| Objective: Use of trimetazidine (TMZ) pretreatment bone marrow mesenchymalstem cells (BMSCs), in the acute phase of myocardial infarction, revascularizationcombined cell transplantation therapy, study its effect on cardiac function improved, and toexplore the mechanism of action initially.Methods: In vitro parts: take6-8weeks tibia and femur SD rat isolated bone marrowmesenchymal stem cells, with whole bone marrow adherent culture differences andpurification of BMSCs, and take the3rd generation BMSCs used in this experiment. Theuse of SD rats1-3days in vitro heart isolated neonatal rat ventricular myocytes (NRVMs),taking the first three days of primary cultured myocardial cells were collected normal andhypoxia/reoxygenation (H/R) treatment myocardial cell supernatant, its supernatantlactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD)levels were detected. The P3BMSCs were divided into the following four groups:①Control group: BMSCs+normal cell culture medium;②H/R group: BMSCs+H/Rsupernatant;③TMZ group: TMZ pretreated BMSCs+normal cell culture medium;④TMZ+H/R group: TMZ pretreatment BMSCs+H/R supernatant. The use of CCK-8assay cell activity above groups of BMSCs, RT-PCR detection of Bcl-2expression in thecell, Bax ’s.Vivo parts: Before cell transplantation with a fluorescent dye CM-Dil labeled BMSCs,established myocardial ischemia-reperfusion (I/R) model32, were randomly dividedinto four groups:①Sham group: coronary wearing without ligation;②I/R group:1h after coronary artery ligation open blocked coronary artery(simulationrevascularization), injecting cell-free supernatant;③BMSCs group:1h after coronary artery ligation open blocked coronary artery, injecting stem cells without drugpretreatment;④BMSCs+TMZ groups: ligation open blocked coronary arteries aftercoronary1h, injecting drug pretreatment stem cells. Each group collected3h rat serumLDH, MDA and SOD in the determination of I/R. After one week of each group weresacrificed two rats to detect the number of survivors of their implanted BMSCs. Theremaining rats were bred to accept the transplanted heart function was measured fourweeks after the examination were killed, including three in each group used to calculatethe infarct size, and the remaining three take heart tissue for protein extraction, WesternBlot analysis of the expression of Bcl-2and Bax.Results: In vitro parts:1. The activity of LDH,MDA in H/R group was increasedsignificantly compared with the control group (P <0.05), and the activity of SOD decreasedsignificantly compared with the control group (P <0.05).2. In BMSCs were culturedmyocardial cell supernatant with each group, cell viability of H/R group decreasedsignificantly (P <0.05) than the Control group, but after TMZ pretreatment before culture,cell viability increase significantly (P <0.05) compared to the H/R group3.RT-PCRresults showed that: H/R group cell gene expression levels of Bcl-2is slightly reduced,but there is no significant difference compared with the control group (P>0.05), but Bcl-2cells gene expression levels of TMZ+H/R group is higher than H/R group, there wassignificantly difference between them (P <0.05); The gene expression level of Bax in H/Rgroup were slightly increased,there is no significantly difference compared with the normalgroup (P>0.05), but the gene expression level of Bax in TMZ+H/R group wassignificantly lower compared to H/R group (P <0.05).In vivo parts:1. It is easy to established rat model of myocardial ischemiareperfusiom with the " second tier II knot" method.2. Compared with Sham group afterreperfusion3h, the activity of LDH, MDA of the rest groups were significantly increased(P <0.05), activity of SOD was significantly decreased (P <0.05), there is no significantdifference between the rest three groups (P>0.05).3. After one week, will be organizedfor quick frozen section, labeled BMSCs double counting under a fluorescence microscope and found that BMSCs+TMZ group of high magnification, the number ofdouble-labeled cells was significantly higher than BMSCs in each group (P <0.05).4. Afterfour weeks, BMSCs+TMZ group and BMSCs group than I/R group improved cardiacfunction (P <0.05), but BMSCs+TMZ group compared BMSCs improve heart functionmore obvious (P <0.05).5. Compared with the I/R group, BMSCs group and BMSCs+TMZ group could reduce infarct size (P <0.05), but BMSCs+TMZ group than in infarctsize BMSCs group to reduce more (P <0.05).6. BMSCs+TMZ group of Bcl-2proteinexpression levels were significantly higher I/R and BMSCs group (P <0.05), BMSCs+TMZ group Bax protein expression levels were significantly lower than the I/R andBMSCs group (P <0.05).Conclusion:1. In vitro cardiomyocyte H/R and myocardial I/R can make LDH,significantly increased MDA, significantly lower SOD (successfully simulated ischemiaand reperfusion microenvironment).2. Ischemia-reperfusion microenvironment existenceof serious cytotoxicity and oxidative stress is not conducive to the survival of transplantedcells.3. TMZ pretreatment in ischemia-reperfusion compared BMSCs microenvironmentnot pretreatment significantly increased the activity of its cells, which may be relatedpretreated cells upregulate the expression of Bcl-2and Bax down on.4. Revascularizationcombined TMZ pretreated BMSCs transplantation, the transplanted cells compared tonon-pretreated group, further reducing infarct size, cardiac function improved moreobvious. |
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