| ObjectiveTo investigate the role and mechanism of TNF-αsecreted by circulating mononuclear cells isolated from SD rats post myocardial infarction on migration and the effect on myocardial differentiation of MSCs.Methods1. A tri-chamber co-culture system was established using self-made trans-well chambers. Bone marrow was flushed, then MSCs were isolated and cultured through density gradient centrifugation and adherence screening method.14 clear healthy SD rats were randomly divided into MI group[with left anterior descending coronary artery ligation, N=8] and Sham group[with no ligation, N=6]. Blood samples were aspirated from the heart chambers on 7th day , and circulating mononuclear cells were isolated by density gradient centrifugation. Cardiomyocytes from neonatal SD rats were isolated by combination of mechanical method and trypase and collagenase digestion. Mononuclear cells, cardiomyocytes and DAPI-labeling MSCs were inoculated into the lower, middle and upper chamber of the tri-chamber co-culture system respectively. According to the different additives into the lower compartments, the whole experiment was divided into MI group[2×10~6 mononuclear cells from MI rats were added],TNF-α-NA group[2×10~6 mononuclear cells from MI rats and final concentration of 50ng/L TNF-αwere added at the same time], Sham group[2×10~6 mononuclear cells from Sham rats were added] and BC group[culture medium was added]. 12 bores were set in each group and co-cultured away from light.2. 48 hours later, six bores in each group were randomly selected, migrating cells were identified by CD44 and CD34 immunocytochemistry staining and were counted under the fluoroscope. Vitality of MSCs transmigration to undersurface of poly-carbonic acid membranes was detected by viola crystalline staining. Concentration of TNF-αin the supernatant was measured by ELISA and its correlation to the number of migrating MSCs was analyzed. Expression of VCAM-1 in MSCs was evaluated by immunofluorescent staining and western blot.3. Medium in the upper chambers of the rest six bores in each group was changed every four days. 14 days later, double-labeling immunofluorescent staining was conducted to detect the expression of cardiac markers cTnI andα-actin in MSCs transmigrating to the middle compartments.Results 1.48 hours later, blue-light-emitting nucleus of MSCs transmigration to the middle chambers were visualized under the fluorescent microscope in MI, TNF-α-NA and Sham group. Compared with Sham group[12.28±2.74], number of migrating cells per visual field increased significantly in MI group[23.72±4.28](p<0.05).In TNF-α-NA group where TNF-αwas neutralized, number of migrating cells per visual field decreased significantly[8.89±2.25] (p<0.05). But no cell was visualized in BC group. All migrating cells were positive for the expression of CD44 and negative for the expression of CD34, which is consistent with MSCs. Migrating cells also expressed VCAM-1 positively. MSCs transmigration to undersurface of poly-carbonic acid membranes were vital cells proved by viola crystalline staining.2.Compared with Sham group, concentration of TNF-αsecreted by mononuclear cells was higher in MI group[11.12±6.02 and 23.95±8.44 ng/L respectively](p<0.05). And a positive correlation between TNF-αconcentration and number of migrating MSCs was observed, with r=0.976 and r=0.969 respectively (p<0.05).3.Compared with Sham group, expression of VACM-1 in MI group was significantly higher[0.78±0.12 and 0.97±0.07 respectively] ( p<0.05),while it was significantly reduced by TNF-αneutralization[0.66±0.05] (p<0.05),but neutralization of TNF-αcould not completely inhibit expression of VCAM-1. 4. 14 days later, double-labeling immunofluorescent staining was conducted to detect the expression of cardiac markers as cTnI andα-actin in migrating cells in the middle chambers, which indicated that partial MSCs transmigrating to contact with cardiomyocytes expressed cTnI andα-actin positively, while those noncontact with cardiomyocytes did not express cTnI andα-actin.Conclusion1.Mononuclear cells isolated from rats post-MI could promote migration of MSCs, which might be attributed to the enhancement of adhesiveness of MSCs by increasing secretion of TNF-αto some extent..2. In our experimental model, biological factors secreted by circulating mononuclear cells isolated from rats post-MI could not interfere the potential of myocardial differentiation of MSCs migrating to directly contact with cardiomyocytes.
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