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Pre-eclampsia is one of the main causes of maternal morbidity in the world. At present, it may be related to insufficient recasting of uterine spiral arterioles, excessive activation of inflammation and immunity, damage of endothelial vascular cells, and so on. Recent studies have shown that microparticle can increase the expression of NF-κB in patients with pre -eclampsia, and promote the formation of neutrophil extracellular trap, resulting in inflammation at the maternal -fetal interface and vascular endothelial cell dysfunction, and participate in the pathophysiological process of pre-eclampsia. [ABSTRACT FROM AUTHOR]

Objective: To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients., Methods: The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF + MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations., Results: The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF + MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF + MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF + MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF + MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05)., Conclusion: The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF + MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

Objective: To explore the correlation of endothelial microparticles and progression of advanced lung cancer, and its predictive value in therapeutic effect. Methods: The data of patients with advanced lung cancer in the Oncology Department of Frist Medical Center of Chinese PLA General Hospital from October 2018 to May 2019 were collected. Blood routine, lactate dehydrogenase (LDH), tumor markers, and circulating endothelial microparticles (CD105+ EMPs) were measured before treatment. Flow cytometry was used to detect the number of CD105+ EMPs, and multivariate regression analysis was used to study the predict factors of advanced lung cancer progression. Results: A total of 88 patients were recruited in the study, including 60 in the objective response (OR) group and 28 in the disease progression (PD) group. There were no significant differences in gender, age, basic diseases, tumor stage, cancer type and therapeutic intervention between two groups, while there were significant differences in tumor marker, LDH, total microparticles (MPs), and endothelial microparticles (CD105+ EMPs) between two groups ( P <0.05). In the multivariate regression analysis, CD105+ EMPs ≥70 events/μl ( OR =3.623, 95% CI =1.345~9.761, P =0.011) and LDH ( OR =1.008, 95% CI =1.001~1.015, P =0.032) were able to predict the progression of advanced lung cancer. A predictive model of advanced lung cancer progression was established based on the multivariate regression results. The area under the receiver operating characteristic curve (AUC) was 0.729 (95% CI =0.620~0.837, P =0.001), the sensitivity was 32.1%, the specificity was 91.6%, the positive predictive value was 64.2%, and the negative predictive value was 74.3%. Conclusion: Circulating endothelial microparticles are associated with the progression of advanced lung cancer, it combined with LDH can predict the therapeutic effect of advanced lung cancer.

Objective: To explore the appropriate procedures for preparing extracellular microvesicles (MV) derived from human mesenchymal stem cells (MSC)., Methods: Human MSCs from umbilical cords were cultured in a serum-free medium and maintained in a basal medium for 72 hours after the cell confluence reached to 80%. The supernatants of cultured cells were collected and MVs were enriched. MVs were identified by flow cytometry and electron microscopy. The total protein amount in MVs was used as a parameter for the content of MVs. The supernatants were adjusted to different pH values, and the output of MVs was detected. The supernatants were also collected for enriching the MV and detecting the protein content of MV after the cells were maintained in the basic medium for different time., Results: Flow cytometric analysis showed that the MVs expressed CD9, CD63 and CD81, morphologically presented round under an electron microscope and the diameter of MV was around 100 nm. After enrichment of MV, the protein content of MVs in the supernatants was 416.8±128.1, 255.4±77.9 and 142.8±46.4 μg per 10 7 MSC，respectively at pH of supernatant 3, 7 and 9 (P＜0.05). The protein content of the supernatants per 10 7 MSC was 173.6±44.5, 262.4±49.6 and 364.2±37.8 μg respectively after starvation culture for 48, 72 and 96 hrs (P＜0.05)., Conclusion: MVs can be readily collected after MSCs were starved for 96 hours, and the pH of the supernatants is adjusted at 3.0.

