| Background:Acute myocardial infarction(AMI)has been a leading cause of morbidity and mortality in human,but at present how to prevent and treat it is still a difficult problem. With the development of study on marrow stem cells,technique of marrow stem cells transplantation has provided us a new hope to treat AMI.Preliminary results of animal studies and clinical trials supported the security and effectiveness of marrow stem cells transplantation in AMI.However,the mechanisms underlying the improvement in cardiac function are incompletely understood.It is necessary to further investigate the mechanisms.There is an emerging body of data to suggest that bone marrow mesenchymal stem cells(BMSCs)have unique immunologic properties,making them ideal for immuno-related diseases treatment.As other immuno-related diseases,AMI can trigger an intense immuno-inflammatory reaction.As a major factor,immuno-inflarnmatory response can lead to ventricular remodeling and heart failure.Whether BMSCs could modulate the immuno-inflammatory response is unclear.The immuno-modulation could be a completely new mechanism of BMSCs transplantation for AMI.Objective:(1)To investigate the treatment effects of BMSCs transplantation in the inflammatory period after AMI.(2)To investigate the modulation effect of b BMSCs on the immuno-inflammatory reaction in AMI:the lymphocyte cytotoxic activity against cardiac myocytes and the expression of inflammatory cytokines in myocardium.Methods:1.Preparation of rat BMSCs(1)Rat BMSCs were isolated from marrow by density gradient centrifugation. Then cells were cultured and propagated.(2)Isolated cells were induced to differentiate into osteocytes and adipocytes in vitro.(3)Passage 3 cells were incubated with fluorescence-labeled antibodies of CD29, CD34,CD45 and CD105.Cell fluorescence was evaluated by flow cytometry.2.Effect of BMSCs on AMI(1)Cultured cells were labeled with BrdU,and then suspended in PBS at a density of 1×107cells per milliliter for transplantation.(2)AMI were induced in SD rats by left anterior descending coronary artery ligation,and the animals were randomly assigned into the Sham group,MI+PBS group and MI+BMSCs group.BrdU-labeled cells or PBS was transplanted into myocardium by direct myocardial injection.Finally,24 rats were enrolled in the study with 8 rats in each group.(3)Evaluation of cardiac function:echocardiography and hemodynarnic exams were performed respectively at 1 day and 4 weeks after surgery to evaluated the cardiac function.(4)After hemodynamic studies were finished,the rats' hearts were obtained. Simultaneously,the spleens were harvested for the lymphocyte cytotoxic activity against cardiac myocytes study.(5)HE stain and Masson trichrome stain were performed to evaluate the myocardial infarction.And infarcted area,infracted free wall thickness and infarcted expansion index were calculated.(6)Anti-BrdU and anti-cTnI were used to detect the living condition and transdifferentiation of the transplanted cells. (7)The expression of collagens in infarcted heats was evaluated by RT-PCR and western blot.3.Effect of BMSCs on the lymphocyte cytotoxic activity against cardiomyocytes(1)Cardiomyocytes and non-cardiomyocytes were isolated from 2-day-old neonatal SD rats and spleen lymphocytes were isolated from above-mentioned experimental animals.(2)Cardiomyocytes or non-cardiomyocytes were co-cultured with spleen lymphocytes for 24 hours,and then the cytotoxic activity assay was performed according to crystal-violet method.(3)HE stained slides were investigated to evaluated the lymphocytes infiltrating into myocardium.4.Effect of BMSCs on the expression of inflammatory cytokines in myocardium(1)The protein expression of IL-6,IL-10 and TNF-a were investigated by immunohistochemistry and western blot.(2)The mRNA expression of IL-6,IL-10 and TNF-a were evaluated by RT-PCR.(3)The activity of nuclear factorκB was examined by western bolt.Results:1.Isolation and identity of BMSCsThe cells isolated from bone marrow had vigorous proliferation capability and could been induced into osteocytes and adipocytes.The results of flow cytometry exam demonstrated that isolated cells expressed CD29(80.10%)and CD105(85.20%)highly, and expressed CD34(15.86%)and CD45(10.50%)lowly.2.Effect of BMSCs transplantation on AMI(1)After labeled with BrdU,more than 85%cultured cells showed BrdU labeling.(2)Four weeks after transplantation,the injected cells were detected in the peri-infarct area of MI+BMSCs group.No neonatal cardiomyocytes from BMSCs transdifferentiation were detected. (3)BMSCs transplantation could decrease the infarcted area[MI+BMSCs vs. MI+PBS:(38.39±2.77)%vs.(45.83±3.08)%,P＜0.001],increase the thickness of infracted free wall[(2.3±0.3)mm vs.(1.5±0.5)mm,P＜0.01],inhibit ventricular remodeling and merely attenuate the deterioration of cardiac function without improvement[MI+BMSCs vs.MI+PBS,LVEDD(mm):(6.45±0.47)vs.(7.81±0.31), P＜0.05;heart weight/body weight(mg/g):(2.99±0.10)vs.(3.21±0.06),P＜0.01; infracted expansion index:(1.37±0.24)vs.(2.20±0.22),P＜0.001;EF%:(30.76±3.39)vs. (24.06±4.71),P＜0.05;FS%:(29.33±4.87)vs.(23.05±3.94),P＜0.05;LVEDP(mmHg): (12.35±4.87)vs.(19.83±4.91),P＜0.01;+dp/dtmax(mmHg/s):(4591.63±415.38)vs. (3887.75±275.62),P＜0.01;-dp/dtmax(mmHg/s):(4242.13±273.93)vs. (3480.88±346.44),P＜0.01].(4)BMSCs could promote collagen synthesis in infarcted area and decrease the synthesis in non-infarcted area.3.Effect of BMSCs transplantation on the lymphocyte cytotoxic activity(1)Four weeks after surgery,rat's spleen lymphocytes could kill the neonatal rat's cardiomyocytes specificly,and BMSCs transplantation could decrease the cytotoxic activity[MI+PBS:(47.20±5.13)%,MI+BMSCs:(34.28±6.13)%,P＜0.05].(2)HE staining examination showed that lymphocytes infiltrated into both infarcted region and non-infarcted region in groups of MI+PBS and MI+BMSCs.4.Effect of BMSCs transplantation on the expression of inflammatory cytokines(1)Four weeks after surgery,the expression of IL-6,IL-10 and TNF-a and the activity of nuclear factorκB were significantly increased in MI+PBS group and MI+BMSCs group.(2)Comparing with injection of PBS solution,BMSCs transplantation could decrease the expression of IL-6 and TNF-a,increase the expression of IL-10 and inhibit the activation of nuclear factorκB.Conclusions: (1)Isolating bone marrow stem cells by density gradient centrifugation and adhering culture can harvest relatively homogeneous bone marrow mesenchymal stem cells.(2)BMSCs injected into myocardium immediately after AMI can survive in the hearts at least for 4 weeks.BMSCs transplantation can inhibit ventricular remodeling and alleviate the deterioration of cardiac function.(3)BMSCs transplantation can improve the collagen synthesis in infarcted area, and decrease the deposition of collagen in non-infarcted area.This may be one of the mechanisms by which BMSCs inhibit the ventricular remodeling.(4)BMSCs transplantation can inhibit the activation of cytotoxic lymphocytes, modulate the expression balance of pro-inflammatory and anti-inflammatory cytokines. BMSCs can alleviate the immuno-inflammatory response in AMI.
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