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重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响
引用本文:周霞,余姣,李琪,钱伟,徐可树. 重组转化生长因子β3基因对大鼠肝星状细胞Ⅰ型胶原表达的影响[J]. 中华肝脏病杂志, 2008, 16(1): 43-48
作者姓名:周霞  余姣  李琪  钱伟  徐可树
作者单位:华中科技大学同济医学院附属协和医院消化内科,武汉,430022
基金项目:国家自然科学基金(30570820)
摘    要:目的观察转化生长因子D3基因(TGFβ3)对大鼠肝星状细胞株(HSC—T6)Ⅰ型胶原合成的影响。方法TGFβ3表达质粒[pcDNA3.1(+)-TGFβ31和TGFβ1表达质粒[pcDNA3.1(+)-TGFD11的构建。通过脂质体介导方法,将pcDNA3.1(+)-TGFβ1、pcDNA3.1(+)-TGFβ3分别及共同转染体外培养的HSC—T6细胞,荧光定量PCR法及Westernblot法分别检测转染后TGFβ1、TGFD3、Ⅰ型胶原mRNA及蛋白质的表达。将pcDNA3.1(+)-TGFD1转染HSC—T6细胞,经G418筛选建立高表达TGFD1的HSC~T6细胞克隆,pcDNA3.1(+)-TGFD3转染克隆细胞,荧光定量PCR法检测转染后TGFβ3、TGFβ1及Ⅰ型胶原mRNA的表达,Westernblot法检测TGFβ1、Ⅰ型胶原蛋白的表达情况。结果构建的pcDNA3.1(+)TGFD3、pcDNA3.1(+)-TGFD1质粒可转染HSC—T6细胞,转染率28.2%。pcDNA3.1(+)TGF侈3转染细胞后,Ⅰ型胶原mRNA及蛋白的表达较空白组及对照组增加,以72h增高最为明显(P〈0.05);共转染组Ⅰ型胶原mRNA及蛋白质的表达较pcDNA3.1(+)-TGFβ1转染组明显降低(P〈0.05)。TGF侈3转染克隆细胞后,TGFD1mRNA表达较克隆组无明显改变(P〉0.05),而蛋白质表达明显下降(P〈0.05),Ⅰ型胶原mRNA及蛋白质表达均较克隆组明显降低(P〈0.05)。结论TGFD3基因转染正常培养的HSC—T6细胞,增加Ⅰ型胶原的表达;转染高表达TGFβ1的克隆组HSC—T6细胞,Ⅰ型胶原表达明显降低,提示TGFβ3对肝纤维化的发生有抑制作用。

关 键 词:转化生长因子β3 肝星状细胞 Ⅰ型胶原
收稿时间:2007-07-23

Effects of transforming growth factor-beta 3 gene transfer on type I collagen synthesis of hepatic stellate cells
ZHOU Xia,YU Jiao,LI Qi,QIAN Wei,XU Ke-shu. Effects of transforming growth factor-beta 3 gene transfer on type I collagen synthesis of hepatic stellate cells[J]. Chinese journal of hepatology, 2008, 16(1): 43-48
Authors:ZHOU Xia  YU Jiao  LI Qi  QIAN Wei  XU Ke-shu
Affiliation:Department of Gastroenterology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Abstract:OBJECTIVE: To observe the effects of transforming growth factor-beta 3 gene transfer on type I collagen synthesis of cells of HSC-T6. METHODS: Transforming growth factor-beta 1 expression plasmid and transforming growth factor-beta 3 expression plasmid were constructed. The recombinant expression plasmids pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 were respectively transfected and cotransfected into cultured HSC-T6 cells; expression of TGF betav1, TGF beta 3, type I collagen mRNA were detected by real-time quantitative PCR, expression of TGF beta 1 and type I collagen protein were detected by Western blot. The recombinant expression plasmid pcDNA3.1(+)-TGF beta 1 was transfected into cultured HSC-T6 cells; positive clones were selected by G418. The positive clones were transfected by the recombinant expression plasmid pcDNA3.1(+)-TGF beta 3; expression of TGF beta 1, TGF beta 3 and type I collagen mRNA were detected by real-time quantitative PCR; expression of TGF beta 1 and type I collagen protein were detected by Western blot. RESULTS: HSC-T6 was transfected by recombinant expression plasmid pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 and the transfection efficiency was 28.2%. After the cells were transfected with pcDNA3.1-TGF beta 3, type I collagen mRNA and the protein expression in the cells were higher than those in the untransfected cells (control group) (P < 0.05). The increase reached to the maximal at 72 h after the transfection. Expressions of type I collagen mRNA and the protein in the cells transfected by pcDNA3.1(+)-TGF beta 1 were higher than in those cotransfected by pcDNA3.1(+)-TGF beta 1 and pcDNA3.1(+)-TGF beta 3 (P < 0.05). TGF beta 1 protein, type I collagen mRNA and type I collagen protein expression significantly decreased in the clones transfected by recombinant expression plasmid pcDNA3.1(+)-TGF beta 3 (P < 0.05), but the changes of TGF beta 1 mRNA were not significant (P > 0.05). CONCLUSIONS: Expression of type I collagen increased after the cultured HSC-T6 cells were transfected by TGF beta 3 gene. The significant decrease of the expression of type I collagen of the TGF beta 3 gene transfected positive clones suggests that TGF beta 3 could inhibit the occurrence of liver fibrosis.
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