Study On Change Of Myocardium Microenvironment Of Heart-Blood Stagnation Syndrom And Effects Of BMSCs Transplantation

Objective:To study the function about cardiac blood stasis syndrome cardiac microenvironment towards BMSCs cardiac differentiation as well as the influences about the active principle region of Yangxin Tongmai Formula(apr-YTF).Methods:(1)Theoretical discussion:Through revieve the concept, development history and current academic tendency of microenvironment, to explore its exact concept academic development tendency and change characteristics of cardiac blood stasis syndrome and its influences on the bone marrow mesenchyma stem cell(BMSCs) transplant. To provide theoretical basis for TCM interferences on BMSCs transplant into cardiac differentiation.(2)Experimental research:①Established the model of rats acute myocardial infarction (AMI) cardiac blood stasis syndrome, use ELISA method to observe the expression about matalloprotease-2 (MMP-2) and matalloprotease-9 (MMP-9);use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K and p38, meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC;Use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the index of cellular differentiation as well as the MMP-2 and MMP-9 and associativity of signal transduction PI3K,p38.②Established the model of rats AMI cardiac blood stasis syndrome and transplanted BMSCs in infarctus cardiac muscule, use ELISA method to observe the expression about MMP-2 and MMP-9, use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K and p38;meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC, use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the associativity between the index of cellular differentiation as well as the MMP-2 and MMP-9 and content of signal transduction PI3K,p38.③Simulated the environment about AMI cardiac blood stasis syndrome in vitro, to cultivate the BMSCs and interfere by apr-YTF, use ELISA method to observe the expression about MMP-2 and MMP-9, use Western-Blots method examine the change about the protein of cellular differentiation signal transduction path PI3K,p38, meanwhile use the immunohistochemistry double-dyed method to measure the Desmin and MHC;Use RT-PCR method to observe the expression about GATA-4 mRNA, and analyze the ssociativity between the index of cellular differentiation as well as the MMP-2 and MMP-9 and content of signal transduction PI3K,p38.Results:(1)After BMSCs transplant there was simultaneous expression of Desmin-Brdu,MHC-Brdu in the cardiac blood stasis infarctus edge area. There was no expression in the sham operation group and health control group. GATA-4 mRNA in the cardiac blood stasis group was higher than that in the sham operation group(P＜0.05),while there was no expression in the health control group.(2)Every part of no BMSCs transplant of MMP-2 cardiac blood stasis syndrome was higher that of sham operation group and health control group. After BMSCs transplant there was statistical difference of rats cardiac tissues between each groups (P＜0.05). The transplant group's expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). Every part of no BMSCs transplant of MMP-9 had statistical differences between each group. Cardiac blood stasis syndrome group was the highest, sham operation group was the next one and health control group was the last one. After BMSCs transplant, the cardiac blood stasis syndrome group was lower than that of health control group and sham operation group. The transplant group's expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). Every part of no BMSCs transplant of p-Akt had no statistical differences between each group.After BMSCs transplant, the cardiac blood stasis syndrome group was lower than that of health control group and sham operation group, the expression of transplant group's was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05).Every part of no BMSCs transplant of p-p38 had no statistical differences between each group. There was no difference between each group's rats cardiac tissues after BMSCs transplant. The transplant group's expression was higher than that no BMSCs transplant of cardiac blood stasis in the same group(P＜0.05). The expression of GATA-4 mRNA,MMP-9 and p-p38 was positive correlation. The incidence coefficient were 0.49and 0.605 while there was no obvious associativity of MMP-2,p-Akt.