[Influence of recombinant transforming growth factor-beta3 on collagen synthesis and deposition: experiment with rat cell model of liver fibrosis].
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.


CONCLUSIONS
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.
Department of Gastroenterology, Union Hospital Attached to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
Qi
Q
Li
Xia
X
Zhou
Jiao
J
Yu
Wei
W
Qian
Ke-shu
KS
Xu
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D008106:Liver Cirrhosis, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D053782:Transforming Growth Factor beta3 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D024042:Collagen Type I; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D053782:Transforming Growth Factor beta3; D049452:Green Fluorescent Proteins; D003094:Collagen; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9