Recombinant Adenovirus Ad-hMMP-1Transfect Bone Marrow Mesenchymal Stem Cells Of Rat In Vitro

Liver fibrosis is the pathological basis of chronic liver disease and it must lead to thecirrhosis. There is still no effects of drugs to reverse the liver fibrosis. In recent yearsnumerous studies have confirmed that bone marrow-derived mesenchymal stem cells in thethe body and outward have the potential of differentiation to liver cell, it is expected torepair or reverse the process of liver fibrosis. The genetically modified stem cells maintainthe directly differentiation characteristics, while corresponding factor can improve theefficacy and is compensate for the lack of simple MSCs transplantation therapy. Matrixmetalloproteinases in the liver was expressed and secreted by hepatic stellate cells (HSC)and Kupffer cell. It was zinc-calcium-dependent family of endogenous proteolyticenzymes involved in extracellular matrix degradation. It was the only enzyme that breaksdown collagen fiber and almostly breaks down the ECM components outside thepolysaccharide,it plays an important role in physiological and pathological processes.MMP-1was also known as fibroblast cell type, it was a major human interstitialcollagenase that can degradate type I collagen-based extracellular matrix constituents(ECM), so as to reverse the process of liver fibrosis.Objective:The BMSCs of rat were isolated by plastic adherence in vitro and identified by theflow assay， then transfected by recombinant adenovirus Ad-hMMP-1carrying greenfluorescent marker in vitro， observeing the GFP expression by fluorescence microscopyand decting the transfection efficiency by flow cytometry. the3rd–passage BMSCs weredivided into three groups for vitro transfection: Group A: the BMSCs, Group B: theBMSCs transfected by Ad-eGFP, Group C: the BMSCs transfected by Ad-hMMP-1-eGFP;the gene and intracellular protein of hMMP-1was detected by RT-PCR and Westeron Blot，the Elisa assay supernatant protein expression， the hMMP-1activity was measured byfluorescent quantification kit. So this study can lay the experimental foundation for thetreatment of liver fibrosis jointing hMMP-1gene transplantation. Method:1.The bone marrow mesenchymal stem cells of rat were isolated and cultured byplastic adherence. Being proficient in the cell culture technology, observed cellmorphology and growth characteristics daily, changed solution and passaged on time, cellsof good growth state were identified in the immune phenotype of stem cells using flowcytometry, the immune phenotype were including CD45、 CD90、 CD105、 CD14、CD34andCD79a.2.Recombinant adenoviral vector Ad-hMMP1-eGFP building, identification andpackaging, the hMMP-1gene was amplified by PCR reaction, building the expressioncloning of pAd-hMMP-1-eGFP by the Gateway technology. The linear pAd-hMMP-1-eGFPcutted down by endonuclease Pac I transfect into HEK293A cells to packaging theAd-hMMP-1-eGFP. The transfected situation was observed under a fluorescencemicroscope, the target protein expression was detected by Western-Blot assay.3. The BMSCs were transfected by recombinant adenovirus Ad-hMMP-1carryinggreen fluorescent marker in vitro, observeing the GFP expression by fluorescencemicroscopy and decting the transfection efficiency by flow cytometry, determining theoptimal multiplicity of infection (multiplicity of infection, MOI). The cell proliferationafter transfection in vitro was dected by MTT assay. The gene and intracellular protein ofhMMP-1was detected by RT-PCR and Westeron Blot, the Elisa assay supernatant proteinexpression, the hMMP-1activity was measured by fluorescent quantification kit.Results:1. The bone marrow mesenchymal stem cells of rat in primary culture grew well, andthere was a large number of cells, growing adherent, forming a single, being fusiform,arraying in polarity and growing whorled. It showed the3rd generation BMSCs highlyexpress the specific marker of CD90(99.6%)andCD105(99.8%), don’t express thesurface marker of hematopoietic stem cell of CD45(0.1%), CD14(0.1%), CD34(0.3%),CD79a (0.1%) by the flow cytometry identification results.2. It was confirmed that the entry vector and the destination vector both containhMMP-1target gene by restriction analysis and sequencing. The green fluorescent proteinwas observed in the293A cells transfected by the Ad-hMMP-1-eGFP4days later. Thefluorescence intensity is the highest10days later. the virus was collected12days later, the viral titer was determined as4.84×1010PFU/ml, the target protein was efficient expressionvia Western-Blot assay.3. The green fluorescent was observed in BMSCs transfected by recombinantadenovirus at24hours after transfection; the fluorescence intensity was highest at72hours;and the optimum MOI was200. The cells of3groups entered the logarithmic growth phaseon the3days and reached plateau phase on the7days by MTT assay; no significantdifference was found in the cell prol iferation rate among3groups (P>0.05). RT-PCR,Western blot, and ELISA assay showed high expressions of the hMMP-1gene and proteinin group C, but no expressions in groups A and B. The MMP-1activity was1.24nmol/(mg min) in group C, but MMP-1activity was not detectable in groups A and B.Conclusion:1. The bone marrow mesenchymal stem cells of rat can be isolate and culture byplastic adherence, the purity of cells is high, the cells growing in good condition, can beused for subsequent cell transfection studies;2. The recombinant adenovirus vector containing human matrix metalloproteinase-1(hMMP-1) was successfully constructed by using the Gateway technology, it was moreefficiency and specificity comparing with the traditional building methods;3. The exogenous gene hMMP-1was successfully transfected into rat BMSCs andhighly expressed via recombinant adenovirus, and there was no significant effect on cellproliferation, laying the experimental foundation for the treatment of liver fibrosis jointinghMMP-1gene transplantation.In summary, the recombinant adenovirus Ad-hMMP-1can be successfully transfectedinto rat of BMSCs in vitro, the target gene and the target protein in transfected cells wereexpression efficiently and sustainedly.The cells are the good seed cells to treatmenting ofhepatic fibrosis in rats in next step.