| [Background] Ovarian cancer is a malignant ovarian tissue occurred in clinical practice, there may be lower abdominal discomfort, abdominal pain, abdominal mass, menstrual disorder, oppression and other symptoms, Because ovarian carcinoma frequently remains clinically silent, most women are diagnosed with advanced diseases , so it is a serious threat to women's health and life. For these patients, although the highest standard of multimodality therapy with surgery and cytotoxic chemotherapy, long term survival remains low and the 5-year survival remains less than 30%. Conventional surgery and radiotherapy and chemotherapy are difficult to significantly improve the long-term survival rate of patients, especially survival rate of advanced stage patients, it is hightime to looking for new methods of treatment of ovarian cancer.A large number of studies have shown that cell-mediated immunity in anti-tumor immunity play a major role ,so adoptive immunotherapy (AIT)has received considerable attention. Adoptive T cell therapy is the focus of adoptive immunotherapy, but it have no cytotoxicity of tumor cell lines which are negative for MHCI class I molecules, while the NK cells express MHCI class I molecules as inhibitory receptors - killer cell inhibitory receptor (KIR) , it can kill the tumor-negative MHCI class I molecules. Thus, NK cells for tumor immunotherapy has received consider.The NK cells were established from the peripheral blood lymphocytes of a patient with rapidly progressive non-Hodgkin's lymphoma, and the cells are similar to NK cells with respect of surface receptor expression and functional characteristics, and display a much higher cytolytic activity against a broader spectrum of tumor targets. However, there is no killing effect on ovarian cancer research, therefore, we have chosen the ovarian cancer cells as target cell to detect the cytotoxicity of NKG , results show that the NKG cells have higher cytotoxic activity than any other has been to build a successful NK cell lines, including the NK-92, NKL, and YT cells. based on the status of Treatment of ovarian cancer and seek new therapeutic strategy, Obstetrics and Gynecology of Anhui Province Hospital Anhui Province approved a significant special issues in common with the Science and Technology Institute of China.NKG cells are clonal and growing NK cell line, which upon adoptive infusion to immunocompromised patients potentially harbors the risk to graft in these patients. One possible meathod is the use of radiation to control the NKG cells proliferation and within retain a maximun of cytotoxic activity.In this study, we introduce the large scale expansion of NKG cells ,describe the cytotoxicity of irradiated NKG cells against human ovarian cancer cell line in vitro, and confirmed the effective of NKG cells by using mouse ovrian tumor model with HO-8910 implantation in vivo.[Objective] To evaluate the expansion, proliferation and anti-tumor activity of NKG cells against human ovarian cancer cells in vitro and in nude mice with human ovrian ascise cancioma.[Methods]①The gas-permeable culture bags were used to expansioned the NKG cells;②the fresh ovarian tumor cells were isolated by the human tumor cell isolation and purification kit according to the manufacture's instructions;③Gamma irradiation instrument was used to irradiate NKG cells, the irradiated NKG cells were used for proliferation assay,cytotoxicity assay and adoptive transfer in vivo;④The proliferation of iradiated NKG cells was quantitated using a standard thymidine incorporation assay;⑤The cytotoxic activity of NKG against target cells was measured by a standard 4-h 51Cr release assay;⑥Ho8910 cells (5×105,1×106,2×106,5×106,1×107) were injected i.p into the nude mice in 200μl RPMI 1640 medium, The body weight and circumference of abdomen were determined twice every week to show the establishment of tumor model;⑦2×106 Ho8910 cells alone or mixed with 1×107 NKG cells irradiated at dose of 8 Gy (E/T: 5:1) were injected i.p. into the nude mice in a volume of 200μl. The injected mice were monitored twice every week for the body weight, abdomen circumference, palpable tumor and the survival;⑧To observe the therapy effect of NKG cells on the already formatted ovarian tumor, At indicated day 21 or 35 of tumor cell implantation, irradiated NKG cells (6.67×107/ml) at dose of 800cGy inα-MEM medium were injected i.p. into the selected mice to determined the effect of NKG cells.[Results]①the concentration of NKG cells was 1.1×106/ml, and the viability≥90%;②The 3H- thymidine riboside incorporation assay confirmed that there was no proliferation of the irradiated NKG cells which were irradiation at dose of 800cGy;③the cytotoxicity of the irradiated NKG cells at the dose of 400cGy and 800cGy against the standard cell line K562 was as high as the untreated NKG cells; while the cytotoxicity would be decreased significantly at the dose of 1200cGy, there was significantly difference. the cytotoxicity of irradiated NKG cells at does of 800cGy against the Ho8910 cells was the highest Compared with the established NK cell lines NK-92, and the cytotoxicity of NKG cells at does of 800cGy against primary ovarian cancer cells was 61.1-15.5%;④Ho8910 cells (2×106) were injected i.p into the nude mice, and the mice developed palpable tumor between days 25~45 after tumor cell implantation with the 100% tumorgenesis incident, The tumor nodules in the liver and spleen, and tumors in the peritoneal cavity were shown, The histological examination confirmed the tumor cell infiltration in the spleen and liver, and the tumor cell growth in the peritoneal cavity with the tumor tissue and ascites;⑤When the irradiated NKG cells were adoptively transferred into the mice together with Ho8910 cells (2×106) with ratio of 5/1, the tumorgenesis (%) was significantly decreased from 100% to 50%, and the palpable tumor developed time was delayed from 25~45 days to 90~135 days, the mean survival time was obviously prolonged from 61.5 to 210.2 days.⑥When the mice were challenged with 2×106 Ho8910 cells for 21 days, NKG cell treatment was then performed. NKG cell treatment could significantly inhibit the tumorgenesis. Until the 140 day under observation, 40% mice were still survival without palpable tumor in the NKG cell treatment group. The mean survival time was obviously prolonged from 64.6 to 119.6 days.⑦When mice with palpable tumor were selected, which has been challenged with 2×106 Ho8910 cells for 35 days, The mean survival time was obviously prolonged from 64.2 to 93.6 days although the mice were dead with tumor progress ultimately. These results demonstrated the therapy effect of irradiated NKG cells on the already formatted human ovarian tumor.[Conclusion]An appropriate radiation dose of 800 cGy was choosed for NKG, which could control cell proliferation and at the same time retain a maximum of cytotoxic activity for HO-8910 cells; Adoptive transfer of NKG cells (800cGy) can significantly attenuates the ovarian cancer progression in tumor-bearing mice and prolong survival time. |
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