Zinc Finger Protein Transcriptional Activator Limb Ischemia And Research On Muscle Fiber Remodeling Treatment

Part I Construct the Engineered Zinc Finger Protein-Activating Transcription FactorObjective:To synthesize the Zinc Finger Protein-Activating Transcription Factor (ZFP-ATF) combining with the Vascular Endothelial Growth Factor (VEGF) promoter. Methods:Firstly, the purposed genes of ZFP binding domain were constructed according to the VEGF promoter sequences with ZFP tools and were cloned into the PVAXI plasmid with P65activating domain. After that, the accuracy and validity were verified with the Endonuclease digestion method and the DNA sequence. Results:The structure of ZFP-ATF contained the DNA binding and activation domain, of which the DNA binding domain was the genes of six ZFP binding domains; the transcription activation domain was from p65unit of the NF-κB in human nuclear factor. Besides, endonuclease digestion results showed the multiple cloning site located between the BamHI and XholI; mealwhile, the DNA sequence results suggested the length of the purposed gene of purposed gene was1000bp, the plasmid was about3.0kb. Conclusion:It is important to choose the correct DNA binding domain and activation domain when constructing the engineered ZFP transcription factor successfully. The innovative and potential point in ZFP-ATF was the specificity of combining with VEGF promoter to stimulate the endogenous VEGF expression.Part II The effect of ZFP-ATF stimulating the VEGF expression with cell experiment in vitroObjective:To test the effect of ZFP-ATF influencing the VEGF gene expression with cell experiment in vitro. Methods:the relative amount of VEGF mRNA and VEGF protein were tested after.transfecting the ZFP-ATF and VEGF165into the HY926cells. Results:ZFP-ATF mainly stimulated the main VEGF isoforms, m(?)reover, the relative amount of VEGF protein in ZFP-ATF group was dramatically higher than the sole VEGF165and control groups (P<0.05).Conclusion:ZFP-ATF can promote the main VEGF splice variants and higher VEGF protein with the HY926cell experiment, which illustrated that the powerful effect in endogenous VEGF expression may promote potential and effective therapeutic angiogenesis.PartⅢ The rat mesenteric angiogenesis experimentObjective:To research the capillary density and maturity of angiogenesis in ZFP-ATF group in vivo with mesentery. Methods:The ICR rats were randomly divided into three groups(N=8). The mesentery were taken out from abdominal cavity and photoed under stereomicroscope, which was performed again after14days when injected the ZFP-ATF, VEGF165and saline, respectively. In addition to, the same mesenteric artery beds were resected and stained with fluorescence antibody (NG2and Ki-67) to observe the maturity of the neovessels. Results:the capillary density and fractional vessel area in ZFP-ATF group were obviously higher than the VEGF165, saline groups (P<0.05).In mesenteric immunofluorescence experiment, the neovessels in ZFP-ATF group had less branch, integrated pericyte coverage and wide diameter, however, the neovessels in VEGF165group had more branch, incomplete pericyte coverage and narrow diameter.Conclusion:The ZFP-ATF may dramatically stimulate the increase of the neovessels; more importantly, the maturity of the neovessels in ZFP-ATF group was optimal and effective because of the intergrated pericyte coverage.Part IV The effect of PLGA nano material as a better carrier and ZFP-ATF stimulating the therapeutic angiogenesis in ischemic limb mice and influencing the skeletal muscle fiber remodeling.Objective:To study the effect of the PLGA nanomaterial as a better carrier and ZFP-ATF stimulating the angiogenesis in ischemic limb mice model and influencing the skeletal muscle fiber remodeling. Methods:The unilateral ischemic limb mice model were performed with the resection and ligation of femoral artery. Double emulsion-solvent evaporation method was used to synthesize PLGA-heparin nanoparticles. Then test the surface morphology, the average diameter, the loading efficiency and the release time in vitro; then inject the PLGA-heparin into mouse ischemic limbs to observe the perfusion recovery with LDPI at the time of post-ischemic7,14,21, and28days; and finally, test the expression of VEGF and HGF and the number of the neo-vessels in ischemic limbs. After that, the mice were randomly divided into three groups (N=8). The ZFP-ATF, VEGF165and saline were respectively injected into the ischemic muscle7days after the femoral artery resection operation. The relative amount of VEGF protein was evaluated7days after injection, the capillary density was analyzed with immunohistochemical experiment including the CD31and PCNA14days after injection. Besides, the skeletal muscle fiber type was firstly observed21days after receiving the injection according to the MHC mRNA Real-time PCR and ATPase staining. Results:The surface morphology of the PLGA-heparin was smooth, the average diameter was290nm, the loading efficiency was5.35%, and the release period maintained for14days. In animal experiment, the perfusion recovery, VEGF and HGF levels, and capillary density in PLGA-heparin group were significantly higher than the control group. The relative amount of VEGF protein in ZFP-ATF group was higher in ischemic muscle than the VEGF165, salinel groups (P<0.05). The immunohistochemical experiment results showed the capillary density in ZFP-ATF group was obviously more than the VEGF165, saline groups (P<0.05). Besides that, the relative amount of MHC isoforms mRNA showed the increase of MHC-I isoform and decrease of MHC-IIb isoform in Gastrocnemius and Soleus in both ZFP-ATF and VEGF165groups compared with the salinel group(P<0.05), of course, the ATPase staining suggested the constituent ratio of muscle fiber type-I was increased.however, the muscle type-II was reduced by ZFP-ATF and VEGF165groups compared with the salinel group (P<0.05) both in Gastrocnemius and Soleus, though the differences between the ZFP-ATF group and VEGF165group had no statistical significance.. Conclusion:Nanoparticle encapsulating heparin could be successful and efficient in ischemic disease with prolongation its therapeutic effects and stimulating growth factors expression. The ZFP-ATF was of importance in therapeutic angiogenesis in terms of the ischemic disease according to supplying enough neovessels and promoting mature neovessels. Moreover, it was the first research on skeletal muscle fiber type remodeling with the gene therapy. The changes from the type-II to type-I indicated a potential clinical therapy for development of the muscle tolerance activity for the ischemic limb.