Study On The Role Of Connective Tissue Growth Factor(CTGF) In Collagen Synthesis Of Swim Bladder From Nibea Coibor

The fish glue(dried product of fish swim bladder)of Nibea coibor is the edible treasure of aquatic products with high nutritional value.Collagen is a vital nutrient component and quality evaluation index of fish glue.Tapping into the collagen synthesis molecular mechanism of swim bladder can provide targets and strategies for the technology development to improve the quality of swim bladder.However,the knowledge of regulatory mechanisms of collagen synthesis in swim bladder is minimal.In mammals,connective tissue growth factor(CTGF)plays an essential role in the fibrosis process associated with collagen deposition.Still the specific role of CTGF in fish collagen synthesis and its regulation mechanism has not been systematically studied.In this research,the ctgf gene and its promoter sequence were cloned from N.coibor,and tissue expression,promoter activity,and crucial transcriptional regulatory elements were analyzed.We further established the N.coibor swim bladder cell line(NCSB),and studied the effect of ctgf on collagen synthesis of swim bladder in vitro.The main results are as follows:1)The ctgf gene of N.coibor ORF was 1041 bp in length and encoded a secreted protein containing 346 amino acids with a molecular weight of 38.29 k Da and isoelectric point(p I)around 8.42.Gene structure of ctgf gene constituted by five exons and four introns,which was similar to other species.Amino acid sequence alignment showed high similarity with other species(94.81% and 92.22% homology of Acanthochromis polyacanthus and Cynoglossus semilaevis respectively).The ctgf gene expressed significantly higher in gonads,spleen,gills and swim bladder than others,followed by kidney,skin,heart,brain and muscle,meanwhile the lowest expression tissue was liver and intestine by q RT-PCR(P<0.05).And it is similar to the expression pattern of type I collagen genes(col1a1 、 col1a2),which revealed that expression levels were higher in the swim bladder and gill,but lower in the liver and brain(P<0.05).2)About 3185 bp upstream of ctgf initiation codon was cloned in research.According to the bioinformatics analysis,five deletions were constructed in the range of candidate promoters(-1877～+166bp)by PCR amplification,including D0(-378～+166)、 D1(-785～+166)、D2(-1049～+166)、D3(-1544～+166)、D4(-1877～+166).Dual-luciferase assay3)showed that D1 has the highest activity.Thus the region of-785～+166 bp was thought as the core promoter of ctgf,and incubation with 10 ng/m L TGF-β that D1 activity up-regulated significantly.Mutations and deletions were constructed to tested ctgf promoter activity,and dual-luciferase results showed that Sp-1 binding sites mutation led to an increase in promoter activity,while Smad-3 and NF-κB binding sites mutations led to a decrease in promoter activity,suggesting that these transcription factors are the critical ctgf promoter cis-acting elements.4)The N.coibor swim bladder cell line(NCSB)was established by the tissue block adherent method.The cell growth rate was stable,and morphology was fibrous,which has been continuously passed over 50 generations.Cells cultured in DMEM complete medium with 5%,10%,15% and 20% serum,respectively,and proliferated rapidly along with serum concentration.The p EGFP-N1 plasmid was transfected into NCSB cells by liposome method,and expressed EGFP successfully.5)Overexpression of ctgf gene up-regulated col1a1 and col1a2 gene expression and collagen I content in NCSB cells significantly(P<0.05).The results were similar to overexpression of ctgf(P<0.05),when NCSB was cultured in 293 T cell culture medium.Meanwhile,CTGF si RNA interference showed that down-regulated col1a1 and col1a2 gene expression and collagen I content in NCSB cells significantly,especially under the condition of TGF-β incubation(P<0.05).These results demonstrated that ctgf can promote collagen synthesis in the NCSB cells.