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,以探究其在肝星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罗格列酮组。用MTT法检测细胞的增殖情况;采用RTPCR方法检测其中PPARγ、TGF-β1及Ⅰ型前胶原mRNA表达;用Westernblot法检测PPARγ、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罗格列酮组或10μM罗格列酮组与3μM罗格列酮组或对照组相比,PPARγ 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);20μM罗格列酮组与10μM罗格列酮组之间,3μM罗格列酮组与对照组之间,PPART和Ⅰ型前胶原mRNA的表达差异无显著性(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—PCRI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:20μM罗格列酮组或10μM罗格列酮组与3μM罗格列酮组或对照组相比,α-SMA表达明显降低(P〈0.05)。20μM罗格列酮组与10μM罗格列酮组之间,3μM罗格列酮组与对照组之间,差异无显著性(P〉0.05)。(4)ELISA:20μM罗格列酮组或10μM罗格列酮组与3μM罗格列酮组或对照组相比,细胞的培养上清中Ⅰ型胶原表达明显降低(P〈0.01)。20μM罗格列酮组与10μM罗格列酮组之间,3μM罗格列酮组与对照组之间,差异无显著性(P〉0.05)。结论PPARγ配体罗格列酮能够在促进PPARγ的合成表达的同?
Objective To study the effect of rosiglitazone, a specific ligand of peroxisome proliferato-activated 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, 10μM rosiglitazone 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 Ⅰ pro-collagen was detected by RT-PCR. The expression of proteins of PPARγ, TGF-β1, type Ⅰ and Ⅲ collagen 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 ELISA. Results 1)The expression of PPARY 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 type Ⅰ 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 and 3 μM rosiglitazone group(P〉0.05). 2)The expression of proteins of PPARγ, TGF-β1 and typeⅠcollagen 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 20μM and 10 μM rosiglitazone group than that of control and 3 μrosiglitazone group(P〈0.05). There was no significant difference between 20 μM and 10 μM 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 20 μM and 10 μM rosiglitazone group markedly decreased than that in control and 3 μM rosiglitazone group(P〈0.01). There was no signific