重组转化生长因子β3基因对大鼠肝纤维化细胞模型胶原合成及沉积的影响  

Influence of recombinant transforming growth factor-β3 on collagen synthesis and deposition：experiment with rat cell model of liver fibrosis

作　　者：李琪[1] 周霞[1] 余姣[1] 钱伟[1] 徐可树[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院消化内科,武汉430022

出　　处：《中华医学杂志》2008年第18期1273-1278,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目（30570820）

摘　　要：目的 观察重组转化生长因子艮（TGF-β3）基因对大鼠肝纤维化细胞模型胶原合成及沉积的影响。方法 （1）构建质粒pcDNA3．1（＋）-TGF-B]和pcDNA3．1（＋）·TGF-β1。（2）将pcDNA3．1（＋）-TGF-β1，转染大鼠肝星状细胞（HSC）-T6，经G418筛选建立稳定高表达TGF-β1的HSC-T6细胞阳性克隆。（3）pcDNA3．1（＋）-TGF-β3转染HSC-T6阳性细胞克隆，荧光实时定量PCR法检测TGF-β3 mRNA的表达；荧光实时定量PCR法及Western印迹法分别检测TGF-β1、I型胶原、基质金属蛋白酶（MMP）-2、MMP-9及TIMP-1的mRNA和蛋白表达情况。结果 （1）pcDNA3．1（＋）-TGF-β3和pcDNA3．1（＋）-TGF-β1质粒均可转染HSC-T6细胞，以转染48h时转染效率最高，达28．2％。（2）pcDNA3．1（＋）-TGF-β3转染HSC-T6细胞阳性克隆后，与单纯阳性克隆组相比，TGF-β1，mRNA表达无明显改变，而蛋白表达明显低（0．48±0．07 vs 0．66±0．14，P=0．023）；I型胶原mRNA及蛋白的表达明显低（mRNA：7．2±1．0 vs 13．7±0．4，P=0．010，蛋白：0．45±0．21 vs 0．82±0．13，P=0．041），MMP-2 mRNA及蛋白较低，但差异无统计学意义（均P〉0．05），MMP-9mRNA及蛋白表达明显多（均P〈0．05），TIMP-1mRNA及蛋白表达明显低（均P〈0．05）。结论 （1）重组TGF-β3真核表达载体构建正确，在转染阳性克隆细胞48h后获得高效表达；（2）稳定高表达TGF-β1，的HSC-T6细胞阳性克隆可作为肝纤维化细胞模型；（3）pcDNA3．1（＋）-TGF-β3转染阳性克隆能减少胶原合成，并通过调节基质金属蛋白酶及其抑制剂的表达对胶原沉积起抑制作用。Objective To investigate the influence of recombinant transforming growth factor-β3 （TGF-β3 ） on collagen synthesis and deposition. Methods Plasmids pcDNA3. 1 （ ＋ ）-TGF-β3 and pcDNA3.1 （ ＋ ） -TGF-β1 were constructed. Rat hepatic stellate cells （ HSCs ） of the strain HSC-T6 were cultured as cell model of fibrosis and divided into 4 groups： blank control group, pcDNA3.1- enhanced green fluorescent protein （EGFP） -transfected group （ negative control group）, pcDNA3.1 （ ＋ ） -TGF-β1 transfected group, and pcDNA3.1 （ ＋ ）-TGF-β3 transfected group. A positive cell clone stably and highly expressing TGF-β1 was established after being screened by G418 medium, pcDNA3.1 （ ＋ ）-TGF-β3 was transfected into the positive clone. Real-time PCR was used to detect the mRNA expression of TGF-133. Western blotting was used to detect the protein expression of TGF-β1 , collagen I , matrix metalloproteinase （MMP）-2, MMP-9, and tissue inhibitor of metallo-proteinase （TIMP）-I. Results There was no significant difference in the TGF-β1 mRNA expression between the TGF-β1 positive clone group and the TGF-β3 transfected group. The mRNA and protein expression levels of TGF-β1 , collagen I , MMP-2, and TIMP-1 of the TGF-β1 positive clone group were all significantly higher than those of the blank control, negative control groups （ all P 〈 0. 05 ） , and the MMP-9 mRNA expression of the TGF-131 positive clone group was significantly lower than those of the blank control and negative control groups （ all P 〈 0.05 ） ; and the mRNA and protein expression levels of MMP-9 of the TGF-β1 positive clone group were significantly lower than those of the blank control and negative control groups （ all P 〈 0. 05 ）. The TGF-β1 and MMP-2 mRNA expression levels of the TGF-β3 transfected group were not significantly different from those of the positive clone group （all P 〉0.05） , the mRNA expression levels of collagen I and TIMP-1 of the TGF-β3 transfected group were s

关 键 词：转化生长因子Β 肝硬化 胶原合成 

分 类 号：R686[医药卫生—骨科学]