[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.
Li
Qi
Q
Department of Gastroenterology, Union Hospital Attached to Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China.
Zhou
Xia
X
Yu
Jiao
J
Qian
Wei
W
Xu
Ke-shu
KS
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