Li Qi, Zhou Xia, Yu Jiao, Qian Wei, Xu Ke-shu
Department of Gastroenterology, Union Hospital Attached to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Yi Xue Za Zhi. 2008 May 13;88(18):1273-8.
To investigate the influence of recombinant transforming growth factor-beta3 (TGF-beta3) on collagen synthesis and deposition.
Plasmids pcDNA3.1 (+)-TGF-beta3 and pcDNA3.1 (+)-TGF-beta1 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-beta1 transfected group, and pcDNA3.1 (+)-TGF-beta3 transfected group. A positive cell clone stably and highly expressing TGF-beta1 was established after being screened by G418 medium. pcDNA3.1 (+)-TGF-beta3 was transfected into the positive clone. Real-time PCR was used to detect the mRNA expression of TGF-beta3. Western blotting was used to detect the protein expression of TGF-beta1, collagen I, matrix metalloproteinase (MMP)-2, MMP-9, and tissue inhibitor of metallo-proteinase (TIMP)-1.
There was no significant difference in the TGF-beta1 mRNA expression between the TGF-beta1 positive clone group and the TGF-beta3 transfected group. The mRNA and protein expression levels of TGF-beta1, collagen I, MMP-2, and TIMP-1 of the TGF-beta1 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-beta1 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-beta1 positive clone group were significantly lower than those of the blank control and negative control groups (all P < 0.05). The TGF-beta1 and MMP-2 mRNA expression levels of the TGF-beta3 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-beta3 transfected group were significantly lower than those of the positive clone group (both P < 0.05), and the mRNA expression level of MMP-9 of the TGF-beta3 transfected group was significantly higher than that of the positive clone group (P < 0.05). The protein expression levels of TGF-beta1, collagen I, and TIMP-1 of the TGF-beta3 transfected group were all significantly lower than those of the positive clone group (P < 0.05), the protein expression level of MMP-9 of the TGF-beta3 transfected group was significantly higher than that of the positive clone group (P < 0.05), and the protein expression level of MMP-2 of the TGF-beta3 transfected group was not significantly different from that of the positive clone group.
Recombinant TGF-beta3 eukaryotic expression vector reduces the synthesis of collagen and inhibits the collagen deposition by adjusting the expression of matrix metalloproteinases and their inhibitors.
探讨重组转化生长因子-β3（TGF-β3）对胶原合成与沉积的影响。
构建质粒pcDNA3.1(+)-TGF-β3和pcDNA3.1(+)-TGF-β1。培养大鼠肝星状细胞株HSC-T6作为肝纤维化细胞模型，并分为4组：空白对照组、pcDNA3.1-增强型绿色荧光蛋白（EGFP）转染组（阴性对照组）、pcDNA3.1(+)-TGF-β1转染组和pcDNA3.1(+)-TGF-β3转染组。经G418培养基筛选后建立稳定高表达TGF-β1的阳性细胞克隆。将pcDNA3.1(+)-TGF-β3转染至该阳性克隆中。采用实时荧光定量PCR检测TGF-β3的mRNA表达。采用蛋白质印迹法检测TGF-β1、Ⅰ型胶原、基质金属蛋白酶（MMP）-2、MMP-9和金属蛋白酶组织抑制剂（TIMP）-1的蛋白表达。
TGF-β1阳性克隆组与TGF-β3转染组之间TGF-β1 mRNA表达无显著差异。TGF-β1阳性克隆组的TGF-β1、Ⅰ型胶原、MMP-2和TIMP-1的mRNA和蛋白表达水平均显著高于空白对照组和阴性对照组（均P<0.05），且TGF-β1阳性克隆组的MMP-9 mRNA表达显著低于空白对照组和阴性对照组（均P<0.05）；TGF-β1阳性克隆组的MMP-9蛋白表达水平也显著低于空白对照组和阴性对照组（均P<0.05）。TGF-β3转染组的TGF-β1和MMP-2 mRNA表达水平与阳性克隆组无显著差异（均P>0.05），TGF-β3转染组的Ⅰ型胶原和TIMP-1的mRNA表达水平显著低于阳性克隆组（均P<0.05），且TGF-β3转染组的MMP-9 mRNA表达显著高于阳性克隆组（P<0.05）。TGF-β3转染组的TGF-β1、Ⅰ型胶原和TIMP-1的蛋白表达水平均显著低于阳性克隆组（P<0.05），TGF-β3转染组的MMP-9蛋白表达水平显著高于阳性克隆组（P<0.05），且TGF-β3转染组的MMP-2蛋白表达水平与阳性克隆组无显著差异。
重组TGF-β3真核表达载体通过调节基质金属蛋白酶及其抑制剂的表达，减少胶原合成并抑制胶原沉积。
