| Ovarian cancer is female genital organs common one of three malignant cancer mortality home first, Gynecologic malignancy is seriously endanger women's health and life of primary tumor. Ovarian cancer is a malignant neoplasm of hormone-dependent, research confirmed in epithelial ovarian cancer may have a 70%-80% of the men of gonado-tropin-releasing hormone LHRH of binding sites. For treatment of ovarian cancer remains surgical, supplemented by radiotherapy and chemotherapy regimen. Ovarian cancer chemotherapy sensitivity, nearly 80% of primary epithelial ovarian cancer patients to Platinum-based chemotherapy had a distinct clinical efficacy, after multiple course of chemotherapy, obvious inhibiting tumor recurrence, improve survival. But with the emergence of drug resistance in patients treated with chemotherapy the side effects of intolerance, makes a ten-year survival rate of ovarian cancer is very low, that is, the first complete remission of patients, there is still a 50% risk of recurrence. In order to remedy the existing shortcomings of the chemotherapy drug, recent molecular targeted drugs, immune drug, endocrine therapy increase gradually.1,BackgroundLHRH is studied in recent years, it was released by the hypothalamus, neural hormones which gene expression mainly in the reproductive system and the relevant organs and organizations, but in the important human and almost all are found in organs LHRH receptor mRNA. LHRH receptor in addition to normal tissues in the distribution, in the cancer has spread, and in some research shows that some tumors of the LHRH receptors on the surface of distribution far exceeds the normal quantity of organs and tissues of the body weight, at the same time, the cancer of LHRH receptor and normal tissue is different, the difference is that cancer cell surface receptor expression not only high, but can be mediated apoptosis, and have different signal transduction pathway. You can think the difference is the LHRH receptors on cells and normal cells. Currently, based on the LHRH analogue and recombinant neurotoxin in recent research on a wide range of progressive cancer used to on in vitro and animal experiments.Based on previous clinical observation, including breast, ovarian and endometrial cancer and prostate cancer, corticosteroid-dependent tumors with hormone therapy is feasible. But including LHRH, hormone therapy, although can significantly improve cancer symptoms, but therapy relapse rate than first-line chemotherapy and hormone treatment failure of patients average life time of less than 1 year, and for non-steroid-dependent patients, the effect is even worse. Reorganization of the toxin is emerging in recent years, some cancer cells produce orientation on specificity of killing ability, and without prejudice or rarely damage normal body cells of organisms targeting anticancer drugs. LHRH recombinant toxin is one of more of a class.LHRH-PE40 is put through protein fusion of Exotoxin PE40 and LHRH chimeric of chimeric proteins. LHRH-PE40 is the perfect localization provider to identify the ovarian cancer cells upper LHRH receptor-when entering the body carried PE40 LHRH, the first LHRH receptor expression and rich ovarian cancer cells, while the PE40 membrane translocation, PE40 was swallowed into cancer cells within the cell, the protein disorder, or a direct result of targeting cell apoptosis achieve the purpose of targeting resort. Its strong cytotoxicity, immune-derived low stability, can continue to play. At the same time, due to its specific recognition, making recombinant immunotoxin on normal tissue of injury is very small, for resistant ovarian cancer and refractory ovarian cancer, there will be a very good outcome of treatment. In addition LHRH-PE40 molecular weight difficult through the blood-brain barrier, brain tumor treatments other than to avoid damage to the pituitary gland. So other than for the brain tumor treatment providing practical factors. At the same time, the LHRH-PE40 itself side more chemotherapy drugs much cancer patient's tolerance will be very good, the application will be very broad.2. Purpose:By in vitro culture LHRH-PE40 SK on ovarian cancer cell proliferation and apoptosis, to verify whether LHRH-PE40 can be used for the treatment of ovarian cancer.3. Method:1, in vitro cell culture technology combined with cytotoxic experiment that takes a look into the LHRH-PE40 on ovarian cancer cell lines in vitro. Will LHRH-PE40 for eight gradient dilution, then join the holes after the drug after 1-5 days inverted microscope cell suppression rates; at the same time further cell culture plates for Crystal Violet dye, A570nm determination process and control of optical density values, calculate LHRH-PE40 on SK cells IC50, select dosing 48h of protein synthesis inhibition of dose-effect relationship of observation.2, immunohistochemical methods on lug/ml LHRH-PE40 effect concentrations of cultivating 48h SK cells for immunohistochemical staining, Ki-67, bcl-2 drug use before and after the positive rate changes.3. use Cytometry apoptosis after dosing rate and the rate of change in the cell cycle.4.Results:1. LHRH-PE40 to inhibit cultured epithelial ovarian cancer cell growth, SK LHRH-PE40 SK on ovarian cancer cell proliferation age, concentration of dependencies. LHRH-PE40 minimum effective dose is 0.625μg/m 1, maximum effective dose 5μg/m 1; the best of times for dosing 48-72h, median lethal concentration is 0.558μg/m 1.2. in the reagent concentrations after 1μg/m 1, medication after cancer Ki-67 (73%, before using medication after 25%) and BCL-2 (92% before using,43% of medication after) positive cells rates are declining, increasing the ratio of apoptotic cells (from before using 0.8% increase to medication after 3.6%), the tip LHRH-PE40 after cancer cell proliferation activities inhibited and apoptosis activities strengthened. PI staining by flow cytometry inl.Oμg/ml concentrations cell G1/GO,s-phase cell ratio increases and decreases the G2/M-phase.5. conclusions:This experiment by in vitro cell culture techniques to study LHRH-PE40 SK on ovarian cancer cell proliferation. After adding LHRH-PE40 SK with a significant inhibition of cell growth, and with the drug concentration increases, the rate of upward trend of suppression, dose-effect, aging figure, you can see in 24—72h, drugs on cell suppression of more stable, so we select the suppress drug action 48h rate changes as research subjects, find the lowest effective dose to 0.625μg/m 1, maximum effective dose for the 5μg/m 1, and the best of times in 48h-72h, computed SK cells half lethal IC50 is 0.558μg/m 1, drug suppression rates and function of time and concentration of the dependencies; in the LHRH-PE40 concentration as lug/ml, by observed SK cells Ki-67, BCL-2 in medication, positive rate than before using decreased ovarian cancer cell proliferation, apoptosis increase; flow cytometry medication before and after the change of the cell cycle, cell after the discovery of drugs than before using GO/G1 ratio increases, S ratio increased ovarian cancer cell line SK cell mitosis is block, cell G1 is stuck in the GO/. At the same time, we found that apoptosis rate by not drug 0.8%, to 3.6% after drug use, the rate of increase apoptosis. This shows the effect of LHRH-PE40 in protein synthesis in cancer cells while promoting tumor cell apoptosis, which in turn inhibits cancer cell proliferation.To sum up, LHRH-PE40 can be targeted against ovarian cancer cells; not destroyed the cell apoptosis by startup programs, LHRH-PE40 cause ovarian cancer cell cycle change and allows cells stuck to the GO/G1 phase of the cell apoptosis proportional increase. LHRH-PE40 ovarian cancer cells the solid dose and time-dependent. This experiment to provide LHRH-PE40 for the treatment of ovarian cancer in experimental basis. |
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