| The diseases relate to retinal neovascularization include diabetic retinopathy , retinal periphlebitis, retinopathy of prem aturity ,etc.This kind of diseases may cause the damage to the blood-retinal barrier, blood microcirculation and vessel permeability. They lead to retinal anoxia and edema. Then retinal tissue has a series of compensatory response to the anoxia and neovascularization. Are the principal causes of blindness.Current treatments including laser photocoagulation and vitrectomy are of limited efficacy and cause significant adverse effects. but poor results in clinical trails of some of the antiangiogenic strategies.The key to treating these diseases is to inhibit the development of the neovascularization.ActivinβA, a member of the transforming growth factor(TGF-β) superfamily, Activin(ACT) are a kind of multifunctional factors,they have tissue specificity,and have critical regulatory contribution in the tissue formation,cell growth,cell differentiation,development and maturation. Espeeially in the dividing cell multiplieation,and apoptosis the functional concern. also are the important regulatory factor in organism during physiology and pathology. A recent study shows that heavy elements through regulation of VEGF expression in vaseular generation.Materials and Methods:1.The activinβA gene was amplified by PCR with the recombinant plasmid pcDNA3.1/ activinβA as the template and the fragment was cloned into pMD18-T vector. gene was activinβA released from pMD18-T activinβA and then was inserted to plasmid pEGFP-Nl with double enzymatic digestion.2. Microvascular endothelial cells of human skin (HMEC) was cultured and then trasfected by liposomes mediated pEGFP-N1/ activinβA .The expression of EGFP was observed under fluorescence microscope.3.HMEC were cultured and then trasfected by liposomes mediated pEGFP-ActivinβA. The effect of cell proliferation was determined by methyl thiazolyl tetrazolium(MTT) method. The effect on cell cycle was examined by flow cytometric analysis.4.Cell death was determined by DAPI staining,Hoechst33342 staining and action's of Caspase-3.5. ACT's action on Smads pathway was examined in HMEC by western blot analysis using primary antibody against the phosphorylated form of Smad2 and Smad3 signal molecules.Results:p1. The eukaryotic expression vector pEGFP-N1/activinβA was successfully constructed. DNA sequencing and blast showed that activinβA completely matched the DNA sequence listed in GenBank.2. ActivinβA protein was expressed in HMEC and secreted in the culture supernatant. With the time prolonging,they became stronger and stronger.3. MTT results showed with the time prolonging,the proliferation of HMEC was inhibited by pEGFP- activinβA transfection; flow cytometric analysis results showed with the ratio of the S phase decreased.4. Hoechst33342 staining showed that there was sifnificant difference in inducing apoptosis between control group and EGFP- activinβA trasfecting group.5. western Blot showed that it was sifnificant increasing in Smad2/ Smad 3 protein express between control group and pEGFP- activinβA trasfecting group. (P<0.01)Conclusion:1. The successful construction of the eukaryotic expression vector pEGFP-activinβA .2. The eukaryotic expression vector pcDNA-F-ARIPzip is conctructed and expresses in HEK293 cells successfully, which establish the foundation for future research on ARIPzip gene function.3. ActivinβA can be transferred to endothelial cells by cationic liposome mediating and inhibit the proliferation of endothelium cells.4. ActivinβA can be transferred to endothelial cells by cationic liposome mediating and induced apoptosis of HMEC in vitro.5. ActivinβA can be transferred to endothelial cells by cationic liposome mediating and showed a decrease in the phosphorylation of Smad2/Smad3 compared to control group.ActivinβA is a member of the family TGF-β,as in adenohypophysis FSH can stimulate the release function was separated out and surrounded by external naming.Recent research shows that activin,it in a variety of biological systems play an important role,especially in the dividing cell multiplication,and apoptosis the functional concern.Even activin from model and divided into three different types,namely,activinβA A ,B and enlivening activinβA. Beinging quality ,are widely distributed in human tissues.A recent study shows that heavy elements through regulation of VEGF expression in inhibiting of vascular. This results indicated that activinβA could inhibit the proliferation of HMEC by stimulating the synthesis of DNA and mitosis ,and the effet of proliferation on HMEC via Smad2/Smad3 signaling pathway.In summary, this study indicates that activinβA inhibit the proliferation of HMEC via Smad2/Smad3 signaling pathway.Thus may play a vital role in the prevention of neovascularization .This study should suggest that there may be some new options to need to be explored in the treatment of the diseases relate to retinal neovascularization.
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