The Effect Of Umbilical Cord Mesenchymal Stem Cells On Allogeneic Lymphocytes And The Proliferation And Activation Of Lymphocytes

This dissertation includes two parts:(1) Based on atomic force microscopy (AFM). combined with laser scanning confocal microscopy, flow cytometry and inverted microscope, the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes were being preliminarily investigated. (2)Using the high-resolution and force spectrum of the AFM, detect the changes of the morphology, adhesion force and Young's modulus properties of the different T lymphocytes by stimulation. the cell surface antigen receptor molecules recognition was performed by laser scanning confocal microscopy.In the first part, atomic force microscopy, laser scanning confocal microscopy combinationed with quantum dots, flow cytometry and inverted microscope were used to investigate the effects of umbilical cord mesenchymal stem cells on proliferation and activation of allogeneic lymphocytes. The morphology and biophysical properties of the resting, PHA activated, coculture with hUC-MSCs were analyzed and compared. Lymphocyte cells intercontact with hUC-MSCs, and adhesion on them. HUC-MSCs had a dose dependent inhibitory effect on lymphocyte proliferation induced by PHA was found by CCK-8. FCM showed hUC-MSCs could inhibit CD69 expression on PHA activated T lymphocyte cells from 52.5±4.7%to 37.9±3.4%. Laser scanning confocal microscopy further illustrated that T lymphocytes adhesion on hUC-MSCs.The second part of this paper, on the base of lymphocytes, AFM, flow cytometry, laser scanning confocal microscopy, quantum dots mark, fluorescence microscope were used to study the biophysical properties(the parameters including morphology, membrane nanostructure, membrane pore, adhesion, distribution of antigen receptor molecules and so on) of T lymphocytes stimulated different time and different stimulation, the main results are the following(1):Studyed the cellular surface ultrastructures and nanomechanical properties of acute T lymphoblastic leukemic cell (Jurkat cell) which was treated with staphylococcal enterotoxin A (SEA) at different time and compared the adhesive force of Jurkat cell at different states. The morphosructure and cell membrane of Jurkat cell exposed to SEA changed significantly and the ultrastructures became more complex with the time prolonging. The adhesion force values of Jurkat cells treated with SEA at 24 and 48 h were five times than those of Jurkat cell treated with SEA at 6h. The changes of the ultrastructures and membrane structure of Jurkat cell have caused the change of mechanical function. (2)BCL 11B were inserted into PIRES-EGFP eukaryotic expression vector to construct recombinant PIRES-EGFP-BCL 11B plasmid, which was transfected into human Naive T cell by electroporation. the morphology, ultrastructure, Young's modulus and stiffness of different four Naive T cell changed greatly after transfection, and BCL 11B promote the proliferation of Naive T cell of human.(3)The differences of morphology and surface antigen receptor molecules of resting, PHA or SEA activated human lymphocyte were compared. PBMCs formed colony after stimulation of PHA, and scattered after stimulation of PHA.Volume of activated lymphocytes in both groups were greater than resting group, and the activation polarization occurs during lymphocyte migration to form a membrane protrusion. the mean relative of CD69 expression after stimulation of PHA (39.5±8.7%) were higher than stimulation of SEA(8.3±1.8%), also illustrate that CD3 and CD69 receptors inhomogenously distributing on cell membrane, and the surface receptors CD3 and CD69 of T-lymphocytes activated by SEA forming micro-domain in space.