| Objective:To investigate the isolation, purification and culture of human umbilical cord blood (HUCB) mesenchymal stem cells (MSCs) in vitro, and built stable culture system to satisfy experimental and clinical needs. then the human umbilical cord blood mesenchymal stem cells was transplanted into the rats of acute myocardial infarction (AMI) to observe its survival and the effect for heart function.Methods:1. Human umbilical cord blood samples (n=42) were collected from healthy mothers. All samples were isolated in 8 hours after collected,2. The cord blood mononuclear cell was isolated by lymphocyte separation medium using the Density-Gradient Technique.ALL samples was divided into 3 groups, groups 1 and groups 2 adopted routine culture method of MSCs (Friedenstein method). The MNCs were inoculated with a concentration of 1 X l08cells/ml in a 25T culture flask which adding Low glucose-DMEM containing 10% fetus bovine serum (FBS).group 1 culture plates did not coated with FBS. group 2 culture plates coated with FBS.group 3 culture medium consisted of Mesencult, and its collection and separation of MNC-derived from HUCB was identical with above. 3. The cells were incubated in a homeothermia culture chamber at 37℃, 5%CO2, in a fully humidied atmosphere. After culture for three days the medium was replaced and non-adherent cells were washed out with the all medium changed. Then the all medium changed every seven days. Once adhered cells reached approximately 80% confluence, cells were harvested and replated at 1:1 under the same culture conditions. Different factors was investigated:culture plates coated with FBS,culture medium.4. Detected the second generation of MSCs' immunophenotypes (CD29, CD44, CD45, CD105) With flow cytometry(FCM).5. 36 SD rats were randomly divided into three groups : MSCs transplantation group(n=12),sham group(n=12) and AMI group(n=12). Myocardial infarction(MI)was created by occluding the left anterior descending artery(LAD)in SD rats. One weeks after MI, the labeled MSCs were transfused into MI rats through caudal vein。6. 28 days after transplantation. all the rats were performed invasive left ventricular (LV) hemodynamic measurement. then to detect differentiation of stem cell and by using immunityhistochemistry. FactorVlll was identified by immunohistochemical staining to evaluate the angiogenesis in the injured heart area.Results1. Our results show: using the standard method of culture the MSCs, most of the derived adherent cells morphology was heterogeneous and no obvious CFU were observed. They reached confluence after about 2 weeks, large, round-shaped cells and fibroblast-life cells were prevalent, although these heterogeneous adherent cells could reach confluence in primary passage, their proliferation capacity was limited, they were never passaged beyond the third passage; Through our exploration, HUCB-derived mononuclear cells of full-termdeliveries when cultured in the suitable medium and higher density seeding with coating culture flask, were able to generate adherent cells, which exhibited either an osteoclast- or mesenchymal-like phenotype. With the conditional medium and subculture, the adherent cells can be purified, and gave rise to a well-established layer of MSCs. They sustain prolonged self-renewal,but can not maintaining homogeneous characteristics for over 2 passages. The results showed that among the 42 human umbilical cord blood samples that carried out original MNCs culture, only 8 succeeded in subculture. The original HUCB-derived MSCs had a 2-8days incubation period, and cells became adhesive gradually, but without obvious enlargement; cells came intologarithmicrapidly at this period; then entered :phaseplacid after 10 to 14 days, and proliferated phase about 4 weeks. The amplification of the third generation grew obviously faster than the original ones, which doubled every 2-4 days and with logarithmic phase at 5-10 days and placid phase at 10-14 days.2. The second generation of HUCB-derived MSCs' immunophenotypes were detected with flow cytometer. The results showed the 2thgeneration of HUCB-derived MSCs did not express CD34,CD45 hematopoietic cell marks or weakly expressed, but highly expressed CD29, CD105 mesenchymal cell's relative surface antigen marks. Which proved that HUCB-derived MSC was a kind of undifferentiated stem cel如rogenitor cell existing in human umbilical cord blood, and they were different from hematopoietie cells with identical surface antigen marks of the MSCs.3. After 4 weeks, The invasive left ventricular (LV) hemodynamic measurement result revealed Left heart functions in MSC transplantation group was more obviously improved compared with those of Myocardial infarction group. Immunochemical staining revealed that labeled cells were viable in the host hearts,but the engrafted stem cells can not expressed Troponin-T and connexin43,and immunochemical staining revealed that MVD in MSCs transplantation group was more obviously improved compared with those of myocardial infarction group and Sham group .In the myocardial infarction tissue zone .Conclusions1.Our study demonstrates that it is possible to obtain MSCs from HUCB with remarkable MSCs-specific immunophenotype. , In the condition of a culture medium with mesencult ,a culture flask coated with FBS and an account of more than 1x108 MNC, MSCs in HUCB can be cultivated successfully in vitro with high achievement ratio and purity.2.HUCB-MSCs can survive, migrate into infracted areas of rats with AMI through vein transplantation.3.Transplantation of MSCs stimulates neovascularization and improved heart function on rat heart of AMI .
|
PDF Full Text Request