中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2008年
11期
786-789
,共4页
郭晏同%赵景明%柏楠%朱峰%周迈%焦岗军%钟朝辉%李涛%冷希圣
郭晏同%趙景明%柏楠%硃峰%週邁%焦崗軍%鐘朝輝%李濤%冷希聖
곽안동%조경명%백남%주봉%주매%초강군%종조휘%리도%랭희골
肝硬化%过氧化物酶体增殖物活化的受体%配体%罗格列酮%肝星状细胞
肝硬化%過氧化物酶體增殖物活化的受體%配體%囉格列酮%肝星狀細胞
간경화%과양화물매체증식물활화적수체%배체%라격렬동%간성상세포
Liver cirrhosis%Peroxisome proliferator-activated receptor gamma%Ligand%Rosiglitazone%Hepatic stellate cell
目的 研究不同浓度的过氧化物酶体增殖物活化的受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)特异配体罗格列酮对肝星状细胞(hepatic stellate cell,HSC)生物学特性的影响,以探究其在肝旱状细胞活化中的作用.方法 设立对照组,3μM罗格列酮组,10μM罗格列酮组,20μM岁格列酮组.用MTT法检测细胞的增殖情况;采用RT-PCR方法检测其中PPARγ、TGF-β1及Ⅰ型前胶原mRNA表达;用Western blot法检测PPARy、Ⅰ、Ⅲ型胶原及TGF-β1蛋白表达;用免疫细胞化学方法测定α-SMA表达的变化;ELISA法检测细胞培养上清中的Ⅰ型胶原表达的变化.结果 (1)RT-PCR:20μM罗格列嗣组或10μM罗格列酮组与3 μM罗格列酮组或对照组相比,PPARY mRNA表达显著增高(P＜0.01),Ⅰ型前胶原mRNA表达显著降低(P＜0.01);20 α-SMA罗格列酮组与10 α-SMA罗格列酮组之间,3 α-SMA罗格列酮组与对照组之间,PPARγ和Ⅰ型前胶原mRNA的表达差异无显著件(P＞0.05).而各组之间的TGF-β1 mRNA的差异无显著性意义(P＞0.05).(2)Western blot:PPARγ及TGF-β1蛋白表达所得结果与RT-PCR结果相一致.Ⅰ型胶原表达与RT-PCR Ⅰ型前胶原mRNA表达结果相一致.各组之间的Ⅲ型胶原表达差异无显著性意义(P＞0.05).(3)免疫细胞化学:20α-SMA罗格列酮组或10 α-SMA罗格列酮组与3 α-SMA罗格列酮组或对照组相比,α-SMA表达明显降低(P＜0.05).20 α-SMA岁格列酮组与10 α-SMA罗格列酮组之问,3 α-SMA罗格列酮组与对照组之间,差异无显著性(P＞0.05).(4)ELISA:20 α-SMA罗格列酮组或10 α-SMA罗格列酮组与3α-SMA罗格列倒组或对照组相比,细胞的培养上清中Ⅰ型胶原表达明显降低(P＜0.01).20 α-SMA罗格列酮组与10 α-SMA罗格列酮组之问,3 α-SMA罗格列酬组与对照组之间,差异无显著性(P＞0.05).结论 PPAR?配体罗格列酬能够在促进PPAR?的合成表达的同时,抑制细胞的增殖及胶原合成,抑制α-SMA的表达,减少细胞分泌Ⅰ型胶原,对肝星状细胞的活化有明显的抑制作用.
目的 研究不同濃度的過氧化物酶體增殖物活化的受體γ(peroxisome proliferator-activated receptor gamma,PPARγ)特異配體囉格列酮對肝星狀細胞(hepatic stellate cell,HSC)生物學特性的影響,以探究其在肝旱狀細胞活化中的作用.方法 設立對照組,3μM囉格列酮組,10μM囉格列酮組,20μM歲格列酮組.用MTT法檢測細胞的增殖情況;採用RT-PCR方法檢測其中PPARγ、TGF-β1及Ⅰ型前膠原mRNA錶達;用Western blot法檢測PPARy、Ⅰ、Ⅲ型膠原及TGF-β1蛋白錶達;用免疫細胞化學方法測定α-SMA錶達的變化;ELISA法檢測細胞培養上清中的Ⅰ型膠原錶達的變化.結果 (1)RT-PCR:20μM囉格列嗣組或10μM囉格列酮組與3 μM囉格列酮組或對照組相比,PPARY mRNA錶達顯著增高(P＜0.01),Ⅰ型前膠原mRNA錶達顯著降低(P＜0.01);20 α-SMA囉格列酮組與10 α-SMA囉格列酮組之間,3 α-SMA囉格列酮組與對照組之間,PPARγ和Ⅰ型前膠原mRNA的錶達差異無顯著件(P＞0.05).而各組之間的TGF-β1 mRNA的差異無顯著性意義(P＞0.05).(2)Western blot:PPARγ及TGF-β1蛋白錶達所得結果與RT-PCR結果相一緻.Ⅰ型膠原錶達與RT-PCR Ⅰ型前膠原mRNA錶達結果相一緻.各組之間的Ⅲ型膠原錶達差異無顯著性意義(P＞0.05).(3)免疫細胞化學:20α-SMA囉格列酮組或10 α-SMA囉格列酮組與3 α-SMA囉格列酮組或對照組相比,α-SMA錶達明顯降低(P＜0.05).20 α-SMA歲格列酮組與10 α-SMA囉格列酮組之問,3 α-SMA囉格列酮組與對照組之間,差異無顯著性(P＞0.05).(4)ELISA:20 α-SMA囉格列酮組或10 α-SMA囉格列酮組與3α-SMA囉格列倒組或對照組相比,細胞的培養上清中Ⅰ型膠原錶達明顯降低(P＜0.01).20 α-SMA囉格列酮組與10 α-SMA囉格列酮組之問,3 α-SMA囉格列酬組與對照組之間,差異無顯著性(P＞0.05).結論 PPAR?配體囉格列酬能夠在促進PPAR?的閤成錶達的同時,抑製細胞的增殖及膠原閤成,抑製α-SMA的錶達,減少細胞分泌Ⅰ型膠原,對肝星狀細胞的活化有明顯的抑製作用.
