Studies On The Expression And Function Of Annexin Ⅱ In Multiple Myeloma And Other Neoplastic Hematologic Disorders

Studies on the expression and function of annexinⅡin multiple myeloma and other neoplastic hematologic disordersAnnexins are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family with members of the family being expressed throughout animal and plant kingdoms. AnnexinⅡ(AnxA2) belongs to the family of annexins, which was discovered by Rade and Martin in 1979. It distributes in endothelial cells, mononuclear macrophages, nerve cells and some tumor cells. It exists as either a membrane-associated, cytosolic or soluble form in serum. AnxA2 presences inside cells in the two forms of monomer and tetramer. The monomer molecular structures include the conserved core and a second principal domain, which NH2-terminally precedes the core, is unique for a given member of the family and most likely specifies individual annexin properties in vivo. The monomer can form the tetramer with the protein of P11(S100A10), and carry out a series of function.AnxA2 can act as a membrane scaffold protein, and is involved in membrane organization, membrane fusion, endocytosis and exocytosis. It also partakes in some pathological processes and is associated with cardiovascular diseases, leukemia, infection and cancer, etc. Wherever inside or outside cells, AnxA2 has extensive function.The research about it has got more and more attention.Preparation and function study of monoclonal antibody against AnxA2 By using our established method, the recombined protein was purified by Ni-NTA agarose column and Balb/c mice were immunized. After the spleen cells of the mice and SP2/0 hybridoma cells were hybridized, positive clones was screened by ELISA assay and the selected hybridoma cells were used to prepare ascites. Western blot was used to detect whether the prepared anti-AnxA2 antibody can bind AnxA2 protein. The results show that: After the spleen cells and hybridoma cells SP2/0 were hybridized, we got two strain of cell that can last secreting anti-AnxA2 antibody named SZ-135, SZ136. The selected hybridoma cells was used to prepare ascites, the titers of anti-AnxA2 antibody in ascites was detected by indirect ELISA. The concentrations of purified anti-AnxA2 antibody were purified by proteinG-Sephrose4B affinity chromatography column. The antibodies could identify 36kD recombinated protein of AnxA2 and the antibody of SZ-135 can inhibite HMEC-1 angiogenesis in vitro effectively.The expression and significient of AnxA2 in multiple myelomaThe abnormal expression of AnxA2(A2) has been associated with the development of many tumors, however, it's expression and function in multiple myeloma(MM) patients and cell lines is less known. We compared the expression of AnxA2 in myeloma cells from MM patients with that in plasma cells from normal person and found that myeloma cells from patients had high expression of AnxA2.At the same time,we compared the expression of AnxA2 in MM cell lines U266 and RPMI8226 with other hematologic tumor cell lines and found that the myeloma cell lines U266 and RPMI8226 had also high expression of AnxA2. By transfecting U266 and RPMI8226 cells with the small interfering RNA(siRNA) that targets human AnxA2, AnxA2 expression was significantly downregulated. This resulted in the decreased proliferation, invasive potential, and increased apoptosis of U266 and RPMI8226 cell lines. Silencing AnxA2 gene by siRNA can also inhibit the expression of pro-angiogenic molecules including VEGF-C, VEGF-R2, MMP-2, MMP-9, MT1-MMP and TIMP-2 in the two cell lines. Our data suggested that the AnxA2 has high expression in MM patients and myeloma cell lines U266 and RPMI8226. The AnxA2 played a critical role in the proliferation, apoptosis and invasive potential of MM cell lines U266 and RPMI8226. The Relationship of AnxA2 and The Thrombosis Induced by Thalidomide and Dexamethasone in Multiple MyelomaMy major is to investigate the effect of thalidomide and Dexamethasone(DXM) on gene of AnxA2(A2) of RPMI8226,HMEC-1 and EAhy926 cell lines in vitro, and compare the change of AnxA2 with thrombosis induced by thalidomide and DXM in multiple myeloma. We cultivated RPMI8226, HMEC-1 and EAhy926 cell lines in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influence of thalidomide and DXM on the expression of mRNA of AnxA2, Flow Cytometry(FCM) and Confocal microscope were used to detect the protein expression on the cells surface after the samples were stimulated with thalidomide and DXM. Substrate chromatography was used to measure the plasminogen activation. The results showed that the mRNA and protein level of AnxA2 in RPMI8226, HMEC-1 and EAhy926 cells line treated by thalidomide in the different concentration of 12.5μg/ml, 25μg/ml, 50μg/ml and DXM was decreased compared with the unblocked (P<0.05). The plasminogen activation can also be decreased by AnxA2 antibody,thalidomide and DXM. Our conclusions was that thalidomide and DXM can inhibite the expression of AnxA2 in RPMI8226, HMEC-1 and EAhy926 cell line,then inhibite the plasminogen activation.It's possibly one of the reason of thrombosis production induced by thalidomide and DXM in multiple myeloma.Arsenic trioxide inhibits invasive potential of NB4 cells through down regulation of AnxA2My objective is to investigate the effect of arsenic trioxide (As2O3) on inhibiting invasive potential of NB4 through down regulation of AnxA2(A2). We cultured NB4 cells in vitro. Flow Cytometry (FCM) was used to detect the protein level on the cells surface after the samples were incubated with As2O3 in different concentrations.The influence of anti-AnxA2 antibody and As2O3 on the invasive potential of NB4 cells was investigated by using transwell plates. The results showed that the protein level of AnxA2 in NB4 cells treated by As2O3 in the different concentrations of 0.31, 0.63, and 1.25μg/ml was decreased compared with the unblocked (P<0.05). the invasive potential of NB4 cells treated with anti-AnxA2 antibody in the different concentrations of 0.01, 0.02, and 0.04μg/ml as well as As2O3 in the different concentrations of 0.31, 0.63, and 1.25μg/ml was decreased compared with the blank (P<0.05). Our data suggested that the down regulation of AnxA2 can inhibit invasive potential of NB4, As2O3 can inhibit invasive potential of NB4 cells through down regulation of AnxA2.