摘要
目的研究Wnt3a对肝星状细胞（HSC）的增殖、活化以及转化生长因子（TGF）p／Smad表达的影响。方法重组Wnt3a刺激HSC后，用EDU法测定其对HSC增殖的影响；用Westemblot法检测HSC表达a-平滑肌肌动蛋白（a-SMA）、TGFD1、Smad3和Smad7的蛋白水平并比较四者之间的关系，多组间数据的比较用单因素方差分析，双变量相关分析研究数据间相关性。结果200ng／mlWnt3a刺激HSC24h后，其增殖率达到最大（63．00％±2．3嘶），显著高于未处理组、50ng／ml、100ng／ml及150ng／ml组（P值均〈0．05），而和250、300ng／ml组的差异无统计学意义（P值均〉0．05）；随着Wnt3a浓度的增加和刺激时间的延长，HSC表达a-SMA，TGFp1和Smad3的蛋白水平也随之升高，均在300ng／ml刺激48h后达到最大值（与3一磷酸甘油醛脱氢酶的灰度比值分别为1．0860±0．0101、1．0346土0．0118、1．0306士0．0122），而Smad7的蛋白水平却随之降低，在300ng／mlWnt3a刺激48h后达到最小（与GAPDH的灰度比值为0．7736±0．0139），差异均具有统计学意义俨值均〈0．05），并且a-SMA的蛋白表达与TGFD1、Smad3的蛋白表达呈正相关（r=0．968，JD〈0．05；r=0．997，P〈0．01），而和Smad7的蛋白表达呈负相关（r=0．960，P〈0．05）。结论重组Wnt3a能促进HSC的增殖、活化，并上调TGFp／Smad的表达，可能对肝纤维化的形成具有促进作用。
Objective To observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells （HSCs） and their the expression of the transforming growth factor beta （TGF[3） and/Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line. Methods Sychronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a （50, 100, 200, 250 and 300 ng/mL）. Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU （5-ethynyl-2- deoxyuridine） incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a ,and theEffects on the protein expression of TGFI3/Smad signaling factors was assessed by western blot detection （gray-value analysis） of alpha-smooth muscle actin （a-SMA）, a-SMA, TGFβ1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase （GAPDH） was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling. Results In general, HSCThe proliferation of hepatic stellate cells increased after the addition ofinresponse to Wnt3a stimulation for 24 h., reaching its peak atThe maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration （63.00±2.30%）, and it increased dramatically compared with those inwhich was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mL1 group P〈 0.05）, but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mL1 group,it had no obvious increase（P 〉 0.05）.; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of ct-SMA, TGFβ1, and Smad3 increased with the addition of Wnt3a and the extension of time. For all three, The maximal amount of increased protein expressiony all reached to thewas maximal produced by stimulation when hepatic stellate cells were treated bywith 300 ng/mL1 Wnt3a for 48 h hours,and the rations of（normalized gray- values：s of ct-SMA, 1.0860± 0.0101; TGF[31, 1.0346±0.0118; Smad3, to GAPDH were 1.0860±0.010 I, 1.0346± 0.0118, 1.0306± 0.0122）respectively. Howeverln contrast, the Wnt3a stimulation led to concentration- and time- depenent decreases in Smad7 expressionvaried inversely, with to them with the minimal ration of it to GAPDHthe maximal decrease occurring with 300 ng/mL Wnt3a for 48 h （0.7736±0.0139） after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, （P 〈 0.05）.There were positive correlations between ct-SMA expression and was found to be positively correlated to TGF[31, Smad3 （r = 0.968, P 〈 0.05） and; Smad3 （r = 0.997, P 〈 0.01）, but ct-SMA and Smad7 had negatively correlated to Smad7 ion（r = 0.960, P 〈 0.05）. Conclusion Wnt3a can increase thestimulates proliferation as well asand activation of ratthe hepatic stellate cellsHSCs, and upregulate modifies the expression of TGFβ/Smad signaling factors, of the hepatic stellate cells, andwhich may promote the hepatic fibrosis.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2013年第2期111-115,共5页
Chinese Journal of Hepatology
基金
国家自然科学基金（30972607）
关键词
肝硬化
转化生长因子D
肝星状细胞
重组Wnt3a
Liver cirrhosis
Transforming growth factor-beta
Hepatic stellate cells
RecombinantWnt3a
