| The death rate of human ovarian carcinoma is the first in gynecological malignant tumor and most of the patients at advanced stage when they are found. The experts of gynecology all over the world successfully strived and explored the way of cytoreductive surgery and union chemotherapy which based on the operation in the last 30 years. But the survival rate of five years of human ovarian carcinoma patients at advanced stage still rambled about 20%. So to investigate the mechanism of how the human ovarian carcinoma happened and developed and to explore the most valid treatment prescription and elevating the quality of patients who suffered from cancer are great challenge faced by expert of gynecology constantly. Human leukocyte antigen-G (HLA-G) and human leukocyte antigen-E (HLA-E) are both non-classical HLA-I molecules, which discovered in recent years. HLA-G could inhibit human immune function by combining with the rejection capability receptors of NK cells, T cells subtype, antigen presenting cells, B cells and monocaryon cells system. HLA-G could stimulate tumor cells to over-express HLA-E, which could inhibit cytolysis of NK cells and T cells by interacting with the rejection capability receptors of them. Initially HLA-G and HLA-E were observed on the extravillous cytotrophoblast. And the effect on the fetal-maternal immune tolerance has generally accepted by the public. Moreover, the scholar carried out rudiment investigation in the relationship between the antigens and tumor. After the report of being expressed in the melanoma cells in 1998, HLA-G was reported in kidney tumor, breast tumor, colorectal cancer, lung cancer, lymphoma, carcinoma of bladder, spongioblastoma. Most of studies indicated that malignant tumor expressed HLA-G antigen and the expression concerned with immune escape of tumor. But the reports were very few about the expression of the antigens in malignant tumor of ovary and there was no final conclusion to date. It was reported aboard in 2006 that progestin, choriogonadotrophin could achieve the goal of tocolysis by up-regulating the HLA-G level. So we presumed that the mechanism of the inducing abortion drug, such as trichosanthin and mifepristone might concerned with down-regulating the expression of HLA-G and HLA-E. We could also presume the anti-tumor mechanism of these drugs in immune escape aspect, which opened up a new pathway in the immunotherapy and biotherapy of malignant tumor and there was no report about this at present. We tried to analyze the effect of HLA-G and HLA-E in immune escape of tumor by detecting their expression in the tissues of ovarian malignant tumor and cultured cells in vitro, observe the inhibiting effect of trichosanthin, mifepristone and DDP on the ovarian cancer cells and compared the changes from mRNA and protein level of HLA-G and HLA-E. Then we could identify the anti-tumor effect and mechanism of the drugs. We established the models of xenografted tumor by transplanting human ovarian carcinoma OVCAR3 cell into the nude mice and observed the influence of the drugs on the tumor's bulk and the expression of HLA-G, HLA-E. We expected to search out more valid anticancer drugs at this fundament and provide creative thinking and grounds for the generating mechanism of ovarian cancer and its biotherapy.Part One The expression and significance of HLA-G and HLA-E in ovarian malignant tumor tissue.Objective: To investigate the expression of HLA-G and HLA-E in ovarian tumor and normal tissue and its corelation with clinical pathological character.Methods: Reversed Transcript-Polymerase Chain Reaction (RT-PCR) method and flow cytometry were performed to examine the expression of HLA-G, HLA-E mRNA and protein levels in 60 malignant tumor and 30 benign tumor of ovary and 10 normal ovarian tissues.Results:①The positive rate of HLA-G mRNA expression in malignant group,benign group and normal group was 93.33%,73.33%,60.00% respectively, while the positive rate of HLA-G protein expression was 61.67%,16.67%,10.00% respectively. Result ofχ2 analysis showed that the positive rate of HLA-G mRNA and protein expression in malignant group were higher than that in benign group and normal group, while the statistical difference between the benign group and the normal group was not significant.②The positive rate of HLA-E mRNA expression in malignant group,benign group and normal group was 96.67%,83.33%,70.00% respectively, while the positive rate of HLA-E protein expression was 75.00%,36.67%,30.00% respectively. Result ofχ2 analysis showed that the positive rate of HLA-E mRNA in malignant group was higher than that in normal group, and also had higher tendency than in benign group(χ2=3.272, P=0.077); The positive rate of HLA-E protein expression in malignant group was higher than that in benign group and normal group. There was no statistical difference between benign group and normal group .