| Background Myocardial infarction (MI) is a disease which heavily threatening the people’s healthy. In general, One of five adults will suffer from cardiovascular disease in our country, and the mortality is climbing up year by year. At present, the treatment for the MI is mainly depending on the medical therapy and surgery, which could not regenerate the infarcted myocardiocytes and was not sufficient to reverse the progression of heart remolding. Stem cell transplantation emerged as an alternative therapy, which aimed to enhance the regeneration of myocardiocytes and vascular endotheial cells, reduce the cardiac apoptosis and improve the heart function. Adipose derived mesenchymal stem cell (ASC) is a hotspot as it has a bit of incomparable advantages. The safety and effectiveness of ASC in treating MI has been proven by numerous studies, however, there are still some concerns relating with the stem cell survival and its function after transplantation. In order to promote the efficiency of transplantation, precondition before transplantation were studied.Hypoxic preconditioning is a simple and effective way to promote the survival of stem cells. ASC is cultured in the hypoxic condition in order to adapt to the microenvironment of the infarcted heart.The ability of antiapoptosis and hypoxic endurance of ASC would be enhanced which would help it to survive. In addition, the secretion of growth factors would be increased to help to repair or regenerate the injured heart.Cord blood mononuclear cells (CBMNCs) can improve the microcirculation in the infarcted heart, by promoting angiogenesis and vascular endothelialization. New formed microvascular would bring oxygen and nutrients to the blocked region and help the implanted stem cell to survive. Therefore, the combination of hypoxic precondition and microenvironment improvement would be a new way to facilitate the survival and function of ASC.Aim1. To study the culturartion, identification and induction of ASC, as well as the separation and cryopreservation of CBMNCs;2. To investigate the gene expressions of ASC after hypoxic precondition. Furthermore, to analyze the related gene expression after HP-ASC were culture in the simulated microenvironment for 5 days;3. To evaluate if HP-ASC was more efficienct in treating MI rats than ASC;4. To assess whether the efficiency of HP-ASC combined with CBMNCs was superior to HP-ASC or CBMNCs alone in treating MI rats.Method1. Adipose tissue were obtained from the liposuction of a health man. It was digested by collagenase and centrifuged for the sediment of stromal vascular fraction (SVF), then the cell was seeded in the culture bottle. The growth curve was graphed according to the cell densities which was measured by MTT method; The phenotype of ASC was detected by flow cytometry (FCM); With lineage-specific differentiation media, ASC was induced for adipogenesis, osteogenesis and chondrogenesis, which was finally detected by histochemical staining and immunohistochemical (IHC) staining. In addition, CBMNCs were separated from human cord blood, which was collected from a health pregnant woman. Total cells were counted and the viability of the cells were assessed. CBMNCs were cryopreserved for future use.2. ASC (P5) were preconditioned in hypoxic condition (HP-ASC) for 24h, and total RNA were extracted after that. Gene expressions were compared between HP-ASC and ASC; Then, we produce the media which simulated the microenvironment in MI heart. The heart was harvested and grinded 3 days after MI, and blended with serum, essential amino acid and antibiotics. HP-ASC or ASC were cultured in the media for 5 days, and then RNA was extracted for Rt-PCR. Gene expressions were compared between treated HP-ASC, treated ASC and non-treated ASC;3. Thirty two-month old female Wistar rats were randomly assigned into three groups: HP-ASC (n=10), ASC (n=10) and PBS (n=10). The left anterior descending branch of the coronary artery were ligated to establish the MI model. Stem cell (total amount: 2×106) or PBS were injected into the myocardium at the border region of the infarcted site. The hearts were harvested 7d or 15d after cell transplantation to produce cryostat or paraffin section. Among these groups, the general morphological change, the ratio of myocardial infarction area to left ventricular area, the survival rate of the transplanted cells, the apoptotic cell amount at the ischemic region and the density of the new vascular formation were compared.4. Sixty two-month old female Wistar rats were randomly assigned into four groups: HP-ASC (n=15), HP-ASC+CBMNCs (n=15), CBMNCs (n=15) and PBS group (n=15). About 2×106 cells were injected into peri-myocardial infarction area. Echocardiogram were performed at 3d and 30d after cell transplantation. At 7d,15d and 30d, the hearts were harvested to analyze the infarct size by Masson trichrome staining; to assess the myocardium apoptosis by TUN EL staining; and to evaluate the engraftment of stem cells and angiogenesis by IHC.Results:1. The primary ASC began to adheren and grow 6h after seeding, and formed colony forming unit (CFU) 24～48h later. The cell showed a typical fibroblast-like appearance after three passages, with uniform spindle shape, and grew in wiorls. The logarithmic phage of ASC was 3-7d after passaged, and the platform stage started thereafter. FCM detection showed that the phenotype of ASC were negative in CD34 and CD45, but positive in CD90, CD 105, CD73 and CD44. Cell differentiation test showed that ASC can be induced for adipogenesis, osteogenesis and chondrogenesis. The total number of CBMNCs reached 8.9×109 after separation, with the viability of 93.16%.2. Compared with ASC, several growth factors and their receptors were upregulated in HP-ASC. The expression of Klf4 and Oct4 were increased, but Nanog and Sox2 were decreased in HP-ASC. The expression of HO-1 and SDF-1, which were induced by hypoxia, were increased in HP-ASC by 38% and 98%. The immigrating and homing gene, such as CXCR4, ICAM-1 and ICAM-2 were upregulated but TIMP-1 was downregulated in HP-ASC. After cultured in the simulated condition media, HP-ASC-T enhanced the expression of cardiac specific markers (CX43, FABP4 and GATA-4) and angiogenin factors (VEGF). In addition, the growth factors and their receptors were also increased in HP-ASC-T.3. Compared with PBS, HP-ASC and ASC can both reduce the size of infarcted myocardium, decreased cell death and apoptosis of implanted cells, and increased cell survival and angiogenesis. And HP-ASC is more effective than ASC.4. Four weeks after injection, echocardiography showed significant improvements for cardiac functions and left ventricular dimensions in all the stem cell-treated groups (HP-ASC, HP-ASC+CBMNCs and CBMNCs), which were better than the PBS group. Among these groups, HP-ASC was more effective in improving EF and FS than the other groups. Histological detection consolidated that HP-ASC treated rats had better improvement in scar areas and the wall thickness, surviving cell amount and angiogenesis. These data suggested that the transplantation of HP-ASC was superior than the combination of HP-ASC+CBMNCs or CBMNCs alone. It was a feasible and efficacious method for improving infarcted myocardium.Conclusion:1. The phenotype of ASC was CD34 (-)/CD45 (-)/CD90 (+)/CD 105 (+)/CD73 (+)/CD44 (+), which was in accordance with the phenotype of MSCs. Multi-lineage differentiation potential of ASC was proved for adipogenesis, osteogenesis and chondrogenesis.Furthermore, CBMNCs were harvested with high quality and quantity with Ficoll-Paque method.2. The expression of growth factors and their receptors would be upregulated in HP-ASC under hypoxic precondition. And the expression of cardiac specific markers, angiogenin factors, growth factors and their receptors would be increased in HP-ASC under simulating microenvironment.3. Compared with ASC and PBS, HP-ASC was more effective in reducing the size of infarcted myocardium, decreasing cell death and apoptosis of implanted cells, and increasing cell survival and angiogenesis.4. HP-ASC was more effective in improving heart function than the other groups. HP-ASC was superior in improving scar areas and the wall thickness, increasing surviving cell amount and angiogenesis than the combination of HP-ASC+CBMNCs, CBMNCs and PBS. |
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