The Study On The Biological Characteristics Of The Rat Bone Marrow Mesenchymal Stem Cells Which Were Labeled By PKH26 And Differentiated To Vascular Endothelial Cells

BackgroundCell marker is the key issues of mesenchymal stem cells (MSCs) in vitro and in vivo studies. It is reported PKH26 labeling technique is simple, easy to detect, high rate of cell marker, and the fluorescence can be observed after the PKH-26 labeled cells were transplanted into body within 60 days. The similar reports are rare in our country.Systemic vasculitis in connective tissue diseases is easy to violate multiple systems and organs. The rate of incidence is high. If the patients with systemic vasculitis are not treated in time, often their life was often threatened. The main pathogenesis is vascular endothelial cell injury and inflammatory cell infiltration. It is found that MSCs have property to differentiate into vascular endothelial cells under appropriate conditions, immune regulation, to participate in the reconstruction and repair organizations, which for the regulation of vascular inflammation in patients with immune disorders, to repair damaged endothelial cells has brought new hope.Objective1. To explore the biological characteristics of the PKH26 labeled rat bone mesenchymal stem cells (rBMSCs). We establish a simple,practical and rapid method of labeling rBMSCs, which could track the removal, location, distribution and differentiation of MSCs in vivo.2. The rBMSCs were induced to differentiate into vascular endothelial cell, which provided a new method for clinical treatment of rheumatoid arthritis without symptoms of joints and vasculitis.Methods1. The isolation, culture and subculture of rBMSCs Rat bone mesenchymal stem cells were isolated from the bone marrow of Wistar rats with density gradient centrifugation and screening the cells adherent onto the glass surface, and then cultured in L-DMEM medium supplemented with 10% fetal bovine serum. The medium was changed half after 72 hours and then 2 times per week. When cell fusion was to 80%, the rBMSCs were digested by 0.25% trypsin for 2-5min and passaged at a rate of 1 to 3 for expansion.2. Identification of rBMSCs CD45 is hematopoietic stem cell-specific surface marker and CD31 is bone marrow endothelial cell-specific surface marker. The above-mentioned two kinds of antigens were negative in bone marrow-derived MSCs. MSCs do not have specific surface markers, but express CD44 and CD71. Flow cytometry detects rBMSCs'surface antigen CD44, CD71, CD31 and CD45 expression, through the exclusion of the cultured cells for identification of rBMSCs.3. Detection of PKH26 labeling rBMSCs in biological characteristics PKH26 marked the 3rd generation rBMSCs. We observed their shape under fluorescence microscope cytomorphology, the percent of labeling rBMSCs by flow cytometry. MTT Determination decected growth curve of rBMSCs. Then we compared two groups of cell population doubling time. Flow cytometry detected cell cycle and we compared cell proliferation index between the two groups. We compared two group cells'flow surface antigens CD44, CD71, CD31 and CD45.4. RBMSCs induced in vitro differentiation to vascular endothelial cells The cells at passage 3 were induced to differentiate into vascular endothelial cell. After 14 days, we test the expression level of CD31 by Fluorescent Activated Cell Sorting and the factorâ…§in immunohistochemistry.Results1. The surface antigens were detected by Fluorescent Activated Cell Sorting and the results showed that CD44 was positive, CD71 was weakly positive, CD31 and CD45 were negative. The above results proved the cells were MSCs.2. PKH26 marked rBMSCs were spindle, with rules, parallel, vortex-like growth under inverted phase contrast microscope, there was no significant change between PKH26 labelled and non-labelled rBMSCs. By flow cytometry rate of cell marker was 91.03% when the 10th days rBMSCs were labeled, the fluorescence can be observed when 40th days. T test results showed that two group cells'cell population doubling time and proliferation index did not have statistically significant differences(p>0.05). the results of flow cytometry showing two group cells were CD44 positive, CD71 weak positive, CD31 and CD45 negative. 3. The cells at passage 3 were induced to differentiate into vascular endothelial cell in vitro. After 14 days, the expression level of CD31 increased by 37.36% and the factorâ…§was positive in immunohistochemistry.Conclusions1. PKH26 labeling method is simple, the high rate of cell marker, marking time is about six weeks, did not affect the biological characteristics of rBMSCs. For scientific research which needs a large number of labelling rBMSCs, we provide a practical, simple, rapid method of cell marker.2. The cells at passage 3 could be differentiated into vascular endothelial cell in vitro. We provided a new method for clinical treatment of systemic vasculitis.