中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
2期
111-115
,共5页
王燕平%贺琪%吴飞%朱兰兰%刘卫%张雅楠%贺永文
王燕平%賀琪%吳飛%硃蘭蘭%劉衛%張雅楠%賀永文
왕연평%하기%오비%주란란%류위%장아남%하영문
肝硬化%转化生长因子β%肝星状细胞%重组Wnt3a
肝硬化%轉化生長因子β%肝星狀細胞%重組Wnt3a
간경화%전화생장인자β%간성상세포%중조Wnt3a
Liver cirrhosis%Transforming growth factor-beta%Hepatic stellate cells%Recombinant Wnt3a
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)β/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Westem blot法检测HSC表达α-平滑肌肌动蛋白(α-SMA)、TGFβ 1、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 200 ng/ml Wnt3a刺激HSC24h后,其增殖率达到最大(63.00％±2.30％),显著高于未处理组、50 ng/ml、100 ng/ml及150 ng/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α-SMA、TGFβ1和Smad3的蛋白水平也随之升高,均在300 ng/ml刺激48h后达到最大值(与3-磷酸甘油醛脱氢酶的灰度比值分别为1.0860±0.0101、1.0346±0.0118、1.0306±0.0122),而Smad7的蛋白水平却随之降低,在300ng/ml Wnt3a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(P值均＜0.05),并且α-SMA的蛋白表达与TGFβ 1、Smad3的蛋白表达呈正相关(r=0.968,P＜ 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TGF β/Smad的表达,可能对肝纤维化的形成具有促进作用.
目的 研究Wnt3a對肝星狀細胞(HSC)的增殖、活化以及轉化生長因子(TGF)β/Smad錶達的影響. 方法 重組Wnt3a刺激HSC後,用EDU法測定其對HSC增殖的影響;用Westem blot法檢測HSC錶達α-平滑肌肌動蛋白(α-SMA)、TGFβ 1、Smad3和Smad7的蛋白水平併比較四者之間的關繫,多組間數據的比較用單因素方差分析,雙變量相關分析研究數據間相關性.結果 200 ng/ml Wnt3a刺激HSC24h後,其增殖率達到最大(63.00％±2.30％),顯著高于未處理組、50 ng/ml、100 ng/ml及150 ng/ml組(P值均＜0.05),而和250、300ng/ml組的差異無統計學意義(P值均＞0.05)；隨著Wnt3a濃度的增加和刺激時間的延長,HSC錶達α-SMA、TGFβ1和Smad3的蛋白水平也隨之升高,均在300 ng/ml刺激48h後達到最大值(與3-燐痠甘油醛脫氫酶的灰度比值分彆為1.0860±0.0101、1.0346±0.0118、1.0306±0.0122),而Smad7的蛋白水平卻隨之降低,在300ng/ml Wnt3a刺激48h後達到最小(與GAPDH的灰度比值為0.7736±0.0139),差異均具有統計學意義(P值均＜0.05),併且α-SMA的蛋白錶達與TGFβ 1、Smad3的蛋白錶達呈正相關(r=0.968,P＜ 0.05;r=0.997,P＜0.01),而和Smad7的蛋白錶達呈負相關(r=0.960,P＜0.05).結論 重組Wnt3a能促進HSC的增殖、活化,併上調TGF β/Smad的錶達,可能對肝纖維化的形成具有促進作用.
목적 연구Wnt3a대간성상세포(HSC)적증식、활화이급전화생장인자(TGF)β/Smad표체적영향. 방법 중조Wnt3a자격HSC후,용EDU법측정기대HSC증식적영향;용Westem blot법검측HSC표체α-평활기기동단백(α-SMA)、TGFβ 1、Smad3화Smad7적단백수평병비교사자지간적관계,다조간수거적비교용단인소방차분석,쌍변량상관분석연구수거간상관성.결과 200 ng/ml Wnt3a자격HSC24h후,기증식솔체도최대(63.00％±2.30％),현저고우미처리조、50 ng/ml、100 ng/ml급150 ng/ml조(P치균＜0.05),이화250、300ng/ml조적차이무통계학의의(P치균＞0.05)；수착Wnt3a농도적증가화자격시간적연장,HSC표체α-SMA、TGFβ1화Smad3적단백수평야수지승고,균재300 ng/ml자격48h후체도최대치(여3-린산감유철탈경매적회도비치분별위1.0860±0.0101、1.0346±0.0118、1.0306±0.0122),이Smad7적단백수평각수지강저,재300ng/ml Wnt3a자격48h후체도최소(여GAPDH적회도비치위0.7736±0.0139),차이균구유통계학의의(P치균＜0.05),병차α-SMA적단백표체여TGFβ 1、Smad3적단백표체정정상관(r=0.968,P＜ 0.05;r=0.997,P＜0.01),이화Smad7적단백표체정부상관(r=0.960,P＜0.05).결론 중조Wnt3a능촉진HSC적증식、활화,병상조TGF β/Smad적표체,가능대간섬유화적형성구유촉진작용.
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(TGFβ)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 TGFβ/Smad signaling factors was assessed by western blot detection(gray-value analysis)of alpha-smooth muscle actin(α-SMA),α-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 ofin response 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/mLl group,it had no obvious increase(P ＞ 0.05).;The Wnt3a stimulation also led to time-dependent increases in the protein expressions of α-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/mLl Wnt3a for 48 h hours,and the rations of(normalized gray-values:s of α-SMA,1.0860 ± 0.0101;TGFβ1,1.0346 ± 0.0118;Smad3,to GAPDH were 1.0860 ± 0.0101,1.0346 ± 0.0118,1.0306 ± 0.0122)respectively.HoweverIn 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 α-SMA expression and was found to be positively correlated to TGFβ1,Smad3(r =0.968,P ＜ 0.05)and;Smad3(r =0.997,P ＜ 0.01),but α-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.
