The Effection Of TGFβ/Smad Cell Sigal Passway To The Expression Of VEGF/CTGF In Human Retinal Endothelial Cells

Mechanisms of retinal neovascularization and research of treatment has been the core of treating clinical retinal neovascularized diseases, current research focused on the pathway of signal transduction in retinal neovascularized disease such as VEGF,bFGF,TGF. But these research study only single level of signal transduction in the angiogenesis, so it is not enough to understand the whole cascade pathway. VEGF is located in downstream of angiogenesis signal transduction, but it is steal unclear that several signals or single signal eaxist in upstream of VEGF. Documents have reported that TGFβstimulate expression of VEGF protein and has many downstreams to regulate. So are there many upstream signals or single cascade to stimulate VEGF? And the precise regularity of regulation michanisma is still unclear.It is well known that Smad is one of signal in downstream pathway of TGFβ, the role of Smad in retinal neovascularization has been reported involved in the TGFβ-Smad-VEGF cascade and other signals. But this conclusion did not convinced because of the fact that TGFβcould stimulate or induce many downstream signals to promote expression of VEGF such as PKC,MAKP and so on. So it is important to investigate the role of single Smad cascade in retinal neovascularization in two parts that supporting clinical strategy or adding target to treat neovascularization. Part one The effection of TGFβstimulate expression of VEGF and CTGF in human retinal endothelial cellsObjective: to investigate the effection of TGFβstimulate the expression of P—Smad2, 3,VEGF and CTGF in human retinal endothelial cells.Methods: Recovery of human retinal endothelial cells was done at first, then cultured with ECM with TGFβ(10ng/ml) for different time group(1h,2h,61h,12h,24h), then investigate the expression of P—Smad2,3,VEGF,CTGF in human retinal endothelial cells when 1h, 2h, 6h, 12h, 24h after vaccination.Result: Expression of P—Smad2,3, VEGF and CTGF increased when adding TGFβ(10ng/ml), no significant difference of P-Smad2 expression was found between five groups,but significant difference was found in the expression of P-Smad3,VEGF and CTGF between groups. The maximum expression of VEGF occurred at 24h group after stimulating with TGFB(10ng/ml); The maximum expression of CTGF occurred at 2h group after stimulating with TGFβ(10ng/ml).Conclusion:1. TGFβcould stimulate the expression of P—Smad2,3,VEGF and CTGF in human retinal endothelial cells and neovascularization. 2. There is TGFβ/Smad singal pathway in human retinal endothelial cells which is activated by TGFβ.3. Time-dependent in the expression of P-Smad2 was not showed, but in the expression of P-Smad3,VEGF and CTGF has been showed.Part two Co-action relationship between Smad,VEGF and CTGFSection one Construction of Smad2,3 and VEGF Gene Expression VectorsObjective: Construction of Smad2,3 and VEGF Gene Expression Vectors.Methods: Extract total RNA from human fetal liver take the total RNA of hepatocyte as model, use a pair of synthetic primers, adopt RT-PCR technology to obtain Smad2, 3 and VEGF gene. Primer was used to modify the open reading frame of Smad2, 3 in order to complete fusion gene with myc-his. Purify and restriction PCR vector of tragated gene, then joint it with pcDNA3.1/myc-his(-)B,pcDNA3.1(-) gene expression vector.Result:The results of shininglake,PCR,restriction enzyme by ApaⅠand KpnⅠprove that the lengths of the clone of Smad2,3,VEGF cDNA are right.Alignment of Smad2,3,VEGF cDNA is same as that reported by GENE BANK.Conclusion:1. Clone the gene of samd2, 3,VEGF.2. Construction of pcDNA3.1/myc-his(-)B-Smad2, 3 Gene Expression Vector is stable and effective.Section two Relationship between Co-actions on Smad,VEGF and CTGFObjective: To investigate relationship between Co-actions on Smad,VEGF and CTGF.Methods: Construction of pcDNA3.1/myc-his(-)B-Smad2, 3,and pcDNA3.1/ VEGF Gene Expression Vector was made at first, then transfected into retinal vascular endothelial cells with pcDNA3.1/myc-his(-)B-Smad2, 3 and pcDNA3.1/ VEGF Gene by liposome respectively. Different groups were made as control group, liposome group, liposome shamed vector group and liposome+pcDNA3.1/myc-his(-)B-Smad2, 3 orpcDNA3.1/VEGF Gene Expression Vector group. Investigate the expression of VEGF,CTGF protein and mRNA stimulated by Smad2, 3 with Western—Blotting and Real—time PCR., and investigate the expression of Smad2, 3 and CTGF mRNA stimulate by VEGF with Real—time PCR respectively. Results:1. The expression of Smad2 mRNA increased to 41.4 times than control group after tranfecting pcDNA3.1/myc-his(-)B-Smad2, and the expression of CTGFmRNA increased to 2.2 times. But the expression of Smad3mRNA and VEGFmRNA decreased to 33.8 and 40.9 times. The expression of P-Smad2,CTGF protein increased,but the expression of VEGF protein decreased than control.2. The expression of Smad2 mRNA and Smad3 mRNAincreased to 2.2 and 46.1 times respectively, the expression of CTGF increased to 2.6times, but the expression of VEGFmRNA decreased to 27.6 times after tranfecting pcDNA3.1/myc-his(-)B-Smad3. The expression of P-Smad3,CTGF protein increased,but the expression of VEGF protein decreased than control.3. The expression of Smad2, 3 decreased to 7.8 and 19.9 time after transfecting pcDNA3.1/ VEGF Gene into retinal vascular endothelial cells; but the expression of VEGF and CTGF protein increased to 296.6 and 9.0 times.Conclusion:1. Construction of Gene Expression Vector is stable and effective to transfect retinal vascular endothelial cells successfully. The targeted gene could express after transfection finished. Smad signal pathway is actived. 2. Smad2, 3 could down-regulated the expression of VEGFmRNA and protein significantly.3. Smad2, 3 could promoted the expression of CTGFmRNA significantly.4. the role of Smad2,3 is similar to regulate the expression of VEGF and CTGF in human retinal vascular endothelial cells.5. VEGF could decreased the expression of Smad2, 3mRNA, but promote the expression of CTGF mRNA.