(3)Apr-YTF interfered in BMSCs transplant of cardiac blood stasis of Desmin-Brdu, the (rhG-CSF)+BMSCs group's positive cell counting were higher than that of NS+BMSCs group. There was no difference between (rhG-CSF)+BMSCs group and NS+BMSCs group. Through MHC-Brdu antibody dyeing, (rhG-CSF)+BMSCs and (apr-YTF)+BMSCs group's positive cell counting were higher than that of NS+BMSCs group. The semi-quantitative IOD of GATA-4 mRNA, NS+BMSCs group as well as (rhG-CSF)+BMSCs and (apr-YTF)+BMSCs group were higher than that of NS+DMEM group;while the (apr-YTF)+BMSCs group was higher than that of NS+BMSCs.(4)Apr-YTF interfered in BMSCs transplant of cardiac blood stasis of MMP-2 and MMP-9, ELISA test showed that the MMP-2 (apr-YTF)+BMSCs group's expression was lower than that of NS+BMSCs group and NS+BMSCs. There was no difference between each group of MMP-9. Through p-Akt Western-Blot, (apr-YTF)+BMSCs's expression was higher than that of in NS+DMEM group NS+BMSCs group as well as (rhG-CSF)+BMSCs group. The p-p38 (apr-YTF)+BMSCs group's expression was higher than that of NS+DMEM group. There was no difference between each other group.(5)After the induction of BMSCs simulated the environment about AMI cardiac blood stasis syndrome in vitro, there was no MHC positive cell been detected in the normal control group and DMEM group, while the weakly positive immunocomplex had been detected in the cardiac blood stasis syndrome group, apr-YTF group, cardiac blood stasis syndrome+apr-YTF in the Desmin and MHC immunohistochemistry dyeing. The IOD of Desmin expressionin the cardiac blood stasis syndrome group and cardiac blood stasis syndrome+(apr-YTF)group were higher than in the apr-YTF group, but there was no statistical difference between the former two ones。The GATA-4 mRNA IOD in cardiac blood stasis syndrome group and cardiac blood stasis syndrome+(apr-YTF)group had statistical differences compared with the normal control group.(6)The MMP-2's mass concentration in the cell culture of simulated the environment of cardiac blood stasis syndrome+apr-YTF group was the highest which had the statistical difference compared with the normal control group DMEM and apr-YTF group. The mass concentration of MMP-9 in the two cardiac blood stasis syndrome group, and cardiac blood stasis syndrome+apr-YTF group were higher than in the DMEM and apr-YTF group.After 28 days of BMSCs' induction, the p-Akt in the signal transduction path was much higher in the cardiac blood stasis syndrome+apr-YTF group than that in the DMEM, apr-YTF and cardiac blood stasis syndrome group. The p-p38's expression in each group had no statistical difference.Conclusion:(1)The microenvironment of myocardial infarction cardiac blood stasis syndrome's cardiac cell can promote the BMSCs cardiac cell cytodifferentiation. Apr-YTF can promote it.(2)The microenvironment of myocardial infarction cardiac blood stasis syndrome's cardiac cell can increase the expression of MMP-2 and MMP-9, promote the BMSCs migration and differentiation. After BMSCs transplant cardiac blood stasis syndrome's cardiac cell microenvironment's content expression lower and thus mitigate the tissure degradation cicatrization and control the deterioration of MI. Apr-YTF can lower the MMP's expression of AMI cardiac blood stasis syndrome in cardian tissure. (3)PI3K signal transduction path in cardiac blood stasis syndrome's cardiac cell microenvironment has been activated. After BMSCs transplant cardiac blood stasis syndrome's cardiac cell microenvironment's cardiac cell differentiation related to the activation of PI3K and p38 signal transduction. Apr-YTF can promote the BMSCs transplant into cardiac cell differentiation and the activation of PI3K and p38 signal transduction.(4)Simulated the microenvironment about cardiac blood stasis syndrome in vitro can induce the BMSCs cardiac cell differentiation. Apr-YTF can induce the BMSCs cardiac cell differentiation in vitro.(5)Simulated the microenvironment about cardiac blood stasis syndrome in vitro, MMP-2 and MMP-9 can promote the BMSCs cardiac cell differentiation. BMSCs differentiation to cardiac cell and apr-YTF's interferences has related to the activation of PI3K signal transduction path.