The Effect Of 17β-Estradiol On Biological Behaviors Of Cervical Cancer Cell Lines And The Culture Of Lymphatic Endthelial From Epithelial Ovarian Tumor In Vitro

Cervical caner is the most common cancer in all female gyneclogic cancer, whose morbidity increased gradually by years, the younger patient ratio is more. There are about 130,000 new patients in China every, almost to one fourth in the world. Cervical caner is one of the malignant tumors that have effective screening method to find in cancer early stage. Cervical cytological test is the best effective method which used to distinguish cervical caner, cervical intraepithelial neoplasia from normal cervical tissue, althogh with of a little false positive and false negative, the outcome is reliable. According to the outcome of cytologic test, we can choose the more accurate method-cervical biopsy to be got the final diagnosis. So the cervical cancer we found now is early stages, and they have the operative chance to rescue their lives. In present, the clssic operation of cervical cancer is Extensive hysterectomy and pelvic lymphadenectomy, which will lead to woman without ovarian fuction.Ovarian reproductive endocrine fuction is of important role in female phsycologic and phthological process,especially young female. Ovarian endocrine with so important role is for it can synthesis and secrete estrogen, which act an irreplaceable role in female phsycologic and phthological process. But for cervical cancer patients which accept radical operation lost ovarian fuction for life and irreversible. For these patients, whether they can accept estrogen replacement or not, which hasn't a detimined conclusion. The effect of estrogen on cervical cancer remain are controversial. Many reports in home and abroad, some said estrogen increase cervical cancer morbidity, by contrast, some said estrogen have nothing impact in cervical cancer. We read many papers about estrogen with cervical in home and abroad, No one focus on invasion and metastasis.So we design this study which used cell culture technology in vitro, regarded the cervical cell HeLa (from cervical adenocarcinoma) and SiHa(from cervical squamous cell carcinoma) as study objects. Then we dectect the changes of the cell in mRNA and protein expression to exprole the possible effect of cervical caner which induced by estrogen, to know the possible mechanism.Part oneThe effect of 17β-Estradiol on biological behaviors of cervical cancer cell lines HeLa and SiHaObjective:To explore the effect of 17β-Estradiol on biological behaviors in human cervical cell lines HeLa and SiHa, and investigate the potential mechanism.Methods:MTT and flow cytometry were respectively used to dectect the changes of proliferation and appotosis of the two human cervical cancer lines after incubated with 17β-Estradiol for 72 hours.RT-PCR and real-time PCR were used to dected the exepression of MMP2/9 and TIMP1/2 in mRNA while gelatin zymogram were only used to dectect MMP2 and MMP9.Transwell assay was used to the effect of 17β-Estradiol on migration and invasion.Result:After exposure 10nM and 100nM 17β-Estradiol, the proliferation of HeLa cell was significantly increased in a dose-manner, while lnM 17β-Estradiol was not. However, only 100nM 17β-Estradiol slightly increased the proliferation in SiHa cell. The appotosis of HeLa cell was lower in17β-Estradiol treatment group than untreat with 17β-Estradiol.MMP2 was expressed in SiHa while it was empty in HeLa; By contrast only MMP9 was expressed in HeLa cell.TIMPl/TIMP2 were expressed in two cell. After treatment with 10nM 17β-Estradiol, the expression of MMP9 in HeLa cell was higher than untreat with 17β-Estradiol while the TIMP2 was lower. The expression of MMP2 in SiHa cell was not signifcantly different between the 17β-Estradiol treatment group and the control group. After exposure to lOnM 17β-Estradiol 72 hours the migration and invasion of HeLa cell were more than the control group by Transwell assay. But we cann't observe obvious difference between the 17β-Estradiol treatment group and control group.Conclusion:17β-Estradiol can increase HeLa cell proliferation and decrease its appotosis, the higher concentration of concentration can slightly increase the proliferation of SiHa cell; 17β-Estradiol can promote migration and invasion of human cervical cancer cell HeLa cell in vitro, the possible mechanism was through activation of MMP9/TIMP-2.Part two The culture of lymphatic endthelial from epithelial ovarian tumor in vitroObjective:To establish the primary lymphatic endothelial cell culture method which were isolated from human epithelial ovarian tumor.Method:(1) We digested the fresh epithelial tumor with collagenase II and filter out the tissue piece to get the single cell suspension (nine maligant and twelve bordline). We used LYVE-1 antibody as lymphatic endothelial cell surface markers to isolated the positve cell by magnetic tosylactivated Macsbeads.(2) To identify the cell by immunofluorescence with the maker molecule CD31, D2-40 and VEGFR-3. (3) The cell weren't subcultured consecutively until cell aging, and observed its change of cell morphology and proliferation rate. (4)Sumarized with the clinic date to analyze the factors which may affect culture efficiency. Result:(1) We got lymphatic endothelial cell by immunomagnetic beads and cultured successfully in vitro. (2) The identification purity of the cell by CD31, D2-40, VEGFR-3 and LYVE-1 more than 90%. (3) The cell we isolated from human epithelial tumor can be subcultured to seven passage. But when subcultured to four or five passage, the cell proliferation went to down and aged. (4) The key factors of culture are patients' age and tumor histological (p<0.05). Conclusion:We can get high purity lymphatic endthelium from human epithelial cancer by immunomagnetic assay. And the cell are stable before four passage in morphology and proliferation.