Construction Of Adenovirus Harboured CysC And It's Effects On The Proliferation Of Cultured Rat Cardiac Fibroblast And Collagen Synthesis

Objective:To establish a recombinant adenovirus vector for cystatin C (CysC) gene using GatewayTM technique and observe it's expression in rat cardiac fibroblast.At the same time this investigation try to explore the effects of cystatin C on the proliferation of rat cardiac fibroblast and collagen synthesis.Methods:1. The CysC gene of rat Myocardium was chemosynthesis by oligo and amplified by RT-PCR which is directly cloned into entry vector pDONR221 by BP reaction to generate the entry clone pDONR221-CysC.2. The recombinant plasmid pDONR221-CysC was identified by PCR and sequencing,Along with the LR recombintion reactions,the pDONR221-CysC and the target vector Ad/CMV/V5-DEST was recombined together in vitro to create the expression clone(pAd/CMV/V5-DEST-CysC),The ccdB-CmR gene in pAd/CMV/V5-DEST was replaced by CysC gene.3. Cardiac fibroblast from 1～3 days old Sprague-Dawley rats (clean grade, weight 10～15 g) were cultured by the anchorage velocity-dependent separation method. The recombinant adenovirus vector for CysC gene (Ad-CysC) was constructed with GatewayTM technique and transfected into rat cardiac fibroblast in vitro. 4. Rat cardiac fibroblast were randomly divided into normal control group,Ad-GFP group and Ad-CysC group.5. The expression of CysC at protein level was examined by the means of Western-blot.6. The cell proliferation rate was measured by bromodeoxyuridine (BrdU)7. The expressions of Types I and III collagen mRNA in rat cardiac fibroblast were determined by qRT-PCR. Analyses were performed using the statistical software package SPSS 17.0.Results:1. The target gene of CysC was transferred into Ad/CMV/V5-DEST vector correctly with the right orientation and it was identified by PCR and sequencing.The expression clone Ad-CysC was packaged into maturated denovirus successfully with the titer of Ad-CysC was 5.36×1010ifu/ml; Western blot anlalysis indicated that the recombinant Ad-CysC was successfully constructed.2. The efficiency of Ad-CysC infecting rat cardiac fibroblast was above 90% when multiplicity of infection(MOI) was 200. Compared to control group, the protein expression of CysC was increased significantly at 24h after transfection (P<0.05), there was no differences between the normal control group and the Ad-GFP group.3.Compared to the normal control group and the Ad-GFP group,BrdU incorporation rate of Ad-CysC group was much higher at 72h after transfection (P<0.05),But the normal control group and the Ad-GFP group showed no significant differences (P>0.05)4.Compared to the normal control group and the Ad-GFP group, The expressions of Types I and III collagen mRNA in Ad-CysC groups were much higher, and the difference were statistically significant (P<0.05).There was also no evident differences between the normal control group and the Ad-GFP group (P>0.05)。Conclusion:1. The recombinant Ad-CysC was successfully constructed and packaged into maturated denovirus with target gene.Compared to the traditional system,it has advantage of efficiency and flexibility,and which lays a experimental foundation for the future research on physiological function of CysC and the genetic therapy of CysC.2. Over-expression of Ad-CysC could transfected in rat cardiac fibroblast effectively,Compared to the old plastid transfected methods, it has higher efficiency.3. The over-expression of CysC could stimulate the proliferation of cardiac fibroblast and promote the level of expressions of TypesⅠandⅢcollagen mRNA, which could stimulate the myocardial fibrosis.