| Bacground and ObjectiveBone mesenchymal stem cells are able to treat acute myocardial infarction,through paracrine effect and promoting tissue regeneration,but their low migration and homing rate make the therapeutic effect poor.Ultrasound targeted microbubble destruction can promote BMSCs to colonize infarct tissue effectively and accurately through capillaries,but it is challenging to improve the migration from peripheral circulation to infarct tissue.In our previous study,we found that CCN1 gene modification could facilitate BMSCs migration.Therefore,we speculate that CCN1gene modification combined with UTMD technology is capable of increasing the homing rate of BMSCs and improving the prognosis of AMI significantly.In order to test the above hypothesis,this study demonstrates it through the following three parts:Part one:Preparation and characterization of lipid microbubblesObjectiveTo prepare lipid microbubbles(MBs)which could be used for ultrasound imaging as well as vivo treatment,together with evaluating their features.Methods1.MBs all were prepared by membrane hydration mechanical oscillation method and separated by means of centrifugation;2.The distribution,morphology and structure of MBs were observed under light microscope together with electron microscope,and the particle size,potential as well as concentration were detected by Brookhaven laser particle size analyzer;3.CCK8 method was adopted to detect the biological safety of MBs;4.MBs were diluted and injected into rats to analyze the effect of contrast and perfusion.Results1.The MBs solution prepared by film hydration mechanical oscillation method was colorless and transparent,and it was milky white suspension after oscillation;2.MBs had negative charge on the surface,stable potential(-22.33±9.69)m V and single peak particle size,whose diameter was(1.07±0.01)μm.Under the light microscope,the distribution was uniform,the size was homogeneous,and there was no obvious tendency of aggregation.Under TEM,MBs were spherical and regular;3.When the concentration of MBs was less than 1×10~7cells/ml,it had no obvious biological toxicity and could be used as a safe dose;4.After MBs injection,the echo in the cardiac cavity was significantly enhanced,and then gradually weakened,and the echo in the ventricular wall was also enhanced.ConclusionLipid microbubbles were successfully prepared in this study.The particle size of lipid microbubbles was uniform and had good stability and contrast effect.Part two:CCN1 gene regulates BMSCs migration and its molecular mechanism ObjectiveTo study the effect of CCN1 on the migration of BMSCs and its molecular mechanism.Methods1.The BMSCs of SD rats were extracted,separated and cultured by whole bone marrow adherent method.The morphological transformation of BMSCs were observed through inverted phase contrast microscope,and the surface membrane antigen was identified by flow cytometry;2.After the adenovirus carrying CCN1 gene were transfected into BMSCs with different MOI,the transfection efficiency was detected by fluorescence microscope and flow cytometry to determine the optimal concentration;3.Cell scratch and Transwell assay were adopted to investigate the effect of CCN1 on BMSCs migration;4.RT-PCR and Western blot were selected to detect the expression of CCN1 together with its downstream CXCR4,PI3K and AKT molecules,as well as analyzing the molecular mechanism of CCN1 regulating BMSCs migration.Results1.BMSCs could adhere to the wall firmly after 2 days;the adherent cells increased in volume and granules after 4 days,and showed colony growth;after 7 days,the cell bodies were long and narrow,arranged in whirlpool,fish swarm or spindle shape.After passage,the cells were uniform in shape and distribution.Flow cytometry showed that the positive rates of BMSCs surface antigen CD29,CD90,CD34 and CD45 were(99.23±0.49)%,(98.23±1.19)%,(0.87±0.29)%and(1.34±0.50)%respectively;2.After adenovirus infected BMSCs with different MOI,the best MOI was 200,and the positive rate was(90.40±1.42)%;3.Cell scratch and transwell assay showed that the migration of CCN1-GFP-BMSCs group was greater than that of BMSCs group and GFP-BMSCs group(P<0.05);4.RT-PCR and Western blot showed that CCN1 gene and its downstream CXCR4,PI3K and AKT gene expression in CCN1-GFP-BMSCs group were higher than those in BMSCs group and GFP-BMSCs group(P<0.05).ConclusionCCN1 gene promotes BMSCs migration by regulating the expression of CXCR4,PI3K and AKT genes.Part three:UTMD promotes CCN1 gene modified BMSCs to treat acute myocardial infarction in ratsObjectiveTo study the homing rate of BMSCs and its effect on ventricular remodeling and cardiac function by inducing CCN1 modified BMSCs into ischemic myocardium of SD rats with UTMD.Methods1.AMI model was established by ligating left anterior descending coronary artery(LAD)in rats and divided into four groups,including sham operation group,AMI group,AMI-GFP-BMSCs group and AMI-CCN1-GFP-BMSCs group;2.BMSCs were injected into rats,the distribution of BMSCs was observed by fluorescence imaging,and the homing rate of BMSCs was calculated by flow cytometry;3.M-mode echocardiography was selected to measure the cardiac function of rats,and contrast-enhanced ultrasound was used to evaluate the changes of perfusion echo and peak signal intensity of anterior wall of infarcted myocardium in different groups;4.The percentage of infarct size(IR%)was calculated by Masson staining.The apoptosis of cardiomyocytes was compared by TUNEL staining.The expressions of CD31,SDF-1,VCAM-1 and VEGF were detected by immunofluorescence and immunohistochemistry.Results1.The results of fluorescence imaging and flow cytometry showed that the cardiac fluorescence intensity and GFP positive cell content in AMI-CCN1-GFP-BMSCs group were higher than those in AMI-GFP-BMSCs group(P<0.05);2.The cardiac function and peak signal intensity of anterior wall perfusion in AMI-GFP-BMSCs group were higher than those in AMI group,and the cardiac function and peak signal intensity in AMI-CCN1-GFP-BMSCs group were higher than those in AMI-GFP-BMSCs group and slightly lower than those in sham group(P<0.008);3.The IR%of AMI-GFP-BMSCs group was lower than that of AMI group and higher than that of AMI-CCN1-GFP-BMSCs group(P<0.008);4.The expressions of apoptotic cardiomyocytes,CD31,SDF-1,VCAM-1 as well as VEGF in sham group were the least.The positive expression in AMI-GFP-BMSCs group was higher than that in AMI group,but lower than that in AMI-CCN1-GFP-BMSCs group(P<0.008).ConclusionCCN1 can promote the migration of BMSCs to infarcted myocardium in rats,and improve the therapeutic effect of UTMD combined with BMSCs on AMI. |
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