Extracellular vesicles (EVs), including exosomes and microvesicles, are lipid bilayer-enclosed nanovesicles secreted by cells. These EVs are important mediators of intercellular communication by serving as vehicles for transfer of proteins, mRNA, miRNA and lipids between cells. Various visualization methods have been established to explore the characteristics of EVs and their role in physiological and pathological processes. The nanoscale size and high heterogeneity of EVs hamper the identification of their biological characteristics and functions. This review presents a comprehensive overview of EV imaging methods in light of the origin, separation and dynamic tracking of EVs, and the advantages and disadvantages of different imaging strategies are discussed. We believe that studies at the levels of single vesicles and single cells will become the frontier of future researches of EVs.

Extracellular vesicles (EVs), including apoptotic bodies, microvesicles and exosomes, play a crucial role in cell-to-cell communication. EVs derived from various cell types have the potential to deliver complex information to endothelial cells and to induce either pro- or anti-angiogenic signaling.

Objective: To investigate the effect of daunorubicin on the number and procoagulant activity of Microparticles derived from acute promyelocytic leukemia(APL) cells., Methods: APL cells were isolated from bone Marrow of 5 newly diagnosed APL patients, the bone marrow mononuclear cells were collected from 5 patients with iron deficiency anemia as control.APL cells were treated with different concentration of daunorubicin(0.1,0.5,1.0 and 2.0µmol/L) for 24 h. Microparticles were extracted from the cell culture medium for qualitative anaysis of the extracted microparticles.The morphologic features of the microparticles were observed by transmission electron microscopy.The number of microparticles was detected by flow cytometry.The procoagulant activity of microparticles was measured by recalcification time assays., Results: Under a transmission electron microscope, theextracted microparticles took a round or oval morphology with a transparent center,and their diameters were arund 100nm, consistent with the morphological characteristics of microparticles. Compared with bone marrow mononuclear cells-derived microparticles,the counts of the bone marrow APL cells-derived microparticles significantly increased(P<0.05).Daunorubicin increased the shedding of microparticles in a dose-dependent manner(r=0.73,P<0.01).Compared with normal bone marrow mononuclear cells-derived microparticles,bone marrow APL cells-derived microparticles showed higher procoagulant activity(P<0.05).Daunorubicin treatment enhanced the prccoagulant activity of APL cells-derived microparticles which paralleled the increasing drug concentrations(r=-0.78,P<0.01)., Conclusion: Daunorubicin can promote the release of APL cells-derived microparticles and enhance their related procoagulan activity.

Objective: To detect the serum levels of platelet microparticle (PMP), fibronectin (FN), and von Willebrand Factor (vWF) in acute leukemia (AL) patients with thrombocytopenic and to analyze the relationship of the serum levels of PMP, FN and vWF with bleeding degree., Methods: One hundred and one newly diagnosed AL patients from May 2014 to May 2017 were enrolled the AL group. According to the WHO standard of bleeding stratification, 101 AL patients were divided into 5 sub groups: 0, 1, 2, 3 and 4 score groups; 52 normal persons subjected to physical examination were enrolled in control group. The PMP level was detected by flow cytometry; the FN and vWF levels were detected by ELISA. The levels of PMP, FN and vWF were compared between the AL group and the control group. The serum levels of PMP, FN and vWF were compared according to bleeding degree group. The relationship of bleeding degree with the serum levels of PMP, FN and vWF was analyzed., Results: The patients with newly diagnosed acute leukemia aged 18 to 60, and accounted for 61.39%. The degree of bleeding was mainly 1 score, which accounted for 38.61%. The serum levels of PMP, vWF and FN AL groups were significantly higher than those in control group (6.06%±4.38% vs 0.89%±0.50%, 205.82±24.89 vs 58.04±13.35 µg/L, 398.29±46.93 vs 311.37±26.02 µg/L)(P<0.001). The serum levels of PMP, FN and vWF were different among 5 subgroup (P<0.01); the level of PMP and FN were the highest in 0 score group and the lowest in 4 score group; the vWF level was the highest in 4 score groups and the lowest in 0 score group. The bleeding degree in the patients with acute leukemia negatively correlated with PMP level, and positively with NF and vWF levels (r=-0.753, r=0.648, r=0.805)., Conclusion: According to the relationship of the bleeding degree with serum levels of PMP, FN, vWF in patients, the detection of PMP, vWF and FN levels can help to evaluale the bleeding degree in the patients.

