中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2009年
2期
101-104
,共4页
刘珺%徐选福%王兴鹏%牛培勤%杨文娟%郭传勇
劉珺%徐選福%王興鵬%牛培勤%楊文娟%郭傳勇
류군%서선복%왕흥붕%우배근%양문연%곽전용
Hedgehog蛋白质%肝硬化%实验性%肝星状细胞
Hedgehog蛋白質%肝硬化%實驗性%肝星狀細胞
Hedgehog단백질%간경화%실험성%간성상세포
Hedgehog proteins: Liver cirrhosis%experimental: Hepatic stellate cells
目的:探讨Hedgehog通路与肝纤维化及肝星状细胞(HSC)活化的关系.方法:清洁级SD雄性大鼠20只,均分为模型组和对照组.模型组采用腹腔注射四氯化碳(CCl4)和高脂饮食诱导肝纤维化,对照组予以正常饮食.第8周末取模型组存活大鼠与对照组中大鼠各5只处死,取左叶肝脏组织.HE、Masson染色观察两组肝组织病理变化;逆转录-多聚酶链反应(RT-PCR)检测纤维化大鼠肝脏中表达Hedgehog通路成员超音速Hedgehog信号通路(Shh)、膜受体patched(Ptc)、smoothened(Smo)和核转录因子Gli表达;实时荧光定量PCR法检测Hedgehog通路成员及HSC活化标志基因α-平滑肌肌动蛋白(α-SMA)mRNA在两组大鼠肝脏中的表达差异.体外培养HSC-T6细胞,RT-PCR检测HSC-T6细胞株中Hedgehog通路成员的表达;四甲基偶氮唑盐比色分析(MTT)法检测不同浓度环耙明(Cyclopamine,Cyc)对HSC-T6增殖的影响;分别用0、100/μmol/L的Cye干预HSC-T6,实时荧光定量PCR法检测Shh、Smo、Ptc、Gli-1及αSMA mRNA表达差异.结果:模型组大鼠肝脏有大量脂质及胶原沉积,且肝脏组织中均有Shh、Smo、Ptc、Gli-1表达.荧光定量PCR结果示模型组大鼠Shh、Smo、Gli-1及αSMA mRNA表达均较对照大鼠升高(20.45±3.31、12.78±0.53、10.88±2.41、4.91±2.59比1;P值均<0.05).Cyc在体外对HSC-T6有明显的抑制作用,且抑制作用呈剂量依赖性(F=636.81,P<0.01).荧光定量PCR结果示,用Cyc 100 μmol/L干预的HSC-T6中,Ptc、Smo、Gli-1和αSMA表达量分别为0.20±0.11、0.21±0.08、0.28±0.05和0.27±0.10,与Cyc 0μmol/L干预比较差异均有统计学意义(P值均<0.01).结论:肝纤维化过程中Hedgehog通路成员表达增高,抑制Hedgehog通路可抑制HSC活化,推测Hedgehog通路通过活化HSC促进肝纤维化的发生.
目的:探討Hedgehog通路與肝纖維化及肝星狀細胞(HSC)活化的關繫.方法:清潔級SD雄性大鼠20隻,均分為模型組和對照組.模型組採用腹腔註射四氯化碳(CCl4)和高脂飲食誘導肝纖維化,對照組予以正常飲食.第8週末取模型組存活大鼠與對照組中大鼠各5隻處死,取左葉肝髒組織.HE、Masson染色觀察兩組肝組織病理變化;逆轉錄-多聚酶鏈反應(RT-PCR)檢測纖維化大鼠肝髒中錶達Hedgehog通路成員超音速Hedgehog信號通路(Shh)、膜受體patched(Ptc)、smoothened(Smo)和覈轉錄因子Gli錶達;實時熒光定量PCR法檢測Hedgehog通路成員及HSC活化標誌基因α-平滑肌肌動蛋白(α-SMA)mRNA在兩組大鼠肝髒中的錶達差異.體外培養HSC-T6細胞,RT-PCR檢測HSC-T6細胞株中Hedgehog通路成員的錶達;四甲基偶氮唑鹽比色分析(MTT)法檢測不同濃度環耙明(Cyclopamine,Cyc)對HSC-T6增殖的影響;分彆用0、100/μmol/L的Cye榦預HSC-T6,實時熒光定量PCR法檢測Shh、Smo、Ptc、Gli-1及αSMA mRNA錶達差異.結果:模型組大鼠肝髒有大量脂質及膠原沉積,且肝髒組織中均有Shh、Smo、Ptc、Gli-1錶達.熒光定量PCR結果示模型組大鼠Shh、Smo、Gli-1及αSMA mRNA錶達均較對照大鼠升高(20.45±3.31、12.78±0.53、10.88±2.41、4.91±2.59比1;P值均<0.05).Cyc在體外對HSC-T6有明顯的抑製作用,且抑製作用呈劑量依賴性(F=636.81,P<0.01).熒光定量PCR結果示,用Cyc 100 μmol/L榦預的HSC-T6中,Ptc、Smo、Gli-1和αSMA錶達量分彆為0.20±0.11、0.21±0.08、0.28±0.05和0.27±0.10,與Cyc 0μmol/L榦預比較差異均有統計學意義(P值均<0.01).結論:肝纖維化過程中Hedgehog通路成員錶達增高,抑製Hedgehog通路可抑製HSC活化,推測Hedgehog通路通過活化HSC促進肝纖維化的髮生.
