| Objective: To study the effects and mechanisms of klotho gene transfected BMSCs transplantation with heart failure and myocardial remodeling in rats.Methods: Bone marrow mesenchymal stem cells were cultured and amplified using whole bone marrow adherent culture methods.Observe the morphological and growth characteristics of BMSCs,cell surface markers were assessed by flow cytometry,and BMSCs were induced to differentiate into adipocytes and osteoblasts,the above characteristics are used to identify BMSCs and get the 3-5generation BMSCs.The recombinant lentiviral vectors bearing Klotho transfected into rat bone marrow mesenchymal stem cells,identifiy of target gene klotho expression by RT-RCR,acquisite of klotho gene modified BMSCs.SPF SD rats with a model of heart failure created by subcutaneous injections isoproterenol(ISO)were randomly divided into three groups: HF group,BMSCs transfected Klotho gene group,BMSCs transfected EGFP group,Control study of the three groups and the normal group.Cell transplantation respectively for 28 days after treatment,the rats in each group were sacrificed.The heart were isolated to caculate the heart quality index.The myocardial pathological change was detected by HE staining.Collagen deposition weredetected by Masson trichrome,TUNEL technology was performed to detect myocardial cell apoptosis.Additionally,the green fluorescin expression was observed on frozen heart section.Myocardial fibrosis correlated gene expression including Klotho gene,collagen ? a1 and ? a1 was detected by RT-PCR.Results: 1.The primarily cultured BMSC kept adherent within the 48 hours and stretched into polygonal or spindle shape.Three stages of cell growth: a relative inhibition phase,cells proliferation phase,the platform phase,cells kept strong growth.The growth curve demonstrated that BMSCs were consistent with the growth characteristics and good activity of normal cells.At the third passage,BMSCs were nagative for the CD34 but positive for CD44 and CD90.Following induction,Oil red O staining and alizarin red staining produced a strong reaction in cells,indicating BMSCs have potentiality of multi-directional differentiation.2.The lentiviral vectors overexpressing Klotho were constructed successfully.BMSCs were infected with lentivirus bearing Klotho.The transfection efficiency of the cells under the fluorescence microscope for the first 4 days was more than 70%-80%,and the expression was stable for seventh days.3.We could also see the green distributed fluorescin around in myocardial tissues of EGFP-Klotho-BMSCs group and EGFP-BMSCs group.And the number of green fluorescent expression in EGFP-Klotho-BMSCs group was significantly increased compared with the EGFP-BMSCs group,which suggested that the survival rate of BMSCs of transfected Klotho gene was significantly increased.4.Rats with Heart failure rats manifest as faster weight loss,reduced food and water intake,shortness of breath,heart rate and spirit is dispirited,but EGFP-BMSCs group and EGFP-Klotho-BMSCs rats in the cell transplantation therapy 4 weeks after the symptoms of heart failure comparedwith saline group improved significantly,which EGFP-Klotho-BMSCs rats improved the most is obvious.5.In the saline group,EGFP-BMSCs group and EGFP-Klotho-BMSCs group,the heart body mass index was significantly higher than that in the normal group(P<0.01),the EGFP-Klotho-BMSCs group was significantly lower than that in the EGFP-BMSCs group(P<0.01),the EGFP-BMSCs group was lower than the saline group(P<0.01).6.Saline group,EGFP-BMSCs group and EGFP-Klotho-BMSCs rats myocardial tissue by HE staining and Masson staining visible myocardial cell hypertrophy and necrosis,myocardial fiber rupture,myocardial interstitial fibrosis scar tissue formation,disorder of myocardial tissue structure,the normal group rats myocardial tissue did not see the pathological changes.the apoptosis rate of saline group was significantly higher compared with the normal group(P<0.01),That EGFPKlotho-BMSCs group rats myocardial fibrosis and myocardial cell degeneration or necrosis such as the degree of cardiac remodeling were compared with the physiological saline group and EGFP-BMSCs group light,EGFP-BMSCs group myocardial fibrosis remodeling degree were compared with saline group light.7.The apoptosis rate of myocardial cells in EGFP-Klotho-BMSCs group and EGFP-BMSCs group was significantly lower than that in saline group,which was significantly different(P<0.01),and the effect of EGFP-Klotho-BMSCs group was the most significant.8.RT-PCR detection results showed that collagen I and III mRNA Klotho were both expressed in the myocardium of rats in each group.Among them,the normal group,EGFP-BMSCs group and EGFPKlotho-BMSCs group Klotho mRNA in the expression level is higher,compared with saline group,the difference has statistical significance(P<0.05),and EGFP-Klotho-BMSCs group of Klotho mRNA expression levels comparedwith the saline group,EGFP-BMSCs group of high difference has statistical significance(P<0.01),while the normal group and the EGFP-BMSCs group was not statistically significant.Normal group,EGFP-BMSCs group,EGFP-KlothoBMSCs groups of collagen I and III mRNA expression levels were lower than that in the saline group,the difference is statistically significant(P<0.05),and EGFP-Klotho-BMSCs group,EGFP-BMSCs group and saline group differences had no statistical significance(P>0.05).9.There was conspicuous and independent direct negative correlation between the expression of klotho mRNA and Collagen I mRNA ? Collagen ? mRNA,myocardial collagen volume fraction,apoptosis of myocardial cells(r=-0.578,p<0.05;r=-0.558,p<0.05;r=-0.598,p<0.05).10.There was conspicuous and independent direct positive correlation between myocardial collagen volume fraction and apoptosis of myocardial cells(r=0.895,p<0.01).There was conspicuous and independent direct negative correlation between the survival rate of stem cells and and apoptosis of myocardial cells,myocardial collagen volume fraction(r=-0.889,p<0.01;r=-0.949,p<0.01).Conclusions: Models of rat heart failure can be constructed effectively by isoproterenol-induced intraperitoneal injection.Klotho gene combined with bone marrow mesenchymal stem cells could attenuate cardiomyocytes hypertrophy and restrain collagens hyperplasy in the progression of myocardial fibrosis. |
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