35 +/- 1.28), (2.36 +/- 0.12) g/L, had no significant difference (P > 0.05). But Treg of non-intervention group was (16.58 +/- 1.12), (3.75 +/- 0.37) g/L, there was a significant difference in the non-intervention group and the three above groups (P < 0.01). The survival time of CTA in intervention of local and systemic groups were (97 +/- 13) and (63 +/- 10) d, which were significant longer than the non-intervention group [(22 +/- 8) d, P < 0.01]., Conclusions: Donor antigen specific CD4+CD25+ Treg has significantly inhibited B cell and T cell function. It can induce immune tolerance and extend the survival time of CTA; as well local application is better than systemic.

Objective: To study the effect of hirudin on the function of human hyperplastic scar fibroblasts (HSFBs)., Methods: HSFBs were cultured in vitro. Hirudin solution in the concentration of 1, 10, and 50 kU/L was respectively added into DMEM culture medium to form 1, 10, and 50 kU/L hirudin groups, with 9 wells in each group. HSFBs cultured without hirudin were set up as control group. Cell inhibition rate, secretion level of TGF-beta1 from cells, and expression levels of mRNA of type I and III precollagen were determined at 24, 48, and 72 h after culture., Results: Inhibition rates of HSFBs growth was respectively (29.3 +/- 0.9)%, (30.1 +/- 0.3)%, and (45.2 +/- 1.9)% when cultured with 10 kU/L hirudin for 24, 48, and 72 hs, which were higher than those in control group [(0.0 +/- 0.0)%, P < 0.05]. There was statistically significant difference between control group and 1 and 50 kU/L hirudin groups in the inhibition rates of HSFBs at some time points (P < 0.05). Secretion level of TGF-beta1 of HSFBs in 1, 10, 50 kU/L hirudin groups was respectively (228.5 +/- 1.8), (210.5 +/- 11.1), and (168.5 +/- 14.1) pg/mL when cultured for 48 hs, of which the last 2 figures were significantly lower than that of control group [(265.0 +/- 1.5) pg/mL, P < 0.05]. Hirudin in the concentration of 10 and 50 kU/L could inhibit the expression of mRNA of type I and III precollagen in HSFBs., Conclusions: Hirudin solution in the concentration of 10 and 50 kU/L can inhibit the proliferation of HSFBs and secretion of TGF-beta1 and collagen in certain degree.

Objective: To investigate the feasibility of applying NIH3T3 cells transfected by VEGF gene to the treatment of ischemic random skin flaps., Methods: Plasmid PcDNA3.1(-)/VEGF(165) containing VEGF gene was transduced into the mouse NIH3T3 cells by liposome. Immunohistochemistry was used to detect the expression of VEGF protein of mouse NIH/3T3 cells in vitro. The NIH3T3 cells were stained with CM-DiI before the transplantation. Thirty mice were randomized into 3 groups: Groups A, B and C, and were respectively injected with NIH/3T3 cells transfected with PcDNA3.1(-)/VEGF(165) plasmid, NIH/3T3 cells and medium only. On the 4th day after the injection, random dorsal skin flaps with an area of 4.0 cm x 1.5 cm were established. The survival, neovascularization and blood flow recovery of the flaps were detected., Results: VEGF-transduced NIH3T3 cells expressed VEGF highly in vitro and in vivo. The results showed that flap survival rate in group A (95.1% +/- 3.1%) was significantly higher than those in group B (37.4% +/- 6.3%) and group C (26.2% +/- 5.6%). The capillary density and the blood perfusion of the flaps in group A were significantly higher than those in other two groups., Conclusions: VEGF-transfected NIH3T3 cells can improve ischemic flap neovascularization and extend survival areas.

Objective: To investigate the feasibility of transplanted endothelial progenitor cells to ischemic flap with increased neovascularization and augmented the survival areas., Methods: EPCs were isolated from human cord blood, cultured in vitro, identified by immunohistochemistry. Then EPCs were transplanted to ischemic flaps of 9 nude mice's back (experimental group), and 9 nude mice's back flaps was injected with M199(control group). And pedicle division time was 4 days after operation. CM-DiI was used to trace the transplanted cells. The blood perfusion of flaps was monitored by the laser Doppler flowetry, and the capillary density of flaps was detected by CD34 immunohistochemistry., Results: EPCs expressed cell markers CD34, KDR and CD133. Transplanted EPCs survived and was incorporated into the capillary networks in the ischemic flaps of nude mice. The percent of experimental group's flap survival area was (60.3 +/- 2.1)%, significantly higher than the control group[ (34.2 +/- 1.8)%, P < 0.05 ]. The blood perfusion, capillary density of flaps of experimental group was significantly higher than the control group (P < 0.05)., Conclusions: EPCs from human cord blood can increase ischemic flaps neovascularization and augment the survival areas.

