内源性转化生长因子β1对人肝星状细胞胰岛素样生长因子结合蛋白相关蛋白1表达的影响

Effect of endogenous transforming growth factor β1 on expression of insulin-like growth factor binding protein related protein 1 with hepatic stellate cells in vitro

目的探讨内源性转化生长因子(TGF)β1对人肝星状细胞(HSC)胰岛素样生长因子结合蛋白相关蛋白1(IGFBPrP1)的表达的影响及其意义。方法将人肝星状细胞LX-2分为3组:空白对照组(加入等量无血清无抗生素的培养液)、阴性质粒组(转染阴性对照质粒)、TGFβ1过表达组(转染TGFβ1过表达质粒)。转染质粒后48 h,荧光显微镜检测质粒转染效率。转染质粒后72 h,采用RT-PCR法检测TGFβ1、CollagenⅠ、IGFBPrP1 mRNA的表达,采用Western印迹法检测TGFβ1、CollagenⅠ、IGFBPrP1蛋白的表达。采用单因素方差分析比较空白对照组、阴性质粒组、TGFβ1过表达组肝星状细胞TGFβ1、CollagenⅠ、IGFBPrP1 mRNA、蛋白相对表达量差异,进一步采用LSD-t检验进行组间两两比较。结果质粒可转染LX-2细胞,转染效率达40%。TGFβ1过表达组肝星状细胞TGFβ1、CollagenⅠ、IGFBPrP1 mRNA相对表达量分别为38.46±0.19、8.63±0.15、2.56±0.30,均较空白对照组的1明显上调,且差异均有统计学意义(t值分别为126.742、122.489、6.015,P均<0.05);较阴性质粒组的1.05±0.01、1.03±0.02、1.00±0.10也均明显上调,且差异也均有统计学意义(t值分别为124.97、121.176、6.015,P均<0.05)。TGFβ1过表达组肝星状细胞TGFβ1、CollagenⅠ、IGFBPrP1蛋白相对表达量分别为0.27±0.04、1.47±0.02、0.53±0.01,较空白对照组的0.20±0.01、0.76±0.03、0.25±0.02均明显上调,且差异均有统计学意义(t值分别为4.108、35.5、19.33,P均<0.05);较阴性质粒组的0.22±0.01、0.77±0.02、0.27±0.03也均明显上调,且差异也均有统计学意义(t值分别为2.988、35.0、18.388,P均<0.05)。结论内源性TGFβ1可引起肝星状细胞CollagenⅠmRNA及蛋白的表达增加,同时也可以促进IGFBPrP1 mRNA及蛋白的表达,推测IGFBPrP1可能作为TGFβ1的一个重要下游因子起到调控肝纤维化的作用。

Objective To identify if the endogenous transforming growth factor β1 （TGFβ1） can influence insulin-like growth factor binding protein related protein 1 （ IGFBPrP1 ） synthesis with hepatic stellate ceils in vitro. Methods LX-2 cell line was divided into three groups ： blank control group （ added equal amount of medium without serum and antibiotics ）, negative plasmid group （transfected negative plasmid）, TGFβ1 over-expression group （transfected TGFβ1 over-expression plasmid）. The transfection efficiency was observed by fluorescence microscopy 48h later. RT-PCR was used to determine the expression levels of mRNA and Western blot was used to determine the expression levels of protein of TGFβ1 ,collagen I and IGFBPrPI 72 h later. One-way analysis of variance was used to compared with the expression difference of mRNA and protein of the blank control group,negative plasmid group and TGFβ1 over-expression group. And LSD-t test was used to compared between two groups. Results The transfection was successful and the transfection efficiency was 40%. After TGFβ1 over-expression plasmid was transfected, the expression levels of mRNA of TGFβ1 ,collagen I,IGFBPrP1 were 38.46 ±0.19,8.63 ±0.15,2.56 ±0.30. The expression levels of mRNA were significantly higher than those in blank control group（ t = 126.742,122.489,6. 015, all P 〈 0. 05 ）and negative plasmid group（ t = 124.97,121. 176,6. 015, all P 〈 0.05） ; After TGF^I over-expression plasmid was transfected, the expression levels of protein of TGFI31, collagen I , IGFBPrP1 were 0. 27 -＋ 0. 04, 1.47-＋0.02,0.53-0.01. The expression levels of protein were significantly higher than those in blank control group（ t = 4. 108,35.5,19.33, all P 〈 0.05 ） and negative plasmid grou （ t = 2. 988,35.0,18. 388, all P 〈 0.05 ）. Conclusions Endogenous TGFβ1 can increase the synthesis of mRNA and protein of collagen I with LX-2 and also can promote the expression of mRNA and protein of IGFBPrP1. It is speculated that IGFBPrP1 may be as an important downstream factor of TGFIS1 to regulate the process of liver fibrosis.