Objective: Acute respiratory distress syndrome (ARDS) is a serious state threaten human health with a high mortality about 30%-40%. At present, there is no effective treatment for ARDS. Microvesicles derived from mesenchymal stem cells (MSC-MVs) have a heterogeneous subcellular structure secreted by MSCs. It plays an important role in the repair of tissue and organ damage. Recent studies have shown that MSC-MVs, played an important role in repairing lung injury, may replace MSC for cell therapy. Therefore MSC-MVs may bring new hope for ARDS treatment.

Objective: To explore the biological characteristics of microvesicles(MV) derived from bone marrow mesenchymal stem cells (BM-MSC) and their capability supporting ex vivo expansion of hematopoietic stem cells(HSC)., Methods: The MV from cultured BM-MSC supernatant were isolated by multi-step differential velocity contrifugation; the morphological characteristics of MV were observed by electron microscopy with negative staining of samples; the protein level in MV was detected by using Micro-BCA method; the surface markers on MV were analyzed by flow cytometry. The peripheral blood HSC(PB-HSC) were isolated after culture and mobilization; the experiment was diveded into 2 group: in MV group, the 10 mg/L MV was given, while in control group, the same volume of PBS was given; the change of PB-HSC count was observed by cell counting; the change of surface markers on PB-HSC was detected dynamically by flow cytometry; the cell colony culture was used to determin the function change of PB-HSC after co-culture with MV., Results: MSC-MVs are 20-100 nm circular vesicles under electron microscope. About 10 µg protein could be extracted from every 1×10 6 MSC. The flow cytometry showed that CD63 and CD44 were positive with a rate of 96.0% and 50.2%, while the HLA-DR, CD34, CD29 and CD73 etc were negative. When being co-cultured with GPBMNC for 2 days, the cell number of MV groups was 1.49±0.15 times of the control group (P>0.05). When being co-cultured for 4 days, the cell number of MV groups was 2.20±0.24 times of the control group(P<0.05). The CD34 + cell number of MV groups was 1.76±0.30 times the control group after culture for 2 day and 1.95±0.20 times after culture for 4 day., Conclusion: The MV has been successfully extracted from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MV can promote HSC expansion in vitro.

Objective: To evaluate the role of microparticle (MP) derived from acute promyelocytic leukemia (APL) cells and tissue factor (TF) carried by the MP in hypercoagulable state, and the effect of treatment with cytotoxic chemotherapy/differentiating agents on procoagulant activity (PCA) of these MP., Methods: Bone marrow mononuclear cells (BMMNC) were extracted from 5 APL patients and 5 sex- and age- matched patients with iron deficiency anemia as controls. The cells were cultured in vitro for 48 h, then MP-rich culture medium and MP-free culture medium were harvested and MP was further obtained from certain volume of MP-rich culture medium. Subsequently, TF expression on MP was measured by ELISA. PCA of MP-rich culture medium or MP-free culture medium was assessed with thrombin generation assay. The role of TF on MP-related PCA was evaluated using anti-human TF antibody. In addition, APL cells were treated with all-trans retinoic acid (ATRA), arsenic trioxide (ATO) or daunorubicin (DNR) for 48 h, then MP-rich culture medium were harvested and the PCA was determined., Results: No TF expression was found in the MP released from bone marrow MNC in control group, whereas the obvious TF expression was found in the MP originated from BMMNC of APL. MP from both APL and control BM-MNC had obvious PCA. However, compared with the MP derived from control MNC, the MP from APL BM-MNC induced significantly higher PCA. TF played a crucial role in the PCA of APL BM-MNC derived MP, while played no role in that of MP from control MNCs. DNR-treating APL BM-MNC resulted in an increase in the PCA of MP, whereas ATO or ATRA exposure lead to exactly the opposite results., Conclusion: MP derived from APL BM-MNC posseses obvious PCA. TF plays a crucial role in the MP-related PCA. The PCA of MP increases after treating APL BM-MNC with chemotherapy agent DNR and decreases following exposure of APL BM-MNC to differentiating agents ATRA or ATO.

