Ovarian Cancer Patients With Ascites Source Of Exosome Body Against Ovarian Cancer, The Immune Function Of The Experimental Study Of Protein Analysis

Ovarian cancer is the 3rd leading malignant gynecological tumor but itsmortality is the highest. Although the basic research of ovarian cancer has madeprogress there are still lots of work that should be done, including explore therelationship between the tumor and the host immune system. Exosome is a kind ofmicro vesicle released by many kinds of cells such as tumor cell, antigen presentingcell (APC) and epithelial cell. Recent researches revealed that exosome purified fromthe ascites of patient with ovarian cancer contains some immunological-effectmolecules and the information about the tumor. Exosome may play an important rolein the host specific anti-tumor immunologic function. In order to reveal how exosomeplay a role in the specific anti-tumor immunologic function and identify the proteincompositions of the ascites-derived exosome, five parts of researches were performed.PartⅠThe Purification, Identification and Quntitation of the Ascites-DerivedExosome[Objective] To purify and identify the ascites-derived exosome from the patients withovarian cancer and quantitate it.[Methods] Ascites were collected from 41 patients with ovarian epithelia cancer.Exosome was isolated and purified by ultracentrifugation. Exosome derived from asciteswas identified by transmission electron microscope (TEM) and Western Blot. Bradfordassay was used to quantitate the exosome indirectly.[Results] Exosome could be isolated and purified from the ascites in 85.4% (35/41) ofovarian cancer patients successfully, but some large protein-polymers wereco-isolated. The identification results of TEM and Western Blot had the consistency.Bradford assay was a convenient method to quantitate the exosome but the result wasnot very accurate.[Conclusions] Exosome can be isolated and purified from the ascites of most patientswith ovarian cancer. Bradford assay was a convenient but not the best way to quantitate the exosome.PartⅡThe Study of the Anti-Ovarian Cancer effect of the LymphocytesStimulated by Ascites-Derived Exosome[Objective] To observe whether the ascites-derived exosome from the patients withovarian cancer has effect on the APC and lymphocyte (LC) participating specificanti-ovarian tumor cellular immunity ex vivo.[Methods] Tumor cells in the ascites and peripheral blood lymphocytes were isolatedand cultured ex vivo. Dendritic cells (DC) were induced from the cord bloodhemopoietic stem cell by GM-CSF and IL-4. Ascites-derived exosome was added tothe DC and LC co-culture system (exosome+DC+LC group) or LC culture system(exosome+LC group). No exosome co-culture systems (DC+LC group and LC group)were ued as control groups. Autogeneic tumor cell (ATC) and ovarian cancer cell lineSKOV3 were used as target cells. Lymphocytes co-cultured with exosome or not wereused as effect cells. The specific anti-tumor cytotoxicity was determined by mortifiedMTT assay. The IFN-γreleasing level was calculated by ELISA.[Results] The cytotoxity of the LC co-cultured with DC and exosome was (6.8±15.8)% but that of the LC from the DC+LC group was (16.0±25.9)%. Thecytotoxicity of the LCs from the exosome+LC group and LC group was (16.9±30.8)% and (16.0±22.5)% respectively. The IFN-γreleasing level of theexosome+DC+LC group was (30.1±9.8)pg/ml and that of the DC+LC group was(34.9±12.4)pg/ml. the IFN-γreleasing levels of the exosome+LC group and the LCgroup were (33.0±10.0)pg/ml and (29.2±7.1)pg/ml respectively. The IFN-γreleasing level had no change after the LCs were co-cultured with tumor cells for 48hours.[Conclusions] Exosome can down regulate the specific anti-tumor T cell cytotoxicitywhen DC presents but has no effect on the IFN-γreleasing level. PartⅢThe Effect of the Ascites-Derived Exosome on the Growth and Apoptosisof Ovarian Cancer Cell[Objective] To study whether ascites-derived exosome from the patients with ovariancancer has effect on the growth and apoptosis of the ovarian cancer cell ex vivo.[Methods] The growth curve of the ovarian cancer cell line SKOV3 co-cultured withexosome or not was made by CCK-8 kit. And the apoptosis of the tumor cellco-cultured with exosomes or not was analyzed by annexin V-PI staining and flowcytometry (FCM) combined with microscope.[Results] The growth curve of the cell line SKOV3 had no change no matter the cellwas co-cultured with exosome or not. And the percentage of apoptosis in the groupwith exosome or not was similar.[Conclusions] Exosome had no effect on the growth of the tumor cell line SKOV3.And exosome had no effect on the apoptosis of the tumor cell either.PartⅣThe Role of the Ascites-Derived Exosome in the Specific Anti-OvarianCancer Immunity[Objective] To analyze how the ascites-derived exosome from the patients withovarian cancer play a role in the host anti-tumor immunologic function.[Methods] The apoptosis of the DC and LC co-cultured with exosomes or not wasanalyzed by annexin V-PI staining and flow cytometry (FCM) combined withmicroscope. The influence on the markers appearing in the DC induced and maturingprocess by exosome was determined by FCM. The ratio of CD4~+ T cell to CD8~+ T cellinfluenced by exosomes was also determined by FCM. Whether exosome contained FasLand Trail or not was confirmed by Western Blot or immunological electric microscopy(IEM). The anti-human FasL mono-clone antibody was used to block the apoptosis of DCand LC induced by exosome. The number of Trail receptors DR4 and DR5 existing onthe DC and LC membranes was determined by FCM.[Results] Exosome may induce cord blood DC-precursor cell, DC and LC apoptosis, but not every exosome sample could induce those cells apoptosis. The markersappearing in the DC induced or maturing process were not influenced by exosomeexcept CD86 was slight higher in the exosome group than no-exosome group. Theratioes of CD4~+ T cell to CD8~+ T cell in the total LC and in the DC+LC culturesystem were not influenced by exosome. FasL and Trail could be found in theexosome suspension. Anti-human FasL antibody could partially block the DC and LCapoptosis induced by exosome. There was low level of receptor DR5 found in the DCand LC. But about 16% of LCs contained DR4. DC also had very low level of DR4on the cell membrane. Exosome had no effect on the DR4 and DR5 presence on theDC and LC.[Conclusions] Exosome may induce apoptosis of cord blood DC-precursor cell, DCand LC partially by containing FasL. Trail existing on exosome may also contribute toLC apoptosis. Exosome has no influence on the DC induced process and the ratio ofCD4~+ T cell to CD8~+ T cell.PartⅤThe Identification of the Protein Compositions of the Ascites-DerivedExosome[Objective] To identify the protein compositions of the ascites-derived exosome fromthe patients with ovarian cancer and look for the components which may haveinfluence on immune system.[Methods] The protein components of the exosome derived from different patientswere separated by SDS-PAGE and the concordance of the protein bands wasdetermined by eyes. Then the exosome containing proteins were separated by2-dimensional electrophoresis and the protein dots were identified by MALDI-TOF.[Results] The protein bands of different exosomes which separated by SDS-PAGEwere same observed by eyes. 2-dimensional electrophoresis and MALTI-TOF showedthere were high levels of fibrinogen, albumen, immunoglobulin, complementC3 andthe subunits of hemoglobin in the exosome sample. HSP70, typeⅠand typeⅡcarbonic anhydrase, actin, keratin, annexin andα-enolase could also be found in the exosome suspension. MHC classⅠ/Ⅱmolecular and tumor associated antigen werenot found.[Conclusions] There are high levels of fibrinogen, transport proteins,immunoglobulin, complement in the exosome sample. Low levels of HSP70,cytoskeletal proteins and some functional enzymes also exsit in the exosome sample.