Bone Marrow Mesenchymal Stem Cells To Optimize Transplantation In The Treatment Of Ischemic Diseases Basic Research

Objective:To explore the feasibility of the clutivation and identification of rat bone marrow-derived mesenchymal stem cells (MSCs) in vitro and analyse their phenotypical properties, to investigate the possibility of a new cell source for ischemic cardiovascular diseases trasnsplantation.Methods: MSCs were isolated from SD rats and expanded by whole bone marrow adherence culture. The growth and morphology of the primary culture and subculture were observed under inverted phasecontrast microscope. MTT method was employed to characterize the potential of proliferation and self-renewal of MSCs. Preliminarily identification of mesenchymal stem cells was used by flowcytometery. Then, MSCs at passage 3 were cocultured with adipogenic culture medium and osteogenic medium respectively. The differentiated cells were identified by oil red O and alizarin red S immunohistochemistry stain respectively. We observed the survival rate and growth state of MSCs with freeze thawing, and observed the efficiency of 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate(DiI) labeling MSCs and the impact of DiI on MSCs.Results:The purified MSCs showed the morphology of firoblasts and proliferated in the culture medium. The MSCs could differentiate into adipocytes and osteoblasts. Flowcytometry showed that they were positive for expression of CD44 and CD90, negative for expression of CD34 and CD45. MSCs were purified at 3rd generation.The survival rates of MSCs thawed after frozen 1 month and 6 months were 93%and 88%respectively.The efficiency of DiI labeling MSCs was 100%.DiI had no influence on the morphology and growth of MSCs.Conclusion:The purification of MSCs can be harvested by method of differential adherence to plastic.These MSCs have the potential of proliferation and self-renewal. This method is easy to operate and simple. Freeze thawing has no obvious influence on MSCs. DiI Can label the membrane of MSCs effectively and has no obvious influence on the morphology and growth of MSCs. Objective:To investigate the effect of simvastatin at different concentrations on the proliferation, apoptosis, paracrine function, cell migration and tube formation function of mesenchymal stem cells (MSCs) in vitro and explore the possible mechanism.Methods: MSCs were isolated from Sprague-Dawley rats and expanded by whole bone marrow adherence culture method. Cells of passage 3were used in all experiments.MSCs were cultured in serum-free medium for 24 hours,then incubated with various concentrations of simvastatin(0,0.001,0.01,0.1,1.0μmol/l) for 24 hours. After that, proliferation of MSCs was measured by MTT assay,release of VEGF by MSCs was assayed using ELISA. MSCs were treated with 0.01μmol/l simvastatin, or wortmannin (PI3K inhibitor) plus simvastatin( 0.01μmol /l ),or culture medium alone,and the influence of simvastatin on MSCs apoptosis was assayed with FACS assay. After being treated with 0.01μmol/l simvastatins, MSCs migration and tube formation function were evaluated by transwell assay and tube formation assay, respectively.Results: Simvastatin at the concertration of 0.001～0.1μmol/l strikingly improved the ability of proliferation and VEGF realease (P<0.01), and the maximum effect was achieved at 0.01μmol /l concentration. When incubated with 0.01μmol/l of simvastatin, the percentage of apoptotic cells decreased and the viability was visibly upregulated. However, pretreatment with 50 nM wortmannin, a PI3-K inhibitor, diminished the anti-apoptotic effect of simvastatin. 0.01μmol /l simvastatin could increase the migrating cells and tube formation numbers of MSCs. Conclusion: Simvastatin at certain concerntrations(0.010～0.1μmol/l) could improve MSCs proliferation and paracrine ability, low-dose simvastatin (0.01μmol/l) could inhibit MSCs apoptosis, and promote migration and tube formation capacity ,and the mechanism might be involved in the PI3K/Akt pathway. Objective: Transplantation of msenchymal stem cells ( MSCs) has been developed as a new method of treating severe ischemia diseases by therapeutic angiogenesis.Statin is capable of inducing angiogenesis and inhibiting MSCs apoptosis.We postulated that combining high-dose simvastatin with bone marrow derived-mesenchymal stem cells (MSCs) delivery may give better prognosis in a mouse hindlimb ischemia model.Methods: Female C57BL/6 mice (n=40) underwent right femoral artery ligation to create a model of hind limb ischemia and were randomly divided into four groups (n=10,each): (1) vehicle administration plus saline injection (control group), (2) simvastatin administration plus saline injection (simvastatin group), (3) vehicle administration plus MSCs transplantation (MSCs group), (4) simvastatin administration plus MSCs transplantation (combination group). Simvastatin administration and MSCs transplantation were performed immediately after hindlimb ischemia was created. Simvastatin (20 mg/kg/d) or vehicle (saline) was administered every day by gavage for 21 days. MSCs (5×106 cells/50μl per mouse) or 50μl saline was injected into the ischemic thigh muscle with a 26-gauge needle at five different points. The blood flow was assessed by laser Doppler imaging at day 0, 10 and 21 after surgery, respectively. Ischemic muscle was harvested for immunohistological assessments and for VEGF protein detection using western blot assay at 21 days after surgery. The number of incorporated DiI-labeled MSCs into the mouse microvascular were detected by fluorescent staining against vWF. Results:The laser Doppler perfusion index was significantly higher in the simvastatin group, the MSCs group and the combination group than in the control group on day 10 after treatment and showed further improvement afterwards on day 21. The LDPI index was the highest in the combination group among the four groups. Combined treatment with simvastatin and MSCs induced a significant improvement in blood reperfusion,a notable increase in capillary density, a highest level of VEGF protein and a significant decrease in muscle cellapoptosis compared with other groups. In vitro, simvastatin inhibited MSCs apoptosis and increased VEGF releaseby MSCs.Conclusions: MSCs transplantation or simvastatin alone could accelerate angiogenesis,development of the collateral vessels and tissue blood flow recovery.Combined therapy with high-dose simvastatin and bone marrow-derived MSCs could augment functional neovascularization in a mouse model of hindlimb ischemia.