Research Of Differentiation From Bone Marrow Mesenchymal Cell To Myocar-dium Like Cell Induced By Jia Wei Dan Shen Yin

Objective:To explore the differentiation possibility of bone mesenchymal stem cells (BMMSCs) into myocardium-like cell induced by Jia Wei Dan Shen Yin(JWDSY) in vitro as well as therapeutic effects made by JWDSY on rats in cardiac blood stasis syndrome present with myocardial infarction after BMMSCs transplantation.Methods:Test 1:Cultivate BMMSCs of rats by bone marrow adherent method, perform passage culture and amplification, inspect cellular morphology and growth characteristics, analysize phenotypes of CD90, CD11b, CD45 with flow cytometer. Prepare extractive of JWDSY with decoction and alcohol sedimentation technique, test toxic effect and proliferation of BMMSCs affected by different JWDSY concentrations in MTT way.Test 2:Induce BMMSCs in vitro, divide them into 4 groups:normal control group,5-Aza group, JWDSY group as well as combined JWDSY and 5-Aza group, induce their differentiation. Detect expressions of peculiar cardiomyocyte bindin Desmin, myosin MHC along with troponin T by immunohistochemistry approaches, check myocardial transcription factor GATA-4 mRNA expression with RT-PCR.Test 3:Set up acute myocardial infarction model in cardiac blood stasis syndrome of the rats, group 60 successfully molded rats into model set, JWDSY group, BMSCs group and combined JWDSY and BMSCs group beyond 10 normal rats. Observe expressions of myocardial bindin Desmin and myosin heavy chain(MHC) in rats' myocardial tissues with double staning immunohistochemical way 28 days after transplantation. Masson trichrome staning is employed in evaluations of ventricular scar proportion and myocardial angiogenesis in myocardial tissues of the rats, take RT-PCR to measure myocardiocyte expression of GATA-4 mRNA.Results:Test 1:Flow cytometry evaluated that BMSCs fineness by panmyelo-adherent culture was higher, positive cell surface antigen CD90 was above 95% while positive CD11b and CD45 cells were below 5% which fit the experiment requirement. MTT outcomes revealed that 0.625mg/ml JWDSY was optimal concentration for proliferation of BMMSCs.Test 2:MHC, Desmin and CTnT positive cells were absent in normal control group after in vitro BMMSCs induction by JWDSY; whereas positive immunocomplex were present in JWDSY group,5-aza group and combined JWDSY and 5-aza group in immunohistochemical stainig. MHC expression induced by combined JWDSY and 5-Aza group was greater than two single ones, the difference has statistical significance (P<0.05), but there was no statistical significance between the two single groups(P>0.05), so did Desmin and CTnT. Significant statistical differences of relative optical density values of GATA-4 mRNA existed in combined JWDSY and 5-Aza group, JWDSY group and 5-aza group compared to the normal control group (P<0.05).Test 3:Twenty-eight days after transplant, Masson stained slices results suggested that neither infarct locus nor fiber is present in normal control group, while infarctions with different sizes appeared in other groups, ventricle muscular scar areas of combined JWDSY and BMSCs group, JWDSY group and BMSCs group showed notable decreases than model group all(P<0.01); in addition, the combined group performed a more significant scar area proportion decline rather than the two later groups(P<0.05), but the apparent differences were absent between the two single groups(P>0.05).The amount of myocardial blood capillary of every model group elevated compared to the control group with statistical significance (P<0.05). JWDSY group, BMSCs group and the combined group obtained remarkable increases than model group(P<0.01); compared with the two single group, the combined group revealed a significant statistics difference(P<0.05). No distinct significance existed in the two former group. Associated MHC-Brdu and Desmin-Brdu expression was unseen either in the model group or the JWDSY group; but scarlet Bru antibody and brown MHC protein dyes were available in infarct edge of both model group and JWDSY group as well as simultaneous expressions of scarlet Brdu antibody dye and brown Desmin protein. Positive cell count in combined group was greater than BMSCs group (P<.05). Positive stripe was absent in model group or control group after RT-PCR detection for GATA-4 expressions, but present in BMSCs group and the combined group and it was lesser in JWDSY group, what's more,the combined group was higher than the BMSCs group by semi quantitative analysis of IOD value(P<0.05).Conclusions:(1) JWDSY is able to promote myocardium-like differentiation of BMSCs in vitro and subserve 5-Aza to induce myocardium-like differentiation of BMSCs.(2) In rat models present with myocardial infarction and cardiac blood stasis syndrome, JWDSY group, BMSCs group and combined BMSCs and JWDSY group are all capable to promote vascular neogenesis, increase blood supply of transplanted cells, and shrink infarct area. JWDSY can precipitate angiogenesis after BMSCs transplant, and reduce cicatrization.(3)JWDSY is able to promote myocardium-like differentiation of BMSCs in cardiac blood stasis syndrome rat models present with myocardial infarction, Combined JWDSY and BMSCs transplant showed a better therapeutic effect than single use of each approach.