| PART I Impact of methodology for collection, isolation and storage of circulating microvesicles on flow cytometric analysis ABSTRACTObjective: To explore the effect of centrifugation, storage conditions and anticoagulant on MV measurements.Methods: We applied calcein-AM staining to label MVs and assessed how pre-analytical preparation, including centrifugation, storage conditions (PFP stored at-80℃for1or4weeks; MVs stored at4℃for3-4days or1week; MVs frozen at-80℃for1or4weeks) and anticoagulant (heparin, EDTA) affect MV measurements by FCM.Results: There were no statistical differences in MV counts between two centrifugation speeds (16000×g and20500x g) or among three centrifugation times (15,30and60min) we employed. No significant distinction was noted in MV counts between the two anticoagulants (heparin and EDTA). The storage of PFP or MVs from heparin-anticoagulated plasma for different periods markedly affected the MV counts and size distribution. However, the counts and sizes of MVs from EDTA-anticoagulated plasma were only influenced when MVs were frozen at-80℃for4weeks.Conclusion:Calcein-AM can efficiently identify MVs from plasma and may be an alternative to annexin V for MV staining. EDTA preserves the MV counts and size better than heparin under calcein-AM staining. PFP centrifuged at16000×g for15min is sufficient to isolate MVs, which enables the batch processing of samples. PFP, rather than MVs, appears to be the preferable mode of sample storage. Our research provides a methodology for MV collection, storage and isolation, to facilitate further investigation of MVs as biomarkers in disease. PART II Correlation between circulating MVs and clinical characteristics in MM patients ABSTRACTObjective: Microvesicles (MVs) have been regarded as a novel biomarker in many diseases, but little is known about the diagnostic or prognostic value of MVs in patients with multiple myeloma (MM). The aim of this study was to explore the correlation between the counts of circulating MVs from MM patients and the clinical characteristics, including bone lesion, renal injury, staging and typing, or biochemical measurements, such as β2microglobulin (β2-MG) and Lactate dehydrogenase (LDH) value. Furthermore, we investigate MV detection in urine and bone marrow samples from MM patients.Methods:We adopt the methods described in part I to collect blood samples from healthy volunteers and patients with MM. Urine samples and bone marrow samples were also collected. MVs were isolated using differential centrifugation. The counts of MVs and the expression of CD molecules on the surface of MVs were examined by flow cytometer (FCM). Then, correlations between MV counts and clinical characteristics were analyzed.Results: We found that CD138expressed strongly on MVs from MM patients than that from healthy volunteers. We defined particles which were less than1μrn in diameter with calcein-AM+and CD138+-positive staining as multiple myeloma-derived MVs (MM-MVs), and discovered that the counts of circulating MM-MVs were not only positively correlated well with bone lesion and renal impairment in MM patients but also with the value of β2-MG and LDH. The staging and typing of MM were not related with the counts of circulating MM-MVs. CD38, another specific surface marker for MM, were used together with CD138to identify MVs from myeloma cells. The counts of circulating CD138+CD38+Vs and the percent in total MVs from patients with MM increased significantly compared with those from healthy controls. Furthermore, the counts of CD138+CD38+MVs positively correlated with the value of β2-MG and LDH. It is acknowledged that CD138-CD19+or CD38+CD45" are markers of cancer stem cell in MM, we define those MVs that are CD138"CD19+or CD38+CD45" staining as MVs derived from cancer stem cell. We discovered that the counts of MVs from cancer stem cell of MM patients were higher than those from healthy controls. Moreover, we noted that the counts of MVs from cancer stem cell correlated well with β2-MG value. MVs isolated from urine and bone marrow of MM patients expressed these markers mentioned above.Conclusion:MM-MVs can be detected in MM patients with FCM. The counts of circulating MM-MVs correlate well with clinical complications and prognostic markers of MM,so circulating MM-MVs may be a novel biomarker for MM in diagnosis and prognosis. This research provides a new perspective for the study about the pathogenesis of MM complications. Furthermore, we initially identified MVs from cancer stem cell in MM, which broadens the research field about MVs in MM. In addition, MM specific markers are expressed on urinary MVs and those MVs from bone marrow samples. Due to the advantages of rich sources and noninvasiveness, urine will be another study hotspot in MM. PART Ⅲ Effect of myeloma cell-derived microvesicles on human kidney proximal tubule epithelial cells and exploration of the mechanism ABSTRACTObjective: To investigate the effect of microvesicles (MVs) shed by human multiple myeloma cell line RPMI8226and U266cells on human kidney proximal tubule epithelial cellline HK-2cells and to explore the mechanism. MVs derived from human chronic myeloid leukemia cell line K-562cells were used as disease control. Methods: RPMI8226, U266and K-562cells were incubated in serum-free medium for24 h and MVs were isolated from these conditioned mediums by differential centrifugation. HK-2human kidney proximal tubule epithelial cells were incubated in serum-free medium for24h before MVs treatment. CCK-8assay was used to detect the effect of various concentrations (1,5,10,50μg/ml) of MVs from different cells on the proliferation of HK-2cells for24h,48h or72h. The apoptosis or necrosis of HK-2cells treated by different concentrations of MVs (derive from RPMI8226, U266and K-562cells) for48h or72h was detected by Annexin V-PI apoptotic kit. Western blotting was applied to detect the expression of proteins, including caspase-3(indicator for apoptosis), E-cadherin, N-cadherin, Vimentin(indicators for epithelial-mesenchymal transition), p-MLKL(indicator for necroptosis) and LC3(indicator for autophagy) in HK-2cells treated with10ug/mL MVs for48h or72h.Results:The proliferation of HK-2cells was significantly inhibited by MM-MVs after48h treatment, while after treatment with MM-MVs for72h, the inhibitory effect of MM-MVs on the growth of HK-2cells was reduced, observed by CCK-8assay. K-562cell derived-MVs had no effect on the proliferation of HK-2cells. Annexin V-PI apoptosis detection revealed that the apoptosis and necrosis of HK-2cells were inhibited by MM-MVs after48h treatment, but after72h treatment of MM-MVs, the inhibitory effect of MM-MVs on the apoptosis of HK-2cells was strongly reduced, compared with control. K-562cell derived-MVs had no effect on the apoptosis or necrosis of HK-2cells. Western blotting indicated that the expression of caspase-3, pMLKL, E-cadherin, N-cadherin and LC3increased after HK-2cells being exposed to MM-MVs for48h, while the expression of vimentin decreased. K562-MVs brought no changes to the expression of these proteins in HK-2cells. After treatment with various MVs for72h, the expression of proteins was similar to the results of48h. We noted that, compared with those treated with MVs for48h, the expression of caspase-3in HK-2cells decreased after treatment with MVs for72h.Conclusion:MM-MVs inhibit the proliferation and promote the apoptosis of HK-2cells after incubation with HK-2cells for48h. After incubation with HK-2cells for72h, the inhibitory effect of MM-MVs on HK-2cells decreased. K562-MVs cannot induce this effect. These results indicate that the effects of MM-MVs on HK-2cells are disease-specific. MM-MVs may be a novel mechanism of renal injury in MM. |
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