The Research About Bone Marrow Mesenchyma Stem Cells Induced By Astragaloside Into Cardiomyocyto-like Cells

By 2020 year, according to WHO estimation the un-infectious diseases will account for about 79% of death cause in China, especially cardiovascular disease will rank the first place. Such cardiovascular diseases as myocardial infarction, cardiomyopathy, terminal stage heart failure etc, will severely threaten human health. However, the fundamental reason is that myocardial cell is terminal differentiation cell, and that decrease of functional myocardial cells, necrotic myocardial cells can not regenerate, which only be replaced by scarring so as to obstinate heart failure, even death. Chinese material medica or western medication in clinic, intervene therapy and operation seem not to replace necrotic cells, but cardiac transplantation is very embarrassing to spread in clinic due to complicate operation, donor difficulty, and expensive costs. Therefore, cellular transplantation affords a brandnew therapeutic strategy for cardiovascular diseases treatment, so called that stem cells will be transplanted into lesion myocardial tissues by application tissue engineering technique, which be transformed and replaced for original myocardial cells, and reconstruct the myocardial structure, get over failure cardiac function.The research of recent years finds out, another kind of stem cells lies in marrow stroma called as mesenchymal stem cells(MSCs), which can differentiate into multiple sorts of histocytes in certain surroundings and stimulating factors. In tissue engineering, MSCs should be attached great importance as an important seed cells. And MSCs can be induced into myocardial cells transplanted into necrotic myocardial tissue, which will provide new theoretic support for the treatment of cardiovascular disease integrated with Chinese herbs and stem cells.PartⅠTheoretical ResearchMSCs (mesenchymal stem cells) is stem cells capable of multi-directional differentiation arising from mesoblast, which principally lies in stroma of connective tissues and organs, especially abundant in marrow, even divorted from fetal umbilic blood, differentiates into osteocytes, chondrocytes, tendon cells, myoctes, nerve cells, adipose cells and hematopoiesis stromal cells and so on. Such as stem cells, mesenchymal stem cells also have self-regenerative and multi-directional differentiate capabilities. Although it only accounts for 0.001%～0.01% of marrow monocytes, it has remarkable reproductive ability. In-vitro culture scholars at home and aboard induced passage of MSCs into differentiation by 5-azacytidine. They found out ardiomyocyto-like cells ultramicrostructure, expression of myocardial specific genes, continual action potential, which indicated that they successfully induced cardiac cells. Others injected MSCs into autoallergic myocardial tissue directly, observed some caridomyocyto-like cells formation capable of cardiomyocyte specific marking of troponin T and myoglobulin heavy-chain positive staining, which proved that MSCs can be induced into myocardial cells in microenviroment in vivo.In recent years, some researches proved that TCM could induced MSCs differentiated into neuron cells and osteoblasts effectively and the inductivity was high. Prophase research showed that astragaloside could induce MSCs differentiatiated into cardiomyocyto-like cells.Immunohistochemistry identified Desmin and myocardial specific antibody cTnI expression positive, and RT-PCR detectedα-MHC andβ-MHC expression is positive without difference in induced rate compared with 5-aza.In order to approach MSCs differentiation mechanism induced by astragaloside provided theoretic fundamenatals for clinical application, to explore prospect of application about stem cells of TCM, and to provide theoretic base for TCM therapy of myocardial necrosis, and new cut-point for modernized research of TCM, the study chose Chinese herb monomer astragaloside to induce MSCs of rats in vitro. Whether myocardial specific antibody cTnT and glial fiber acidic protein(GFAP) are expressed or not by immunohistochemistry detection, proved AS could induce MSCs into myocardiocyto-like cells by location direction. Cell differentiation condition was detected, to discuss mechanism of TCM induction; Stem cells transplantation treated acute myocardial infarction, to explore TCM clinical application fundamentals by observation of pathological changes about heart function and cardial tissue of rats.Part two. Experimental study