激活素A对人皮肤微血管内皮细胞增殖的抑制作用及对SMADs信号转导通路影响的研究
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摘要
视网膜新生血管性疾病是指由于新生血管生长伴随出血、渗出、增生等病理性改变造成的致盲性玻璃体视网膜疾病,是世界范围内最严重的致盲性眼病之一,包括糖尿病性视网膜病变( diabeticretinopathy,DR)、早产儿视网膜病变(Rerinopathy of prematurity, ROP)等。视网膜新生血管形成过程中,血管内皮细胞发挥着重要作用。血管内皮细胞的分化、增殖、迁移、微血管形成等生命活动是整个过程的中心环节。抑制血管内皮细胞增殖和新生血管生长是治疗这类疾病的关键。本研究首先探讨了激活素A对人皮肤微血管内皮细胞的增殖活力、细胞周期和凋亡的影响,通过MTT,流式细胞术,DAPI及caspase-3活力检测法证实激活素A能够抑制人皮肤微血管内皮细胞的增殖并诱导细胞凋亡。然后通过western blot法检测Smad2和Smad3信号转导通路,发现Smad2和Smad3信号转导通路参与了激活素A对人皮肤微血管内皮细胞增殖的抑制及诱导细胞凋亡的作用。表明激活素A对抑制视网膜新生血管的形成具有重要意义。为视网膜新生血管性疾病的治疗提供了新思路和理论依据。
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.
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.
引文
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[9] Harrison CA, Gray PC, Vale WW and Robertson DM. Antagonists of activin signaling: mechanisms and potential biological applications. Trends Endocrinol Metab, 2005, 16(2): 73-8.
[10] Lau AL, Kumar TR, Nishimori K, etal. Activin beta C and beta E genes are not essential for mouse liver growth, differentiation, and regeneration [J]. Mol Cell Biol, 2000, 20 (18): 6127-6137.
[11] Date M, Matsuzaki K, Matsushita M, et al. Differential regulation of activin A forhepatocyte growth and fibronectin synthesis in rat liver injury. [J]. J Hepatol, 2000, 32(2): 251-260.
[12] Vejda S, Cranfield M, Peter B,et al. Expression and dimerization of the rat activin subunits bC and bE: evidence for the formation of novel activin dimers. J. Mol. Endocrinol, 2002, 28(1): 137–148.
[13] Mellor SL, Cranfield M, Ries R, et al. Localization of activin ?A-, ?B- and ?C-subunits in human prostate and evidence for formation of new activin heterodimers of ?C-subunit. J. Clin. Endocrinol. Meta., 2000, 85:4851–4858.
[14] Massague J. TGF-beta signal transduction. Annu Rev Biochem ,1998, 67:753–791.
[15] Massague J, Chen YG. Controlling TGF-beta signaling. Genes Dev, 2000, 14:627–644.
[16] Heldin CH, Miyazono K, Ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature, 1997, 390:465–471.
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