Objective: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) transfected with VEGF165 gene to free transplanted fat tissue for increasing neovascularization and the survival., Methods: EPCs isolated from human cord blood were cultured in vitro and identified by immunocytochemistry. After transfection by VEGF165 gene, the expression of VEGF was assessed using ELISA. Then EPCs with (VEGF gene transfection group) and without VEGF165 gene transfection (EPCs group) were transplanted to free transplanted fat tissue at 18 nude mice's back, and nine nude mice transplanted with free fat tissue were injected with M199 (control group). CM-DiI was used to trace the transplanted cells. The capillary density of transplanted fat tissue was detected by CD34 immunohistochemistry., Results: EPCs expressed cell markers CD34, KDR and CD133. After transfection, the expression of VEGF was positive. Transplanted EPCs survived and proliferated, and transplanted EPCs were incorporated into the capillary networks in the transplanted fat tissue. The percent of survival volume of transplanted fat tissue of VEGF gene transfection group was (96.2 +/- 8.6)%, significantly higher than that of the EPCs group [(75.3 +/- 6.8)%, P < 0.05) and M199 group [(40.2 +/- 2.5)%, P < 0.05). The capillary density of transplanted fat tissue of VEGF gene transfection group was significantly higher than those of the EPCs group and M199 group (P < 0.05)., Conclusions: EPCs from human cord blood can increase free transplanted fat tissue neovascularization and the survival volume, and the ability of promoting neovascularization of EPCs transfected with VEGF165 gene is more potent than EPCs alone.

Objective: To investigate the feasibility of transplanted endothelial progenitor cells transfected with VEGF165 gene to ischemic flap with increased neovascularization and augmented the survival areas., Methods: EPCs were isolated from human cord blood and cultured in vitro. Plasmid PcDNA3.1(-)/VEGF165 containing VEGF gene was transfected into the EPCs. EPCs transfected with blank plasmid, and EPCs without transfection were used as controls. ELISA was used to detect the expression of VEGF protein in the culture fluids. The EPCs were dyed with CM-DiI 7 days later. Ischemic skin flaps were made on the backs of 27 nude mice. The mice were randomly divided into 3 equal groups with their skin flaps being transplanted with EPCs transfected with 3.1(-)/VEGF165 plasmid, EPCs not transfected with 3.1(-)/VEGF165 plasmid, and injected with M199 medium at the basal part. Four days after the peduncles of the skin flaps were cut. Seven days after the cutting-off of the peduncles the survival rate of skin flap was observed and the blood perfusion was observed with laser Doppler flowmetry, 10 days after the density of capillary arteries were observed with microcirculation microscope. Three specimens of skin flap were taken 7 and 11 days after the skin flaps were made to undergo histological examination to detect the density of capillary arteries by CD34 immunohistochemistry and to observe the proliferation of EPCs with fluorescence microscopy. Peripheral blood samples were collected 1, 4, 7, 14, and 28 days after the skin flaps were made to undergo ELISA to detect the levels of VEGF protein, Results: The VEGF levels in the culture supernatants of the groups A, B, and C were 352 ng/L +/- 35 ng/L, 45 ng/L +/- 5 ng/L, and 0 ng/L respectively with significant difference between any 2 groups (all P < 0.05). The skin flap survival rates of the three groups were 97.2%, 60.3%, and 34.2% respectively with significant difference between any 2 groups (all P < 0.05) and the survival quality of the group A was the best. The capillary density of the group A was greater than those of the groups B and C. The VEGF levels at any time point of the group A were all significantly higher than those of the group B and C (all P < 0.05) and there was not a significant difference between the groups B and C. The capillary density levels at different time points decreased progressively in the order of groups A, B, and C with significant difference between any 2 groups (all P < 0.05). No EPC was shown by fluorescence microscopy in the skin flaps of the group C. The EPC density in skin flap 7 and 11 days after the flaps were made were 136 +/- 10 and 75 +/- 6/mm(2) and 305 +/- 26 and 199 +/- 18/mm(2) respectively with significant differences between the groups A and B. (both P < 0.05)., Conclusion: The EPCs from human cord blood, especially those transfected with VEGF165 gene increases the neovascularization in ischemic skin flaps and augments their survival rate.