Objectives To explore the method for isolation and cultivation of Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs), and prove that ADMSCs can be induced into myocardial cells by 5-azacytidine (5-aza);To prove Rehmannia Glutinosa Oligosaccharides can promote the proliferation of ADMSCs and test the best RGOs concentration which promote the proliferation of ADMSCs at best;To build up the model of swine heart failure after myocardial infarction; and confirm that ADMSCs can survive in vivo and improve heart function after myocardial infarction in swine, and investigate its possible mechanism.
Methods The first part (1) Isolating, culturing and identifying ADMSCs. Third-generation cell washed through phosphate buffer solution (PBS) for surface molecular identification of placebo, CD13, CD44, CD105, CD34, CD45, HLA-DR, VIII; (2) ADMSCs in vitro differentiation and identification: induced by 5-aza, the expression of α-actin, cTnT were examined by immunecytochemical method. The second part: screen out the best RGOs concentration for promoting ADMSC proliferation: put the third generation of ADMSCs and 5-aza induced ADMSCs into different concentrations of RGOs culture medium, measure and draw cell growth curve by counting the number of ADMSCs cells to figure out the best RGOs concentration. The third part: Treatment of heart failure after myocardial infarction with differently treated ADMSCs. ADMSCs marked with fluorescent agent DAPI were injected inside and around the infracted area,respectively. Heart function was checked using UCG in each group 1 week before and 1 and 4 week after the artery ligation. After MI and cell transplantation for 4 weeks, the HE staining, immunohistochemical staining, capillary densities, collagen volume fraction were done.
Results (1) There was a large amount of mesenchymal stem cells in swines adipose tissue. Immunocytochemical staining showed that most of cultured cells were CD13, CD44, CD105 positive. Induced cells became bigger and longer. The connection of the cells was formed. The direction of cell arraying was gradually similar. After the 5-aza treatment, the cells were stained positively for α-actin, cTnT. (2) the best RGOs concentration for proliferation of ADMSCs is 200mg/L. (3) four weeks after transplantation DAPI positive ADMSCs can be seen inside and around the infarction area, and the positive result of α-actin, cTnT immunohistochemical staining indicates the differentiation of transplanted ADMSCs into myocardioyte-like cells; four weeks after ADMSCs transplantation either treated by 5-aza or 5-aza plus RGOs LVEF, LVSP, +dp/dtmax and vascular density increased and LVEDV, LVESV, LVEDP,-dp/dtmax, collagen volume score from infarction and non-infarct District reduced significantly. The effects of latter were even more conspicuous than the former.
Conclusions swines fat tissue contains mesenchymal stem cells, and ADMSCs induced by 5-aza may differentiate into myocardial cells. RGOs can promote the proliferation of the third-generation ADMSCs and 5-aza induced ADMSCs,and the best RGOs concentration are both 200mg/L. After ADMSCs transplanted into the swines, the cells can suviving and differentiate into myocardial cells. Furthermore they can improve heart function after myocardial infarction in canines and the possible reason may be correlate with microenvironment and produce a marked effect by angiogenesis, inhibition MMP and reduction collogen deposition.