Characters Of Tumor Associated Mircovessels Lymphatic Endothelial Cell From Epithelial Ovarian Tumor

PartⅠThe existence and characters of tumor associated lymphaticendothelial cell in epithelial ovarian tumorObjective To study the existence and distribution of lymphatic endothelialcells in epithelial ovarian tumor.Methods (1)Monoclonal lymphaticendothelial cell marker D2-40 antibody was used to immunistain thelymphatic microvessels in the paraffin sections of epithelial ovarian tumor(including 10 malignant and 5 borderline).Antibodies of D2-40 and Ki-67were used together by double immuohistchemistry to detect proliferation oflymphatic endothelial cells.Computer-assisted morph metric analysis wasused to quantitatively analyze lymphatic vessel density (LVD) and lymphaticvessel area (LVA) in epithelial ovarian tumor.(2) Polyclonal lymphatic vesselendothelial HA receptor- 1(LYVE-1) antibody was used to embark epithelialovarian tumor tissue (including 10 malignant,5 borderline,5 benign and 5control) single cell suspension for FCM detection.Results There was no significant difference between LVD in epithelial borderline ovarian tumorand malignant ovarian tumor ((13.23±8.26) vessels/4HP VS(14.22±7.21)vessels/4HP,P＞0.05).However,LVA in malignant ovarian tumor wassignificantly increased than that in borderline group ((3.52±1.61)% VS(2.21±1.42)%,P＜0.05).Lymphangiogenesis of epithelial ovarian cancer wasstill a pending question as the co-existence of D2-40 and Ki-67 was absenceby double immunohistchemostrity technique.LYVE-1 (+) cells can bedetected in both malignant and borderline tissue single cell suspension byFCM.Conclusion The existence of lymphatic endothelial cells in epithelialovarian tumor was sure.It's possible to isolate this cell from its single cellsuspension.PartⅡThe isolation,culture and identification of lymphatic endothelialcells from epithelial ovarian tumor and its biological characters incommonObjective To study the feasibility of culture lymphatic endothelial cells(LECs) which were isolated from epithelial ovarian tumor in vitro,and offerbasic technology for researching development of epithelial ovarian tumor (including borderline and malignant ones).Methods (1) Epithelial ovariancancer single cell suspensions were obtained by digestion with collagenasefrom 26 cases (11 borderline and 15 malignant).LYVE-1 antibody was usedas LEC markers to isolate LEC by magnetic tosylactivated Macsbeads.LECswere culture in both two- and three-dimensional model in vitro.(2) Toidentify the cell by immunostain pan-endothelial cell marker CD31 andseveral LEC markers such as LYVE-1,D2-40,VEGFR-3(Flt-4),Prox-1.(3)LECs were subcultured consecutively until died,summarized the rule ofisolation and culture this new cell.Results (1) By magnetic beadstechnology LYVE-1(+) lymphatic endothelial cell can be harvested andculture successfully in vitro.Most of TLECs were from borderline tumor asthe age agent.(2) The identification purity of this cell by CD31 was 100%,Prox-1 was 99%,D2-40 was 95%,VEGFR-3 was 90%,LYVE-1 was 98%.(3)The most thriving one was cultured more than six passages.The success ratewas related to the age of the patient but other clinicopathologic variables,theyounger (＜30y),the better (P＜0.05).Conclusion By magnetic bead,highpurity lymphatic endothelial cell can be isolated and cultured from epithelialovarian tumor with stable characteristics for the future research. PartⅢThe effect of tumor associated lymphatic microvacsular endothelialcell from epithelial ovarian tumor on ovarian cancer cell lineCAOV-3Objective The aim of this experiment was to detect the effects of LEC fromhuman epithelial ovarian tumor (EOT) on ovarian cancer cell line CAOV-3 invitro.Methods (1) Tumor associated lymphatic endothelial cell (TLEC) andnormal lymphatic endothelial cell (NLEC) were cultured in vitro respectively.Their serum free conditioned mediums were harvested.(2) CAOV-3 cellswere treated with these conditioned mediums;the cells were trypsinized,washed,stained with trypan blue solution and counted.Its ability of migrationand invasion was measured by Transwell.(3) Real-time PCR was used todetect MMPs/TIMPs of CAOV-3 treated by conditioned mediums from TLECand NLEC.(4) MMP-2 and MMP-9 excreted by ovarian tumor cell line whichtreated by conditioned mediums from TLEC and NLEC was detected bygelatin zymography.Results (1) Tumor associated lymphatic microvacsularendothelial cell from epithelial ovarian tumor enhanced migration andinvasion of ovarian cancer cell line CAOV-3 in vitro obviously (P＜0.05).However,the proliferation effect to CAOV-3 cell was still a pending problemas the method was simple.(2) The expression of MMP-9 and TIMP-2 mRNA of CAOV-3 changed obviously by influence of TLECs,the changes of MMPsalso demonstrated by gelatin zymography.Conclusion LECs from epithelialovarian tumor could enhance migration and invasion of human ovariancarcinoma cell line via activation MMP-9/TIMP-2 in vitro.