| Objective: By detecting the count and proportion of specific MVs in different stages of myeloma patients,and to master the changes of MVs in different disease states,and to find a novel biomarker for the diagnosis and prognosis of myeloma.Methods:1 We collected peripheral blood and bone marrow liquid from healthy volunteers and patients with different stages of MM.2 MVs in peripheral blood and bone marrow were isolated by differential centrifugation.3 Morphology of the MVs were observed by transmission electron microscopy.4 Flow cytometry was used to detect the quantity of MVs,and the proportions of plasma cells derived MVs(CD38+ CD138+),endothelial cells derived MVs(CD105+CD45-)and platelet derived MVs(CD41+CD105-)were analyzed.The flow cytometry can clearly distinguish 1.0um,3.0um standard microspheres,according to the literatures,the minimum detection limitation of the flow cytometry is 200 nm,1.0um standard microspheres were applyed in the detection for setting of FSC and SSC,3.0um standard microspheres were used as internal reference for counting.We labeled the MVs with Calcein-AM and set the diameter to less than 1.0um for MVs.5 The count of MVs and the expression of CD molecules on the surface of MVs were collected,statistical signifcance was evaluated across the different groups and subgroups of patient,s including healthy volunteers,multiple myeloma patients in remission,initial treatment multiple myeloma patients and recurrent multiple myeloma patients.The correlations between the proportions of MVs and clinical characteristics were analyzed.Results:1 We used the differential centrifugation method to obtain the precipitate.Morphology of the MVs from peripheral blood myeloma cells observed by TEM,TEM results showed that microvesicles had membrane structures and complex compositions,with diameter of 0.1-1um.This is consistent with the morphology and size of MVs secreted by other cells reported at home and abroad so far.2 Application of BECKMAN flow cytometry can accurately count the number of MVs in the specimen.We found that the number of MVs in peripheral blood and bone marrow of patients with multiple myeloma was significantly higher than that in healthy volunteers(P<0.05),recurrent patients seemed to have a stronger ability to release MVs compared to initial treatment patients(P<0.05),and the number of initial treatment patients was significantly higher than that of remission patients(P<0.05).Compared with peripheral blood,bone marrow aspiration showed a higher level of MVs,the difference was statistically significant.3 CD38 and CD138 were used to label plasma-derived MVs in multiple myeloma cells,the proportion of CD38+CD138+-MVs in peripheral blood and bone marrow fluid of patients with multiple myeloma was significantly higher than that of healthy volunteers(P<0.05),the proportion of CD38+CD138+-MVs in recurrent patients was significantly higher than that in initial treatment patients(P<0.05),and the proportion of CD38+CD138+-MVs in initial treatment patients was significantly higher than that in patients with remission(P<0.05).The ratio of CD38+CD138+-MVs was positively correlated with the value of the tumor load index ?2-microglobulin and LDH(Bone marrow fluid ?2-MG:r =0.601,P= 0.005)(Bone marrow fluid LDH : r=0.720,P=0.000)(Peripheral blood ?2-MG: r=0.729,P=0.029)(Peripheral blood LDH :r=0.621,P=0.018).The stages and types of MM were not related with the proportions of CD38+CD138+-MVs.4 It is acknowledged that CD105+CD45-was markers of endothelial cells secreted by MVs and CD41+CD105-were used to label platelet-derived MVs.We discovered that the proportion of endothelial cells(CD105+CD45-)and platelet-derived MVs(CD41+CD105-)in peripheral blood and bone marrow fluid MVs of patients with multiple myeloma were significantly higher than those from healthy controls(P<0.05),the percentage of MVs derived from endothelial cells and the proportion of platelet-derived MVS in recurrent patients were significantly higher than those in initial treatment patients(P<0.05),and the ratio of endothelial cells derived MVs and platelet-derived MVs were significantly higher in patients with initial treatment patients than those in relieved patients(P<0.05).The tumor burden index ?2-MG,LDH,and MM type,stage were not related with the proportion of endothelial cells(CD105+CD45-)and platelet-derived MVs(CD41+CD105-)in peripheral blood.Conclusions:1 The count of MVs and the ratio of MVs in peripheral blood and bone marrow of patients with multiple myeloma were closely correlated with the disease status of patients with multiple myeloma.2 The proportion and count of endothelial cells and platelet-derived MVs in peripheral blood and bone marrow fluid of patients with multiple myeloma were significantly higher than those from healthy controls.Suggesting that endothelial cell and platelet activation may play an important role in the development of the disease,and as a potential cause of high thrombosis in patients with myeloma. |
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