| Objective:To explore the feasibility of the treatment of dilated cardiomyopathy (DCM) with heart failure by peripheral vein injection of bone marrow mesenchymal stem cells (MSCs) and the effection on collagen remodeling.Methods:1. Establishment and identification of the animal model of DCM with heart failure85inbred male SD rats (SPF degree, weighing240-290g,8weeks old) were divided into2groups:DCM group (n=65). normal control group (n=20). DCM group of rats were given intraperitoneal injection of doxorubicin (adriamycin, ADR)(2.5mg/kg every time. one time every week for6weeks, total dose15mg/kg); normal control group of rats were given the same injection volume of normal saline instead of ADR intraperitoneal injection. In the10th week after intraperitoneal injection, the two groups of rats underwent echocardiography for left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and left ventricular fractional shortening. At the end of the10th week after intraperitoneal injection,10rats from each group were sacrificed and their myocardial tissue underwent HE and Masson pathological staining, analysis of myocardial collagen and immunohistochemical detection of type Ⅰ and type Ⅲ collagen expression.2.MSCs’abstraction in vitro, cultivation and identificationMale SD rats (SPF degree, weighing50-70g.3weeks old) were selected and their bone marrow from bilateral femurs and tibias was collected. MSCs were abstracted and cultivated by adherent culture in vitro and cell morphology was observed. Then MSCs were purified and amplified. Transmitted to the third generation,MSCs were identified by immunofluorescence assay.3.Transplantation of MSCs for the treatment of DCMDCM group rats were divided into cell transplantation group (n=13), transplantation control group (n=13). DCM blank group (n=12). Cell transplantation group received injection of cell suspension with MSCs via the tail vein. Cell number was5×106each rat and the injection volume was0.5ml every time, a total of10times, once every other day. Transplantation control group received injection of serum-free medium excluding MSCs and the injection volume and method was the same as cell transplantation group. DCM blank group received no intervention. Normal control group (n=10) also received no intervention. Four groups of rats were fed under the same conditions. The four groups of rats underwent echocardiography for left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and left ventricular fractional shortening prior to cell transplantation, the5th week and the10th week after cell transplantation respectively. At the end of the10th week after cell transplantation, all four groups of rats were put to death and their myocardial tissue underwent HE and Masson pathological staining, analysis of myocardial collagen and immunohistochemical detection of type Ⅰ and type Ⅲ collagen expression.Results:1.Establishment and identification of the animal model of DCM with heart failureBy intraperitoneal injection of ADR, DCM group of rats were found different level of heart failure performance such as asthma, cyanosis, edema, reduced activity, decreased food intake. Cardiac ultrasound examination showed that expanded heart chamber and diffuse weakening of the wall activity of DCM group of rats. Their left ventricular end-diastolic diameter and left ventricular end-systolic diameter were significantly larger than normal control group [(7.1±0.3) mm vs.(5.7±0.2) mm,(4.9±0.2) mm vs (2.8±0.1) mm. P<0.05], while left ventricular ejection fraction and left ventricular fractional shortening were significantly lower than normal control group [(66.3±1.3)%vs.(88.6±0.6) %.(30.4±0.9)%vs (51.6±0.8)%, P<0.05]. DCM group myocardial tissue HE staining showed that the myocardial fibers arranged in disorder and normal structure of some of myocardial fibers disappeared with hyperchromatic nuclei and infiltration of inflammatory cells; myocardial tissue Masson staining showed a large number of blue-stained collagen fibers arranged with red-stained myocardial fibers. The analysis of myocardial collagen showed that collagen volume fraction of DCM group was (28.24±1.28)%, significantly higher than normal control group (3.16±0.51)%. The difference between the two groups was statistically significant (P<0.05). Immunohistochemical analysis showed that a large number of tan type Ⅰ and type Ⅲ collagen expression in the myocardial interstitium of DCM group of rats;the ratio of type Ⅰ and Ⅲ collagen of DCM group (1.34±0.02) was higher than normal control group (1.10±0.02) and the difference between the two groups was statistically significant (P<0.05).2. MSCs’abstraction in vitro, cultivation and identificationAdherent cells were triangular, fusiform or polygonal at first. By the cell passage, the morphology of adherent cells gradually tended to be uniform, and orderly arranged with a certain direction. Transmitted to the second generation, the growth of adherent cells showed a single form of swirling circling arrangement. The third generation of cells were identified by immunofluorescence assay. It showed that cell surface antigen CD44expression were positive while CD34were negative, according with the cell surface antigen characteristics of MSCs. Combined with adhesion properties and morphological observation of the cells, MSCs could be clear.3.Transplantation of MSCs for the treatment of DCMAfter intravenous injection of MSCs, heart failure performance such as asthma. cyanosis, edema, reduced activity, decreased food intake of cell transplantation group of rats improved significantly. In the10th week after the last cell transplantation, cardiac ultrasound examination showed that left ventricular end-diastolic diameter and left ventricular end-systolic diameter of cell transplantation group [(6.3±0.2) mm.(3.7±0.1) mm] were significantly lower than transplantation control group [(7.1±0.3) mm (5.0±0.2) mm] and DCM blank group [(7.1±0.2) mm.(5.0±0.1) mm](P<0.05). while left ventricular ejection fraction and left ventricular fractional shortening [(79.6±1.7)%,(41.2±1.7)%] were significantly higher than transplant control group [(65.7±0.9)%,(30.0v0.6)%] and DCM blank group [(66.1±0.9)%,(30.3±0.6)%](P<0.05). Myocardial tissue HE staining showed that compared with transplantation control group and DCM blank group, morphological structure of myocardial fibers of cell transplantation group obviously improved. Myocardial tissue Masson staining showed that compared with transplant control group and DCM blank group, blue-stained collagen fibers of cell transplantation group significantly reduced and replaced by red-stained myocardial fibers. The analysis of myocardial collagen showed that collagen volume fraction of cell transplantation group was (15.92±0.99)%, significantly lower than transplantation control group (29.25±1.21)%and DCM blank group (29.28±1.16)%. The difference was statistically significant (P<0.05). Immunohistochemical analysis showed that compared with transplantation control group and DCM blank group, tan type Ⅰ and type Ⅲ collagen significantly reduced in the myocardial interstitium of cell transplantation group of rats;the ratio of type I and III collagen of cell transplantation group was1.23±0.02. lower than transplantation control group (1.35±0.02) and DCM blank group (1.35±0.02). The difference was statistically significant (P<0.05).Conclusion:1. The animal model of DCM with heart failure could be successfully induced by ADR intraperitoneal injection (2.5mg/kg every time, one time every week for6weeks, total dose15mg/kg), which was highly consistent with pathology and pathophysiological process of clinical DCM with heart failure.2. MSCs were abstracted and cultivated by adherent culture in vitro successfully and then were purified and amplified. By imrnunofluorescence assay, adhesion properties and morphological observation of the cells. MSCs could be clear.3. By transplanting allogeneic MSCs cultured in vitro via peripheral vein repeatedly, myocardial collagen network remodeling of DCM rats with heart failure reduced, and then cardiac function improved to a certain extent. |
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