Objective: To investigate the effect of Iptakalim (Ipt) preventing injury of endothelial microvesicles (EMVs) derived from hypoxia/reoxygenation (H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism., Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope (TEM). Thoracic aortic rings of rats were incubated with 10 -7 -10 -3 mol/L Ipt and co-cultured with 10 μ g/ml H/R-EMVs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) was recorded in vitro. The nitric oxide (NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinas (Akt) and phosphorylated Akt (p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting., Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles (100-1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner ( P <0.05, P <0.01). The expression of total eNOS (t-eNOS) and total Akt (t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-eNOS and p-Akt increased after treated with Ipt ( P <0.01)., Conclusions: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-eNOS and p-Akt expression.

Objective: To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism., Methods: To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot., Results: The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/ μ l. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment ( P <0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats ( P <0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment ( P <0.01). In addition, the expression of Bcl-2 was increased ( P <0.01), the expression of Bax was decreased ( P <0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment ( P <0.01)., Conclusions: IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

Objective: To investigate the alteration of microparticles (MP) in the recipients following hematopoietic stem cell transplantation (HSCT) and its significance, and to search the early diagnostic indicators of thrombotic complications after transplantation., Methods: According to the occurrence of transplantation-associated complications, 94 allo-HSCT patients were divided into 4 groups: thrombotic group (VOD n = 7, TMA n = 2), acute graft-versus-host disease (aGVHD) group (n = 27), infection group (n = 41) and non-complication group (n = 17). Alterations of serum concentration of tissue factor positive microparticles (TF(+) MP) and endothelial microparticles (EMP) were analyzed by flow cytometry during the process of conditioning treatment and the early stage after transplantation. The relation of these 2 kinds of MP with complications was analysed., Results: (1) The levels of TF(+) MP and EMP of patients undogoing allo-HSCT before conditioning treatment were obviously higher than those in normal controls, and showed some elevation during different times, but there was no significant statistical difference. Although the levels of TF(+) MP and EMP at the end of conditioning treatment were some higher than those before conditioning treatment, but there was no statistical difference between them. (2)The levels of TF(+) MP and EMP in thrombotic group were obviously higher than those in aGVHD group and infection group (P < 0.05). (3)The levels of TF(+) MP and EMP in thrombotic group at different times were significant differences from those in other groups (P < 0.05), and the levels of TF(+) MP and EMP were no significant difference from those in non-complication group., Conclusion: The increase of the TF(+) MP and EMP levels may be associated with occurrence of thrombosis after transplantation, indicating occurrence of the thrombotic complications, like hepatic vein occulusive disease (HVOD). The dynamically monitoring levels of TF(+) MP and EMP contributes to early discovery of thrombotic complications.

Objective: To analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy adults., Methods: The 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking analysis (NTA)., Results: uEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed that the number of 24-hour uEVs amount ranged from 3.56 × 10¹² particles to 5.12 × 10¹² particles, with a CV of 14.23%. The proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49)× 10⁹ particles; the proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a number range of (1.00-1.77)× 10¹² particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86)× 10¹² particles., Conclusion: The established HFD method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies.

Objective: To exam the role of leukemia cells-derived microparticles in the post-complete molecular response stratification., Methods: Blood samples from 29 patients diagnosed with chronic myeloid leukemia (CML) were collected. Microparticles (MP) were extracted from the peripheral blood. Real-time PCR was performed to measure the level of BCR-ABL mRNA., Results: BCR-ABL mRNA could be stably detected both in MP and peripheral blood cells; BCR-ABL in MP showed significant difference within complete molecular response, major molecular response and complete cytogenetic response (9.1±2.8, 25.2±6.9 and 62.8±6.3 respectively, P<0.05). BCR-ABL was detected in MP even when it was negative in peripheral blood cells (3.7-15.3). For patients with complete molecular response, BCR-ABL in MP but not cells were significantly different between imatinib and stem cell transplant recipients (3.3±2.1 vs 9.1±2.8, P<0.05)., Conclusion: This study indicated that MP may serves as a new target for monitoring of CML. Quantification of BCR-ABL in MP may offer a novel strategy for stratification of molecular response.