oneObjectives: to explore the mechanism of astragaloside induced mesenchyma stem cells(MSCs) into myocardiocyto-like cells by location differentiation.Methods:7-week age of SPF SD rats purebred, without limitation of female or male, the bodyweight of 200±10g, were provided by animal experimental center of Guangzhou university of CM; MSCs of rats were abstracted by density gradient centrifugation and adherent culture. Amplified in vitro, cultured, MSCs were identified by flow cytometer,and went down to the eighth passage by serial passage. Then MSCs were induced in vitro respectively by group AS (end level 250mg/L) and group 5-aza(end level 10μmol/L). MSC were induced respectively at 24h, 48h, and 72h, and control group were set up, induced only by IMDM.After induction, cultured by fresh complete medium replaced( once per 3 days), cytomorphology changes were observed. After 4 week of culture, GFAP and myocardial specific trponin T(cTnT) were identified by SABC immunhistochemistry kits.Results: MSCs of primary culture appeared adherence at 24h, and showed colony growth at 3～4d. Their pathological transformation were mainly short fusiform shape, with nucleolus. Passaged at 3d, the volume of cells was augmented than primary cells, presented cambiform or fibriform, with transparent kytoplasm and evident nulcleolus. MSCs surface antigens were detected by flow cytometry. The expression of CD34 was negative, and that of CD44 was positive. Induced by AS and 5-aza, cellular shape transformed from fibroblast form to coherent alignment long fusiform with augmented volumn. After 4 weeks, cellular periphery stretched out many long prominence, inequality of size, fusiform, presentation of fiberlike structure and myotubes. Immunohistochemistry identified that: Control group could not find out GFAP, cTnT positive cells without induction, and experimental group with MSCs induced by AS and 5-aza showed positive immune complex by cTnT immunohistochemistry staining, meanwhile with expression of GFAP.Conclusions: MSCs from rats marrow could differentiate into myocardiocytolike cells in vitro by Chinese herb monomer astragaloside, and differentiate into neuron-like cells, which proved multipathway and multi-target of Chinese herbs induction mechanism, and added up theory of stem cells with Chinese herbs.Part 3. Experiment 2Objectives: To explore clinical application fundamentals of stem cells transplantation of myocardial infarction, heart function and cardial tissue pathological changes of rats after transplantation were observed. Methods: each 25 of SPF SD rats purebred, with female or male, the bodyweight of 220±10g, were provided by animal experimental center of Guangzhou university of CM;Rats were divided into 3 groups at random, control group (n=10),model group (n=11),stem cells group(n=10). Except for control group, the other two groups were prepared for acute myocardial infarction model of rats. Color of infarction area grow blanc by macroscopic observation, and ECG showed ST-T changes, which proved the success of myocardial infarction model. Model group was injected into 10μml of normal saline 10 at 4 points cardiac muscle, and stem cells group was injected into sixth passage of MSCs 10μml, density of 1×10~6/L at 4 points cardiac muscle. Thorax was close by layers, and penicillin was injected intraperitoneally after operation, rats raised for 4 weeks. After 4 weeks, chromatic ultrasonic of heart detected heart function of rats, N-BT staining detected areas of infarction of rats, and histopathology detected changes of cardiac pathological changes of rat heart.Results:N-BT staining consequence showed that infarction areas of model group were 25.20±2.18mm~2, and infarction areas of stem cells group were 4.12±2.18mm~2. Compared with model group, stem cells group showed statistical difference. Detection of each group of rats by chromatic ultrasonic of heart about LVESD, EF, SV, CO and so on, showed that compared with LVEDD, LVESD, EF AND and CO of model group, stem cells group had significantly statistical difference P＜0.05. Pathological histological of heart observation found out necrotic myocardium was replaced by fibrous tissue, and formed into scar. But necrosis of myocardium in stem cells group was less, without evident scar of myocardium.Conclusions:MSCs transplantation could improve function of myocardial infarction of rats, decrease areas of myocardial necrosis, and alleviate ventricle reconstruction. Furthermore, it was very important to prevent incidence of congestive heart failure.