③There was no statistical difference of the HLA-G mRNA and protein expression among different histological typies. Expression of HLA-G mRNA among different clinical stages also showed no statistical difference, but the expression in clinical I/II stage had lower tendency than III/IV stage (χ2=4.133,P=0.077); The expression of HLA-G protein was increasing according with clinical stage. There was statistical difference of the HLA-G mRNA and protein expression among differentation levels, the positive rate in well-differentiated group was lower than that in poorly differentiated group.④There was no statistical difference of the HLA-E mRNA and protein expression among different histological typies. Expression of HLA-E mRNA among different clinical stages also showed no statistical difference, but the expression in clinical I/II stage had lower tendency than III/IV stage (χ2=4.828,P=0.086); The expression of HLA-E protein was increasing according with clinical stage. There was statistical difference of the HLA-E mRNA and protein expression among differentiation levels, the positive rate in well-differentiated group was lower than that in poorly differentiated group. Conclusion:①The expression of HLA-G and HLA-E were all higher in malignant group than in benign group and normal group.②The expression of HLA-G and HLA-E were not corelating with histological typy of ovarian malignant tumor, but had corelation with clinical stage and pathological differenitation.③HLA-G and HLA-E might participate in the development of ovarian malignant tumor.Part Two The expression and significance of HLA-G and HLA-E in ovarian cancer cells.Objective: To investigate the expression of HLA-G and HLA-E in ovarian cancer cells and provide experimental evidence for immunotherapy of ovarian cancer.Methods: Human ovarian cancer SKOV3, 3AO and OVCAR3 cells were cultured in vitro. RT-PCR and flow cytometry were performed to examine the expression of HLA-G, HLA-E mRNA and protein levels, and using chorioepithelioma JEG-3 cells as positive control.Results: The expression of HLA-G, HLA-E mRNA and protein in SKOV3 cells was negative. The expression of HLA-G mRNA and protein in 3AO cells was negative, while the expression of HLA-E mRNA and protein was positive. The expression of HLA-G mRNA in OVCAR3 cells was positive, protein expression was negative; The expression of HLA-E mRNA and protein in OVCAR3 cells was positive. The expression of HLA-G, HLA-E mRNA and protein in JEG-3 cells showed intense positive. Conclusion: Although different ovarian cancer cells showed different levels of HLA-G,HLA-E, the significance of HLA-G,HLA-E in the immune escape mechanism for ovarian cancer was confirmed.Part Three The effect of trichosanthin,mifepristone, DDP on the proliferation of ovarian cancer cells.Objective: To investigate the influence of trichosanthin,mifepristone, DDP on the proliferation of ovarian cancer cells and identify theirs anti-tumor role.Methods: Human ovarian cancer SKOV3, 3AO and OVCAR3 cells were cultured in vitro. MTT method was performed to observe the influence of trichosanthin,mifepristone, DDP on the proliferation of ovarian cancer cells .Results:①The inhibition ratio of trichosanthin about the concentration of 10μg/ml,50μg/ml,100μg/ml,500μg/ml,1000μg/ml for SKOV3 cell was 0, 3.67%, 18.60%, 34.46%, 46.96%, respectively; For 3AO cell was 0, 2.97%, 9.17%, 35.01%, 48.98%, respectively; For OVCAR3 cell was 1.06%, 9.03%, 14.95%, 39.66%, 58.65%. The higher the concentration, the higher the inhibition ratio.②The inhibition ratio of mifepristone about the concentration of 2.5μg/ml, 5μg/ml, 10μg/ml, 20μg/ml, 40μg/ml for SKOV3 cell was 2.45%, 4.52%, 5.98%, 21.67%, 30.03%, respectively; For 3AO cell was 11.38%, 17.93%, 20.82%, 45.75%, 58.63%, respectively; For OVCAR3 cell was 8%, 24.88%, 47.6%, 61.58%, 76.94%.③The inhibition ratio of DDP about the concentration of 1.5625μg/ ml, 3.125μg/ ml, 6.25μg/ ml, 12.5μg/ ml, 25μg/ ml for SKOV3 cell was 5.68%, 17.27%, 21.75%, 45.32%, 54.98%, respectively;For 3AO cell was 25.55%, 39.81%, 63.07%, 86.93%, 95.28%, respectively; For OVCAR3 cell was 17.82%, 29.25%, 60.15%, 70.57%, 85.67%. Conclusion: Trichosanthin, mifepristone, DDP inhibited the proliferation of ovarian cancer cells obviously , and the inhibition ratio according with the drug concentration, which confirmed the anti-cancer effect of the three drugs for ovarian cancer cells.Part Four The influence of trichosanthin, mifepristone, DDP on the expression of HLA-G and HLA-E in ovarian cancer cells.Objective: To investigate whether the expression of HLA-G and HLA-E can be downregulated by