Microvesicle-based Liquid Biopsy And Hematological Malignancy Cells Derived Microvesicles Effect On Endothelial Cells

Objetive:Annexin V is the marker of micro vesicles.Flow cytometry can effective detect the annexin V-positive microvesicles.The purpose of this experiment is compare the different solutions,water,saline,and phosphate-buffered saline(PBS)dissolved the mircovesicles pelltes,which solution is the more suitable to detect the Annexin V-positive rate and microvesicle counts analyzed by flow cytometry.Method:To extract microvesicles from the supernatant of cells through gradient centrifugation.Using different solutions,water,saline and PBS dissolved the microvesicle pellet,added the Annexin V antibody and Annexin V binding buffer,incubated 30 minutes,flow cytometry analyzed the microvesice counts and Annexin V-positive rate.Result:PBS and saline dissolved the microvesicle,respectively,and the flow cytometry analyzed Annexin V-positive rate was significant higher and the number of nano-sized vesicle was increased significant(p<0.01).Conclusion:There were chemical reaction between PBS and Annexin V binding buffer,the production of insoluble Ca(H2P04)2 or Ca3P04 from the phosphate and calcium in PBS and Annexin V.Such calcium-phosphate microprecipitates could increase non-specific binding of the fluorescent probe leading to false-positive detection.Thus,considering the osmotic pressure of water,we suggest that saline is a more suitable buffer when counting MVs by flow cytometry.Objective:1 Establish the methodologies that flow cytometry analyzed light chain restriction of microvesicles,and to explore the potential clinical value.2 To explore the gene rearrangement of microvesicles from the lymphoma cell line and lymphoma patients and potential clinical value.Method:1 To extract microvesicles from the supernatant of cells through gradient centrifugation.CD 19-positive magnetic beads positive sorting the microvesicles,and labeled the kappa and lambda mixed antibody.Flow cytometry analyze the monoclonal light chain of CD19-positve micro vesicles from the lymphoma cell lines.2 To extract microvesicles from the supernatant of cells and serum of lymphoma ptients through gradient centrifugation.Using qiagen microDNA extract kit extracted the microvesicle DNA,DNA amplification was performed using the Genome Amplification Kit.Amplified DNA was used to gene rearrangement detection.Result:1 After the CD 19 magnetic beads sorting,the CD 19 positive microvesicles have light chain restriction analyzed by flow cytometry.2 Microvesicles DNA extracted from supernatant of lymphoma cell lines and serum of lymphoma patients for gene rearrangement detection,monoclonal peaks were detectd.Conclusion:Micro vesicle may be used as a microcosm of cell.Micro vesicles maybe could instead of cells in disease diagnosis and could provide the clinical date and technical support for further development the microvesicle-based liquid biopsy.Objective:1 Comparison of miRNA sequencing in lymphoma cells and corresponding microvesicles.2 Differentially expressed miRNA were analyzed by bioinformatics tools,the target gene predictin and gene enrichment analyses were performed.Finally to explore the target gene and miRNA associated with tumor growth.Method:To extract microvesicles from the supernatant of cells through gradient centrifugation.RNA was extracted from the lymphoma cell lines and corresponding microvesicles and miRNA sequencing were performed after the RNA quality qualified.Looking for target miRNA,predicting target gene,and gene enrichment analysis were completed by bioinformatics analysis.Result:There were 801,1076,679,and 1031 miRNA were detected in oci-ly1,oci-ly 10,and microvesicles from oci-ly1 and coi-1y10,respectively.There were 24 and 28 miRNA co-expressed in two cell lines and corresponding microvesicles,6 miRNA up regulated and 13 miRNA down regulated in cell lines,and 5 miRNA up regulated and 19 miRNA down regulated in corresponding microvesicles.Up-regulated miRNA and down-regulated miRNA predicted 5790 and 9542 target genes.Using FunRich and David enrichment analysis tool cormpleted the enrichment analysis and found the 4 target gene associated with angiogenesis and proliferation of tumor cells.Conclusion:Lymphoma cell lines and their corresponding microvesicle had similar miRNA expression.Through the enrichment analysis tools,there were four target genes from the cell lines and microvesicles could promote the cells proliferation.We speculated that these miRNA and target genes associated with the proliferation and migration of lymphoma cells.Objective:1 To explore the microvesicles which from the lymphoma cell lines oci-lyl and oci-ly10 and chronic myeloid leukemia cell line k562 affected the invasion,migration,and proliferation of HUVECs,and the preliminary study of the mechanism.2 To observe the green fluorescent zebrafish which were micro injected the microvesicles from the hematological malignance cell lines.Method:1 To extract microvesicles from the supernatant of cells through gradient centrifugation.PKH26 labeled the microvesicles and instantly added to the HUVECs,and observed red microvesicles which were endocytosised by HUVECs at different times(8H,16H,24H).2 Microvesicles added to the HUVECs 24 hours later,FITC-Phalloidin marked the HUVECs as instructions,and observed the morphology and skeletal changed of HUVECs.3 The change of cell cycles of HUVECs after the microvesicles stimulated analyzed by flow cytometry.EdU(fluorescence and flow analysis)detected the proliferation of HUVECs.4 HUVECs which were stimulated by microvesicles,through clone formation experiment,wound healing experiment,transwell experiment,FITC-detrex penetration experiment,cell growth density,observed the invasion,migration,and proliferation of HUVECs.5 Western blot detected the HUVECs which were stimulated by different microvesicles protein level change.6 MEK inhibitor U0126 was added in stimulated HUVECs,clone formation,transwell,and wound healing experiment were performed.7 To observe the number of tumor cell which microinjection of zebrafish yold sac.Result:The quantities of microvesicles endocytosis by HUVECs were different according to the different concentration of microvesicles and different times.After endocytosis microvesicles,HUVECs became slender and appear pseudopodia,the changed HUVECs more conductive to invasion and migration.Cell cycle of HUVECs showed the rate of G2/M increased compare to no stimulated HUVECs.EdU red fluorescence signal and EdU-positive cell increased observed by fluorescence microscopy and flow cytometry,respectively(p<0.05).Microvesicles stimulated HUVECs had higher cell density and more numbers of clone formation than un-stimulated HUVECs(p<0.01).FITC-detrex experiment showed that the stimulated HUVECs had lower green fluorescent signal than un-stimulated HUVECs.Transwell(migration and invasion)and wound healing experiment showed that microvesicle-stimulated HUVECs had stronger ability of invasion and migration(p<0.01).Microinjection of mixture of microvesicles and tumor cells into yolk sac of zebrafish,there were more tumor cells migrated to head and tail of zebrafish compare to microinjection of tumor cells only(p<0.01).Conclusion:Microvesicles from the hematology malignant tumor cells induced morphology and cytoskeleton changes of HUVECs,leading to more cells migration.Microvesicles stimulated HUVECs had stronger ability of proliferation,invasion,and migration,these were favorable factors for tumor cell growth.Invo zebrafish experiment indirect demonstrated that microvesicles could promote tumor cells migration.The mechanism may be microvesicles mediated HUVECs cytoskeleton changed and accelerated the HUVECs proliferation,invasion,and migration,at last induced the HUVECs permeability changed,and then,promoted migration of tumor cells.