NogoB-shRNA表达质粒的构建及其在大鼠原代肝星状细胞收缩中的作用
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摘要
目的构建针对大鼠NogoB基因的shRNA表达质粒,并初步探究其在大鼠肝星状细胞(HSCs)收缩功能中的作用。方法设计并合成针对大鼠NogoB基因3个不同位点的shRNA,通过DNA重组技术,将shRNA插入pSuper质粒中,构建NogoB-shRNA重组质粒,转染至大鼠原代肝星状细胞(HSCs)中,并通过Realtime PCR技术检测基因的相对表达量,筛选出能沉默大鼠NogoB基因的重组质粒,再通过Western blot加以验证,同时观察NogoB基因被抑制后,HSCs中内皮素1(Endothelin-1,ET-1)受体A、受体B(ETA、ETB)表达水平的变化。结果构建的3对重组质粒中,NogoB-shRNA2对NogoB的基因的表达具有明显的抑制作用。NogoB基因被抑制后,大鼠HSCs中ETA的表达无明显变化,ETB的表达升高,ETA/ETB的水平降低。结论成功构建了有效、特异的抑制大鼠NogoB基因表达的shRNA表达质粒,并初步观察到NogoB在肝星状细胞的收缩中可能有一定作用。
Objective To construct shRNA expressing plasmid inhibiting rat NogoB and to observe its possible effect on rat primary hepatic stellate cells(HSCs)contraction.Methods Three pairs of shRNAs targeting different sequence of rat NogoB were designed and constructed into pSuper plasmid by DNA recombination technique.Culture-activated HSCs were transfected with NogoB-shRNA plasmids to scan the effective plasmid which could inhibit NogoB gene expression by Real-time PCR.And this depressant effect was also confirmed with Western blot.After NogoB was knocked-down effectively,ETAand ETB mRNA expression were assessed by Real-time PCR.Results Among the three pairs of recombinant plasmids,NogoB-shRNA2plasmid could inhibit NogoB expression specifically.In HSCs,NogoB knockdown decreased the ratio of ETAand ETB.Conclusion We constructed specific NogoB-shRNA expression plasmid successfully which might be involved in contraction of HSCs.
Objective To construct shRNA expressing plasmid inhibiting rat NogoB and to observe its possible effect on rat primary hepatic stellate cells(HSCs)contraction.Methods Three pairs of shRNAs targeting different sequence of rat NogoB were designed and constructed into pSuper plasmid by DNA recombination technique.Culture-activated HSCs were transfected with NogoB-shRNA plasmids to scan the effective plasmid which could inhibit NogoB gene expression by Real-time PCR.And this depressant effect was also confirmed with Western blot.After NogoB was knocked-down effectively,ETAand ETB mRNA expression were assessed by Real-time PCR.Results Among the three pairs of recombinant plasmids,NogoB-shRNA2plasmid could inhibit NogoB expression specifically.In HSCs,NogoB knockdown decreased the ratio of ETAand ETB.Conclusion We constructed specific NogoB-shRNA expression plasmid successfully which might be involved in contraction of HSCs.
引文
1 Acevedo L,Yu J,Erdjument-Bromage H,et al.A new role for Nogo as a regulator of vascular remodeling.Nat Med,2004;10(4):382-388.
2 McKerracher L,Winton MJ.Nogo on the go.Neuron,2002;36(3):345-348.
3 Wang M,Han Y,Zhang XP,et al.Nogo,a star protein in reticulon family.Neurosci Bull,2006;22(3):183-186.
4 Zhang D,Utsumi T,Huang HC,et al.Reticulon 4B(Nogo-B)is a novel regulator of hepatic fibrosis.Hepatology,2011;53(4):1306-1315.
5 Xu W,Hong W,Shao Y,et al.Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3and increasing MYL-9expression.Respir Res,2011;12 :14.doi:10.1186/1465-9921-12-14.
6 Liu XJ,Yang L,Luo F,et al.Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray.World J Gastroenterol,2004;10(11):1600-1607.
7 Reynolds A,Leake D,Boese Q,et al.Rational siRNA design for RNA interference.Nat Biotechnol,2004;22(3):326-330.
8 Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.Nucleic Acids Res,2004;32(3):936-948.
9 Takashimizu S,Kojima S,Nishizaki Y,et al.Effect of endothelin A receptor antagonist on hepatic hemodynamics in cirrhotic rats.Implications for endothelin-1 in portal hypertension.Tokai J Exp Clin Med,2011;36(2):37-43.
10 Seo YS,Shah VH.Pathophysiology of portal hypertension and its clinical links.J Clin Exp Hepatol,2011;1(2):87-93.
11 Hellerbrand C.Hepatic stellate cells——the pericytes in the liver.Pflügers Arch,2013;465(6):775-778.
2 McKerracher L,Winton MJ.Nogo on the go.Neuron,2002;36(3):345-348.
3 Wang M,Han Y,Zhang XP,et al.Nogo,a star protein in reticulon family.Neurosci Bull,2006;22(3):183-186.
4 Zhang D,Utsumi T,Huang HC,et al.Reticulon 4B(Nogo-B)is a novel regulator of hepatic fibrosis.Hepatology,2011;53(4):1306-1315.
5 Xu W,Hong W,Shao Y,et al.Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3and increasing MYL-9expression.Respir Res,2011;12 :14.doi:10.1186/1465-9921-12-14.
6 Liu XJ,Yang L,Luo F,et al.Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA mircoarray.World J Gastroenterol,2004;10(11):1600-1607.
7 Reynolds A,Leake D,Boese Q,et al.Rational siRNA design for RNA interference.Nat Biotechnol,2004;22(3):326-330.
8 Ui-Tei K,Naito Y,Takahashi F,et al.Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.Nucleic Acids Res,2004;32(3):936-948.
9 Takashimizu S,Kojima S,Nishizaki Y,et al.Effect of endothelin A receptor antagonist on hepatic hemodynamics in cirrhotic rats.Implications for endothelin-1 in portal hypertension.Tokai J Exp Clin Med,2011;36(2):37-43.
10 Seo YS,Shah VH.Pathophysiology of portal hypertension and its clinical links.J Clin Exp Hepatol,2011;1(2):87-93.
11 Hellerbrand C.Hepatic stellate cells——the pericytes in the liver.Pflügers Arch,2013;465(6):775-778.