| OBJECTIVETo observe the influence of the first replacing culture medium time and different methods to the MSCs.And identified its biologic characteristic.Providing an better project for organise engineering. To investigate the significance of Caspase-3 in anoikis induced by preventing mesenchymal stem cells(MSCs) from adherence. And to model MSCs suspending condition in vitro, in order to investigate MSCs anoikis phenomenon and the role of Caspase inhibitor in MSCs anoikis. And the model of MSCs will be investigated oreigienally.MATHODESWe choose specific pathogen free (SPF) rats, 6W, mean weight 150g, disassociate the femoral and shin bone, then wash the medullary cavity of bone with DMEM and resuspended cells in complete culture medium. Our experiment selects second filial generation MSCs. The cultured MSCs are randomly divided into different groups.And the influence of the first replacing culture medium time and different methods to the MSCs was observed with growth curves, morphologic, immunocyte chemistry, superficial expression, clone rate and filial generation. Before experiment, we coat dishes with 1.5% agarose liquor and waiting for its coagulation; in inhibitor group,DVED-CHO and cells are put into procoated dishes at the same time; control group has not any interventional measure. The cells are collected in three groups at 2h, 6h, 12h, 24h. The alteration of caspase-3 activity is evaluated by Caspase-3 Fluorometric Assay and Western blot analysis, and the apoptosis rates are detected by flow cytometry, repeat the experiment twice. RESULTSWhen the first replacing culture medium time is at 6 hour after resuspended, the MSCs was purified faster, and expressing CD44+ and CD71+, butCD34 was negative. Full marrow adherence culture had more clone rates, cells survival rates and shorter filial time than that of the others. Caspase-3 fluorometric assay shows that Caspase-3 activity in anoikis group at 2h, 6h, 12h, 24h are(9.2197, 7.8514), (12.3425, 16.2718), (24.3826, 25.7443), (22.7386, 24.2836); (2.1754, 2.3675), (2.7910, 2.3925), (3.7428, 3.2769), (4.7564, 5.7938 ) in inhibitor group; And(9.1632, 7.7438), (8.2463, 7.3524), (7.2673, 7.8294), (7.2783, 6.1245 ) in control group. The Caspase-3 activity in anoikis group increase significantly along with the apoptosis conditions going on, but they are lower and stable in the inhibitor group and the control group. Western blot analysis discovers that the Caspase-3 relative expression in anoikis group at 2h, 6h, 12h, 24h is (0.3640, 0.5352, 0.7124, 0.8456); (0.2375, 0.3527, 0.4743, 0.5123) inhibitor group; (0.3923, 0.4976, 0.5272, 0.5154) in control group. The flow cytometry indicates that apoptosis peaks appear distinctly in every group. And apoptosis rate increased dramaticly in the anoikis group and the apoptosis rates are(7.22%, 6.23%), (17.97%, 15.22%), (35.98%, 34.25%), (52.95%, 54.12%) at 2h, 6h, 12h, 24h relatively;In inhibitor and control group, they are(4.4%, 5.2%), (6.2%, 5.8%), (8.7%, 7.9%), (9.8%, 8.5%); and(5.3%, 5.5%), (6.7%, 6.3%), (9.6%, 8.5%), (11.3%, 10.4%). The flow cytometry analysis showd that apoptosis peak was appeared significantly in the induced group and increased dramaticly along with the apoptosis conditions going on.The apoptosis rate of the others were lower and stable.Western blotting and fluorometric assay showed the caspase-3 expressive level or activity in induced group was the highest and was related with its apoptosis rate. The fluorometric assay technique was sensitive to the western blotting.CONCLUSIONWe improved the measure of obtaining MSCs,and the MSCs was purified. Argorose can suspended MSCs efficiently. And MSCs will undergo anoikis in suspended condition if they are separated from extracellular matrix. Caspase-3 was play a vital part in suspended culture induced anoikis of MSCs in vitro. But with Caspase-3 activity suppressed, Caspase inhibitor should reduce the apoptosis rate significantly,and should set up the model of MSCs suspending culture.
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