목적 연구불동농도적과양화물매체증식물활화적수체γ(peroxisome proliferator-activated receptor gamma,PPARγ)특이배체라격렬동대간성상세포(hepatic stellate cell,HSC)생물학특성적영향,이탐구기재간한상세포활화중적작용.방법 설립대조조,3μM라격렬동조,10μM라격렬동조,20μM세격렬동조.용MTT법검측세포적증식정황;채용RT-PCR방법검측기중PPARγ、TGF-β1급Ⅰ형전효원mRNA표체;용Western blot법검측PPARy、Ⅰ、Ⅲ형효원급TGF-β1단백표체;용면역세포화학방법측정α-SMA표체적변화;ELISA법검측세포배양상청중적Ⅰ형효원표체적변화.결과 (1)RT-PCR:20μM라격렬사조혹10μM라격렬동조여3 μM라격렬동조혹대조조상비,PPARY mRNA표체현저증고(P＜0.01),Ⅰ형전효원mRNA표체현저강저(P＜0.01);20 α-SMA라격렬동조여10 α-SMA라격렬동조지간,3 α-SMA라격렬동조여대조조지간,PPARγ화Ⅰ형전효원mRNA적표체차이무현저건(P＞0.05).이각조지간적TGF-β1 mRNA적차이무현저성의의(P＞0.05).(2)Western blot:PPARγ급TGF-β1단백표체소득결과여RT-PCR결과상일치.Ⅰ형효원표체여RT-PCR Ⅰ형전효원mRNA표체결과상일치.각조지간적Ⅲ형효원표체차이무현저성의의(P＞0.05).(3)면역세포화학:20α-SMA라격렬동조혹10 α-SMA라격렬동조여3 α-SMA라격렬동조혹대조조상비,α-SMA표체명현강저(P＜0.05).20 α-SMA세격렬동조여10 α-SMA라격렬동조지문,3 α-SMA라격렬동조여대조조지간,차이무현저성(P＞0.05).(4)ELISA:20 α-SMA라격렬동조혹10 α-SMA라격렬동조여3α-SMA라격렬도조혹대조조상비,세포적배양상청중Ⅰ형효원표체명현강저(P＜0.01).20 α-SMA라격렬동조여10 α-SMA라격렬동조지문,3 α-SMA라격렬수조여대조조지간,차이무현저성(P＞0.05).결론 PPAR?배체라격렬수능구재촉진PPAR?적합성표체적동시,억제세포적증식급효원합성,억제α-SMA적표체,감소세포분비Ⅰ형효원,대간성상세포적활화유명현적억제작용.
Objective To study the effect of rosiglitazone,a specific ligand of peroxisome prolif
erator-activatcd receptor gamma (PPARγ),on the biological characters of activation hepatic stellate cells (HSCs).Methods The activated HSCs were divided into four groups:control,3 μM rosiglitazone group,10 μM rosigtitazone group and 20μM rosiglitazone group.The cell proliferation was determined with MTT colorimetric assay.The expression at mRNA level of PPARγ,TGF-β1,and type Ⅰpro-collagen was detected by RT-PCR.The expression of proteins of PPARγ,TGF-β1,type Ⅰ and Ⅲcollagen was detected by Western blot.α-smooth muscle actin (α-SMA) of HSCs was detected with immunocytochemistry.Type Ⅰ collagen in the supernatant of cultured HSCs was measured by EIASA.Results 1)The expression of PPARγ at mRNA level markedly increased in HSCs of 20 μM and 10 μM rosiglitazone group than 3 μM and control group (P＜0.01),while typeⅠ pro-collagen decreased in HSCs of 20 μM and 10 μM rosiglitazone group (P＜0.01).The difference of the expression of PPARγ and type Ⅰ pro-collagen at mRNA level was not significant between 10 μM rosiglitazone group and 20 μmol/L rosiglitazone group(P＞0.05).Neither was it between control and3 μM rosiglitazone group(P＞0.05).2)The expression of proteins of PPART,TGF-β1 and type Ⅰ collagen was accordant with that of mRNA.The expression of type Ⅲ was not significantly different among the 4 groups(P＞0.05).3)The staining of α-SMA in HSCs markedly decreased in 20 μM and 10 μM rosiglitazone group than that of control and 3 μM rosiglitazone group(P＜0.05).There was no significant difference between 20μM and 10μM rosiglitazone group.Neither was it between 10μM rosiglitazone group and control group(P＞0.05).4)Type Ⅰ collagen in the supernatant of cultured HSCs in 20μM and 10 μM rosiglitazone group markedly decreased than that in control and 3 μM rosiglitazone group(P＜0.01).There was no significant difference between 20μM and 10μM rosiglitazone group.Neither was it between 10μM rosiglitazone group and control group(P＞0.05).Conclusion By increasing expression of PPARγ in activated HSCs,roziglitazone,a specific agonist of PPARγ,decreases α-SMA expression and collagen synthesis,inhibits proliferation and attenuates the production of type Ⅰ collagen.These results demonstrated that the activation of HSCs can be suppressed.
