The Paracrine Effects On The Treatment Of Acute Myocardial Infarction By Transplantation Of Bone Marrow Mesenchymal Stem Cells

Objectives:1. To finish the culture and passage of rat MSCs in vitro; to observe whether or not MSCs induced by 5-5-aza in vitro can differentiate into cardiomyocyte-like cells; and to study the effect of radiation on the proliferation and differentiation potency of MSCs.2. To establish the model of AMI in rat, and to investigate the feasibility in transplantation of MSCs after AMI. To evaluate the appearance, structure and function of the heart at the early stage of post-AMI in rats by transplantation of MSCs. To detect the changes of cytokines (VEGF,bFGF,IL-1β) secreted by MSCs in myocardium after transplantation.3. To research the changes of cytokines secreted by MSCs. To evaluate the appearance, structure and function of the heart after AMI by injection of supematant liquid secreted from MSCs, and by intraperitoneal injection of supematant liquid regularly, too.Methods:1. We cultured and passaged MSCs with the method of adherent culture. The MSCs in the third generation was radiated by ⁶⁰Coγwith different dosage(2Gy, 4Gy, 8Gy, 12Gy). The morphous and growth condition were observed by hexamethylpararosaniline stain test. The survival condition of MSCs were observed by trypan blue rejection test. The ability of proliferation was detected by MTT shade selection test. Two groups of MSCs( one group was radiated by 4Gy) were induced by 5-aza. The express of C-TNT andβ-MHC were detected by RT-PCR after 4 weeks.2. Fifty rats were divided into three groups: control group, MSCs group and radiated MSCs(4Gy) group. The heart function were checked by echocardiography pre-operation. The model of AMI was established by ligation of coronary artery with thorax opened. One hour after AMI, different kinds of MSCs were injected into the myocardium, the ECG was recorded synchronizedly. The heart function were checked by echocardiography and hemodynamics after 4 weeks. The cytokines in myocardium were detected by RT-PCR. The histopathology of heart specimens were observed by HE stain, and the differences in densities of small vessels were checked by VIII factor immunohistochemistry stain.3. The culture solution were collected in different irradiated groups and unirradiated group pre-irradiation and post-irradiation. The changes with the contents of VEGF,bFGF,IL-1βin supernatant liquid were checked by ELISA.4. Thirty rats were divided into two groups randomly: the supernatant liquid group and the medium group. The expressions of VEGF,bFGF,IL-1βin myocardium were measured by RT-PCR and ELISA. Other steps and methods were same with the second part.Results:1. The cells in 2Gy, 4Gy groups and unirradiated group grew well. All cells in 8Gy and 12Gy groups were dead after irradiation. The ability of proliferation in 2Gy group was not influenced by irradiation, but in 4Gy group that was depressed , and lasted for more than 3 weeks. We did not detect contractile cells, cell fusion and myotube in both unirradiated group and 4Gy group, which were induced by 5-aza. But C-TNT andβ-MHC were expressed somewhat in unirradiated group, which were not found in 4Gy group.2. The achievement ratio of AMI in rats in second part was 74%, survival rate was 66%. ECG recorded appearance of myocardial ischemia. All indexs postoperation of echocardiogram(LVEF,LVDd,LVDs,FS) in three groups were lower than pre-operation. The cardiac function in MSCs and irradiated MSCs groups improved obviously compare with the control group, the cardiac diameter became smaller than control group. All hemodynamics (LVSP , LVEDP +dp/dt_max,-dp/dt_min,) indexs in MSCs and irradiated MSCs groups were better obviously than control group. Three cytokines in MSCs and irradiated MSCs groups expressed higher than control group. The densities of small vessels in MSCs and irradiated MSCs groups were also higher than control group.3. The contents of three cytokines decreased significantly in all irradiated groups after irradiation. With the increasing in the dosage of irradiation, the secretory volume of cytokines decreased greatly.4. The achievement ratio of AMI in rats in third part was 83.3%, survival rate was 73.3%. All indexs in echocardiogram and hemodynamics in supernatant liquid group were better than medium group. The express of three cytokines in supernatant liquid group increased greatly compare with medium group. We didn't detect three cytokines in the medium group. But with the method of ELISA, these cytokines expressed somewhat in the medium group and control group. The densities of small vessels in the supernatant liqiud and medium groups were different significantly.Conclusions1. Large dosage of radiation exposure(>8Gy) make all MSCs death. Low dosage of radiation exposure will suppress its ability of proliferation for a period of time. Low dosage of radiation expose(4Gy) will suppress or damage MSCs's differentiation potency to myocardial cells.2. Both MSCs with or without the differentiation potency can improve cardiac function obviously after AMI. Transplanted MSCs in myocardium can express many cytokines , such as VEGF,bFGF and IL-1βThose cytokines injected solo can improve the cardiac function, too. So, the paracrine action of MSCs, which should take important role in improving the cardiac function after AMI. The mechanism of prarcrine is complex, neovascularization is the important link.3. MSCs cultured in vitro can excrete many cytokines. ⁶⁰Coγradiation exposure can influence MSCs's ability of paracrine. With augmentation of radiation exposure, the ability of secretory decrease obviously.