목적:탐토Hedgehog통로여간섬유화급간성상세포(HSC)활화적관계.방법:청길급SD웅성대서20지,균분위모형조화대조조.모형조채용복강주사사록화탄(CCl4)화고지음식유도간섬유화,대조조여이정상음식.제8주말취모형조존활대서여대조조중대서각5지처사,취좌협간장조직.HE、Masson염색관찰량조간조직병리변화;역전록-다취매련반응(RT-PCR)검측섬유화대서간장중표체Hedgehog통로성원초음속Hedgehog신호통로(Shh)、막수체patched(Ptc)、smoothened(Smo)화핵전록인자Gli표체;실시형광정량PCR법검측Hedgehog통로성원급HSC활화표지기인α-평활기기동단백(α-SMA)mRNA재량조대서간장중적표체차이.체외배양HSC-T6세포,RT-PCR검측HSC-T6세포주중Hedgehog통로성원적표체;사갑기우담서염비색분석(MTT)법검측불동농도배파명(Cyclopamine,Cyc)대HSC-T6증식적영향;분별용0、100/μmol/L적Cye간예HSC-T6,실시형광정량PCR법검측Shh、Smo、Ptc、Gli-1급αSMA mRNA표체차이.결과:모형조대서간장유대량지질급효원침적,차간장조직중균유Shh、Smo、Ptc、Gli-1표체.형광정량PCR결과시모형조대서Shh、Smo、Gli-1급αSMA mRNA표체균교대조대서승고(20.45±3.31、12.78±0.53、10.88±2.41、4.91±2.59비1;P치균<0.05).Cyc재체외대HSC-T6유명현적억제작용,차억제작용정제량의뢰성(F=636.81,P<0.01).형광정량PCR결과시,용Cyc 100 μmol/L간예적HSC-T6중,Ptc、Smo、Gli-1화αSMA표체량분별위0.20±0.11、0.21±0.08、0.28±0.05화0.27±0.10,여Cyc 0μmol/L간예비교차이균유통계학의의(P치균<0.01).결론:간섬유화과정중Hedgehog통로성원표체증고,억제Hedgehog통로가억제HSC활화,추측Hedgehog통로통과활화HSC촉진간섬유화적발생.
Objective To investigate the role of Hedgehog pathway in hepatic fibrosis and its association with activation of hepatic stellate cells. Methods Twenty male Spragur-Dawley rats were divided into control and model groups with 10 each. The animal models were induced by injection with CCl4 and fed with fat-rich diet. The rats in both groups were sacrified at the 8 week with 5 each and the liver tissues were removed for HSC-T6 culture. The deposition of collagen fiber in liver was detected with HE and Masson staining. RT-PCR was used to detect the expressions of Sonic hedgehog (Shh), smoothened (Smo), patched (Ptc), Gli-1 and α-smooth muscle action (α-SMA) mRNA in HSC-T6 and liver tissues. The influence of cyclopamine (Cyc) and lipopolysaccharide (LPS) on HSC-T6 proliferation were assayed by MTT. The expressions of Shh, Smo, Ptc, Gli-1 and α-SMA mRNA after intervention with Cyc (100μmol/L) and LPS were measured by real-time PCR. Results A lot of lipo and collagen deposited in liver of model rats. The Shh,Smo,Gli-1 and α-SMA mRNA were highly expressed in model rats than those in control group (2-△△Ct were 20.45±3.31 vs. 1, 12.78 ± 0. 53 vs. 1, 10.88 ± 2.41 vs. 1, 4.91 ± 2. 59 vs. 1, respectively, all P value <0. 05). In vitro Cyc inhibited HSC-T6 proliferation in dose dependant manner (F=636.81, P<0.01). Compared to the control group, the mRNA expressions of Smo, Ptc, Gli-1,α-SMA in HSC-T6 were significantly reduced after Cyc intervention (2△△Ct, were 0. 20±0. 11, 0. 21 ± 0. 08, 0. 28 ± 0. 05,0. 27±0.10,respectively, all P values<0.01). Conclusion The expression of members of Hedgehog pathway are increased in the progress of hepatic fibrosis, which may accelerate the hepatiee fibrosis by activating HSC.