Qi Li1, Xia Zhou, Jiao Yu, Wei Qian, Ke-shu Xu. 1. Department of Gastroenterology, Union Hospital Attached to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
Abstract
OBJECTIVE: To investigate the influence of recombinant transforming growth factor-beta3 (TGF-beta3) on collagen synthesis and deposition. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
OBJECTIVE: To investigate the influence of recombinant transforming growth factor-beta3 (TGF-beta3) on collagen synthesis and deposition. METHODS: 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. RESULTS: 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. CONCLUSION: 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.