Experiment On Immunoregulation Of Mesenchymal Stem Cells On Allogeneic T-Lymphocyte

Part I Isolation and Expansion of BM-Derived MSC and Transfection of MSC with pEGFP-C2Materials and Methods:1. Isolation and expansion of MSCHealthy male wistar rat was anesthetized and its tibia was broken. Then bone marrow aspirates were diluted in PBS. Density gradient centrifugation and sticking wall methods were used to obtain rat MSC which were expanded with L-DMEM.2. Examination of surface markers in MSCThe third passage MSC were prepared and flow cytometry assay was used to analyze the surface marker CD34, CD45, CD44 and CD29.3. In vitro adipogenic differentiation of MSCThe third passage MSC were cultured with adipogenic incubation media for 14 d, and then examined by microscope.4. Propagation and identification of pEGFP-C2PEGFP-C2 was propagated in E. coli. DH5 a and then the plasmid DNA was extracted by SDS alkaline lysis method. It was confirmed by restriction enzyme Nhe I and Ase I identification. The tramsfected rat MSC were obtained with pEGFP vector by Tfx-50 and FuGene 6 reagents respectively.5. Transfection of MSC with pEGFP-C2The tramsfected rat MSC were obtained with pEGFP vector by Tfx-50 and FuGene 6 reagents respectively. We compared the transfection efficiencies. After G418 selecting, we obtained the MSC expressing EGFP, which can be detected by confocal.6. MTT testing for cell viabilityThe MTT testing was used to evaluate cell viability 48 h after transfection, so that we could compare two different ways of transfection and find the better way for cell growth.Results:1 , MSC can be successfully isolated and developed in vitro with density gradientcentrifugation method and sticking wall method. The cells grow more slowly but have less non-MSC with the former. At third passage, the purity of MSC is 93% with both two methods2 , According to the flow cytometry assay, MSC at third passage were positive forCD44 and CD29, while negative for CD34 and CD45.3, After incubation in adipogenic inducation media for about 4~5 days, MSC produced lipid droplets. Oil Red O straining revealed red lipid vacuoles and blue nucleus.4, Compared with Tfx-50, transfection by FuGENE 6 showed higher efficiency andcell viability. 5, In the optimum condition: FuGENE 6:DNA=6:1, transfection of rat MSC withGFP displayed a stable and long-term GFP expression in vitro, and untransfectedMSC gradually died in G418 selecting culture. 6, Bright green fluorescence of the transfected cells could be observed underconfocal after 24h of transfection. The transfection efficiency measured by GFPfluorescence was 70% after one month. 7, In culture of one month in vitro, the cells could express high-intensive and stableGFP (for 6 passages), but the fluorescence was obviously weakened after 6passages.Conclusion:1, MSC can be successfully isolated and developed by with density gradientcentrifugation method and sticking wall method. They have their own advantagethemselves. 2, MSC can be identified by virtue of cell morphology, membrane phenotype, anddifferential potential. 3, MSC can be transfected efficiently and safely byliposome reagent FuGENE 6.The transfected cells showed stable and long-term GFP expression in vitro.Part II Experiment on MSC suppressing allogeneic T-lymphocyte proliferationMaterial and methods:1, Preparation of T-lymphocyteRat spleen was removed and ground into homogenate in aseptic condition. The erythrocyte was destroyed by ammonium oxalate and the monocyt was removed by sticking glass method. Finally, nylon wool column was used to get more pure T-lymphocyte.2, Flow cytometry assay of CD25 and CD69 on the surface of T-lymphocyteSample were resuspended and washed with PBS. Then fluorescence-marked monoclonal antibody (anti-CD25 and anti-CD69) was added and incubated for 20 minutes in dark condition before analysis.3, Group of experimentBased on quantity of MSC: 100, 10000, 100000, and control; Based on time of mixed-lymphocyte culture: 24 h, 48 h, 72 h, 96 h and control; Based on different immunosuppressive methods: mixed-lymphocyte culture, culture-conditioned medium, CsA as suppressive method and contr...