| Part OneThe influence of donor-derived mesenchymal stem cells infusion on the immunological status in heterogenous recipientBackgroundThe defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cells types. Traditionally, stem cells have been divided into two major groups: 1) embryonic stem cells, derived from the inner cell mass of fertilized ova which can differentiate any cells tyupe, 2) tissue-specific stem cells, derived from specific organ, which were believed to have differentiation capacity limited to the original organ but now are recognized to be capable of differentiation into cells of other tissues. Mesenchymal stem cells(MSC), also know as bone marrow stromal cells, are capable of differentiation to marrow and non-marrow cell types, including: adipocytes, chondrocytes, osteocytes, myocytes, astrocytes and neurons. MSC are capable of substantial proliferation and expansion in culture.MSC express varieties of epitope, including adhesion molecules, growth factors and cytokine receptors, integrins, additional markers, and produce IL-6, IL-7, IL-8, IL-11 ,G-CSF,GM-CSF, DSF, SCF and so on. By virtue of their potential to differentiate into tissues such as bone, cartilage, fat and muscle, they have been investigating in the field for cellular treatment, tissue engineering or transplantation.Nowadays, a potential role for MSC in immunoregulation is paid further attention. The latest study showed that MSC not only prevent the occurrence of graft failure, but play a role in the complex immunoregulation of T and B cells. However, theimmunological status induced by MSC infusion has not been clearly described.Objective To observe the influence of MSC culture in vitro on the immunological status in heterogenous recipient.Materials and Methods Animal selectionHealthy male guinea pig, weighting between 150g to 180g, were used as donor, And healthy male Wister rat, weighting between 180g to 220g, as recipient. MethodsBone marrow aspirates were diluted in PBS. The mononuclear cell fraction obtained by centrifugation over Percoll was cultured in vitro at L-DMEM. Cell surface epitope were analyzed by flow cytometry technique with the CellQuest Software Program. MSC at passages 3 were incubated at the addition of adipocyte induction media, and stained in Oil Aed O. MSC at passages 5 were observed by fluorescence microscope after stained in AO. 24 Wister rats were randomly divided into two groups after CyP infusion. Group A were infused into MSC and Dexamethasone, Group B were infused into natural saline and Dexamethasone. 1 ml blood was taken out before the infusion and at 12d,15d,18d and 21d. The immunoglobin and competent in recipient serum were assayed. With SPSS 10.0 statistical software, and the differences among the groups were compared with the One-Way ANOVA . A P value less than .05 was considered statistically significant.ResultsAfter density gradient centrifugation, the bone marrow specimen was divided into three layers. The bone marrow mononuclear cell population collected at the Percoll interface. In growth media, MSC are long, flattened cells of a fibroblastic morphology. After passages 10, MSC grew slower than before, gradually stopped.According to the flow cytometry assay, MSC were positive for CD44, CD29 , while negative for CD34,CD45. After incubation in adipogenic inducation media, MSC produced lipiddroplets.Oil Red O straining revealed red lipid vacuoles and blue nucleus.According to the observation of fluorescence microscope at 604nm wavwlengh, membrane and plasm of vigor MSC exhibit red fluorescence, and nucleus green or yellow fluorescence. The concentration of IgG, IgM, C3 at all time point after MSC infusion significantly decreased than before infusion and control, and gradually returned back. The concentration of IgA and C4 have not significantly chranged.Conclusion1 It is feasible that MSC expand in vitro culture.2 MSC can be identified by virtue of cell morphology, membrane...
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