trichosanthin, mifepristone, DDP.Methods: Human ovarian cancer OVCAR3 cells were cultured in vitro, which express both HLA-G and HLA-E. RT-PCR and flow cytometry were performed to examine the expression of HLA-G, HLA-E mRNA and protein levels when being treated with trichosanthin, mifepristone, DDP respectively.Results:①The optical density value of HLA-G mRNA expressed by OVCAR3 cells in control group, 500μg/ml group and 1000μg/ml group was 1.00±0.12, 0.64±0.24, 0.36±0.08, respectively. Trichosanthin could down-regulate HLA-G mRNA level significantly and the optical density value of HLA-G mRNA in 1000μg/ml group was lower than that in 500μg/ml group. Trichosanthin could down-regulate HLA-E mRNA and protein levels significantly, the optical density value of HLA-E mRNA expressed in control group, 500μg/ml group and 1000μg/ml group was 1.14±0.09, 0.82±0.05, 0.65±0.06, fluorescence index was 2.29±0.12, 1.77±0.09, 1.37±0.11, respectively.②Mifepristone could down-regulate HLA-G mRNA level significantly, the optical density value in control group, 20μg/ml group and 40μg/ml group was 1.02±0.14, 0.67±0.14, 0.35±0.12, respectively. Mifepristone could down-regulate HLA-E mRNA and protein levels significantly.③DDP had no distinct influence on the HLA-G mRNA level, the optical density value in control group, 6.25μg/ml group and 12.5μg/ml group was 1.02±0.09, 0.98±0.11, 0.94±0.07, respectively. The optical density value of HLA-E mRNA expressed by OVCAR3 cells in control group, 6.25μg/ml group, 12.5μg/ml group was 0.96±0.16, 0.94±0.08, 0.81±0.11, respectively, and the difference was not significant. DDP could down-regulate HLA-E protein expressed significantly.④At the same concentration, trichosanthin and mifepristone could down-regulate the mRNA and protein levels of both HLA-G and HLA-E, while the difference for DDP was not significantly.Conclusion: The three drugs could rivalry immune escape of ovarian cancer in different degree, combining trichosanthin or mifepristone to DDP might gain the better result.Part Five The influence of trichosanthin, mifepristone, DDP on the growth of xenografted tumor and the expression of HLA-G and HLA-EObjective: To investigate the therapy mechanism of trichosanthin, mifepristone, DDP on ovarian cancer .Methods:①Human ovarian cancer OVCAR3 cells were cultured in vitro.②Raised BALB/c nude mice and infused cell suspension into the right shoulder of nude mice. Everyone of them received 0.2ml, which including 1.2×106 cells. The tumor formed after 7days and the inoculation successful rate was 100%.③When the xenografted tumor growed two weeks and the circumscription of tumor was distinct, the nude mice were randomly divided in five groups, which were control, TCS, MIF ,DDP, and DDP+ MIF group.④The nude mice were killed after 4 weeks'medication, and the volume of tumor were measured. RT-PCR method and flow cytometry were performed to examine the expression of HLA-G, HLA-E mRNA and protein in the xenografted tumor of nude mice in different groups.Results:①The xenografted tumor of nude mice in control, TCS, MIF, DDP and DDP+MIF group was (6100.83±1000.69) mm3, (3500.46±562.04) mm3, (3689.30±1017.66) mm3, (3491.40±820.13) mm3 and (2532.55±762.56) mm3 , respectively. The tumor growth inhibiting rates of treated groups was obviously lower than the control group.②The optical density value of HLA-G mRNA in control, TCS, MIF, DDP and DDP+MIF group was 1.01±0.17, 0.71±0.18, 0.62±0.10, 0.89±0.11 and 0.49±0.10, and the fluorescence index number of the HLA-G protein was 2.38±0.43, 1.45±0.35, 1.26±0.31, 2.29±0.35, 1.08±0.25, respectively. Except DDP group, the expression of HLA-G mRNA and protein in treated group were notably lower than the control group., which of MIF and DDP+MIF groups was lower than DDP group.③The optical density value of HLA-E mRNA in control, TCS, MIF, DDP and DDP+MIF group was 0.99±0.19, 0.67±0.18, 0.76±0.17, 0.96±0.16 and 0.69±0.21, and the fluorescence index number of the HLA-E protein was 2.27±0.19, 2.02±0.17, 1.59±0.15, 2.17±0.19 and 1.36±0.25, respectively. Except DDP group, the expression of HLA-E mRNA and protein in treated group were notably lower than the control group.④There was consistency between HLA-G and HLA-E mRNA level(r=0.388, P<0.05) and protein level(r=0.668, P<0.01).Conclusion: Trichosanthes, mifepristone, DDP could obviously inhibit the growing of the xenograft of nude mice. The depressant effect of DDP+MIF on the tumor was obviously stronger than single drug group. The anti-tumor mechanism of trichosanthin and mifepristone connected with the down-regulating of HLA-G, HLA-E, but DDP had no such effect, which suggested that the therapeutic efficacy would be much better if the DDP combined with trichosanthin or mifepristone.
|
PDF Full Text Request