| Background and Objective:With ischemic heart disease (IHD) as the most frequent type, cardiovascular diseases have been one of the principal diseases which are seriously threatening human health at present, and myocardial infarction (MI) has become the focal point as the most serious IHD. Many treatments such as medications, intravascular intervention and surgery could bring revascularization of occlusive blood vessel and then improve clinical symptoms of myocardial ischemia and congestive heartfailure (CHF) , but they can not reablement the impaired myocardial cells to improve function of the heart at all. Heart transplantation, which could solve myocardial reconstitution thoroughly, is extremely restricted because of the high specification, donator source, high expense, social ethics and so on.Rencently, the technology about abstraction, purification and exosomatic directional differentiation of stem cells has advanced accompanying with the development of tissue engineering and studies about stem cells. Inducing stem cells differentiation into myocardial cells directionally and then to replace the necrotic myocardial cells by cell transplantation could improve function of the heart in abstracto, which provides a original study trend about MI treatment. But not any stem cell can take on the role, for example, embryonic stem cells are limited by source and ethics principles, and skeletal muscle stem cells have problem in the establishment of electromechanical coupling between host cells and skeletal muscle cells after mature differentiation. Bone marrow mesenchymal stem cells (MSCs) are antecedent of marrow stroma cells (MSCs) and locate at dictyointerstitium, which not only have the capability of differentiating into bone cells, cartilage cells, adipose cells and myocardial cells under specified inducting condition in vitro, but also merits such as convenient obtaining and still maintaining the potency of multi-directional differentiation after continuous passage culture and refrigeration. It has been proved that MSCs can used in autoplastic transplantation with no reject reaction. So, inducing MSCs differentiation into myocardial cells and then to treat MI by cell transplantation has been one of the investigative hot spots.Nowadays, chemical induction is the main direction of researches about inducing MSCs differentiation into myocardial cells , with 5-azacytidine (5-aza) being confirmed as the most effective chemical. But it has been confined in clinical application since that it's transformation efficiency is lower than 20%-30% all the time and has cytotoxic effect. Accordingly, finding the more suitable inductor has been one of the hot spots. Several cytokines such as hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF) have been considered significant in directional differentiation of bone marrow mesenchymal, and sufficient theory and experimentations have indicated that using cytokines as inductors to induce stem cells differentiation into myocardial cells has important value in investigation and application. Meanwhile, cytokines have no cytotoxic effect comparing with chemical materials which have been studied such as 5- aza and can become relative ideal stem cell inductor. Hepatocyte growth factor is a multifunctional cytokine secreted by mesenchymocyte with functions that regulating cell growth and movement, promoting occurrence of many kinds of cells and so on. It is also a myocardial nutrition factor which can strong promote caryocinesis of vascular endothelial cell (VEC) to repair impaired endodermis and suppress apoptosis and tissue reconstitution. It has been pointed out that HGF could induce MSCs differentiation into myocardial cells with a higher induction efficiency, but it's not accepted since the deficiency of considerable duplicate test.Recently, inducing researches about MSCs differentiation into myocardial cells have been mainly confined in cell culture and animal experiment, and reports concerning to abstraction, purification, directional induction in vitro of human bone marrow messenchymal syem cells (hMSCs) have been quite few, and then the reports about HGF induced hMSCs differentiation into myocardial cells are awfully few. According to the above study status quo and taking the hMSCs as object, this study was to approach whether or not HGF could induce hMSCs differentiating into cardiomyocyte-like cells and the concentration of HGF could influence the induction through appling density gradient centrifugation and adherence screening method to separate and purify hMSCs, and then added different concentrations of HGF into the amplified hMSCs to induce them differentiating into cardiomyocyte-like cells.Material and Methods:1. The bone marrow samples were extracted from patients sternum of our cardiovascular surgery from 2007 to 2008 who were noncyanosis congenital cardiopathy. All the patients were selected without diseases in hematopoietic system and this process was licensed by Ethics Committee and permissed by patients.2. Bone marrow about 3-5ml was took suction through punctum sternale and send to laboratory after anticoagulation using heparin (100U/ml). Adding Percoll separating medium pro rate (1.073g/ml) to separate mononuclearcell from marrow by density gradient centrifugation and then to purify hMSCs by adherence screening method, and finally undertook serial subcultivation.3. Groups: The third passage hMSCs were cultivated and divided into 6 groups with the group 1 to 5 were separately exposed to different concentrations of HGF (3,5,10,20 and 40μg/L HGF) and the group 6 as the negative control with no HGF at all. All groups were continuously cultured for four weeks with changing the culture media every three days.4. Consequence assessment: Ultramicrostructure change of all groups' inducer cells were successively observed by electronmicroscope and to examine the myocardium-specific cardiac troponin I (cTnI) and connexin 43(Cx43) by immunohistochemistry after 4 weeks' culture . We definited the cells positive with buffy particles in cytoplasm and the ratio of positive cells of cTnI was counted as transformation efficiency.5. Statistical methods: The SPSS 13.0 software was applied to analyse the data. Several sample means were compared by Kruskal-Wallis H test and Student-Newman-Keuls test was used to analyse the difference between groups, while taking 0.05 as the size of test.Results:1. The findings using electronmicroscope: hMSCs in all groups with HGF emerged proliferation and the appearance change of hMSCs took place after 7 days induced by HGF, which showing as tight growth of cells, obvious augmentation of volume, ratio degression of fusiform cells, orbicular-ovate or cylindric cells as the main and flanking cells getting in touch with each other. After 2 weeks , the link between cells become increasingly compact. But the cell multiplication slowed down 3-4 weeks after culture with flanking cells gradually connecting to myotubiform, ratio of caryon to endochylema manifestly steping down and petty particle structure which localised around cell nucleus emerging in endochylema. At the same time, there were no change in negative control group.2. Detecting the cTnI and Cx43 by immunohistochemistry 4 weeks after cell culture, they did not express in negative control (group 6 with no HGF) while positive express in the 5 groups which effected by HGF. The percentage of cTnI positive cells were 14.24±0.46%, 25.32±1.41%, 38.46±1.41%, 27.38±1.32% and 20.36±2.15% in the order of groups with HGF 3μg/L, 5μg/L, 10μg/L, 20μg/L and 40μg/L. The difference between the 5 groups had statistical significance (P<0.05) with the 10μg/L group was the highest.Conclusion:1. There were myotubule type structure formation between cells which were differentiated from hMSCs induced by HGF, and the cells expressed cTnI and Cx43 which can be considered as the specific appearance of myocardial cell, and the above fact hinted that HGF could induce hMSCs differentiation into myocardial cells.2. The transformation rates from hMSCs to myocardial cells were different using different concentration HGF to induce and the 10μg/L HGF group was the highest. This result indicated that the concentration of HGF could influence the inducing effect and 10μg/L would be the best concentration. |
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