Experimental Study On The Immunotherapeutic Effect Of Newcastle Disease Virus Vaccine, Interleukin-12 And Interleukin-15 To Human Ovarian Cancer

Objective: To investigate the effect of autologous tumor cell vaccines-Newcastle disease virus-infected (ATV-NDV), interleukin-12 and interleukin-15 on the growth of human ovarian carcinoma cell strains in vitro.Methods: Normal human ovarian epithelium cell and human ovarian carcinoma cell strains 3AO,SKOV3 and OVCAR3 were cultured in vitro; Peripheral blood lymphocyte was separated from normal individual and autologous tumor cell vaccines-Newcastle disease virus-infected (ATV-NDV) with human ovarian carcinoma cell strain 3AO was prepared(2-4 ×10⁷cells/ml). ATV-NDV, IL-12, IL-15, ATV-NDV+IL-12 and ATV-NDV+IL-15 were cultured for 24h-96h together withhuman peripheral blood lymphocyte(the controls) respectively. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was used to analyze the effect of ATV-NDV, EL-12 and IL-15 with different concentration and different acting time on the proliferation degree of lymphocyte. ATV-NDV(50 μ 1), rhIL-12(50ng/ml), rhIL-15(50ng/ml), ATV-NDV(50 μ 1) + rhIL-12(50ng/ml) and ATV-NDV (50μ 1) +rhIL-15(50ng/ml) were cultured together with human peripheral blood lymphocyte for 48 hours, according to the ratio of effect and target(E:T) which were 50:1, 25:1 and 10: 1, added into normal human ovarian epithelial cells and human ovarian carcinoma cell strains 3AO, SKOV₃ and OVCAR₃ respectively, continued to culture for 24h-96h, the cell growth inhibiting rate was determined using the MTT assay and the change of cell modality was observed by hematoxylin-eosin (HE) staining cultured for 24h⁹6h whose E:T was 50:1.Results: 1. Lymphocyte Proliferation Influence: ATV-NDV showed proliferation effect on lymphocyte at the dose of 10ul, the effect reached peak at the time 24h⁴8h, P<0.01,and promoted with dosage increasing, P<0.05; At the concentration of 1ng/ml, IL-15 and IL-12 showed the proliferation effect on lymphocyte, and the effect of IL-15 went up with the increasing of dosage and acting time, P<0.05. The difference was significant comparing with the controls, P<0.01. At the concentration of 50ng/ml and 100ng/ml, the effect of IL-12 on lymphocyte had no difference, P=0.053, and the effect went up with the increasing of dosage and acting time at other concentrations, P<0.05; There was significant difference between the treatment group with IL-12 and the controls; IL-15, IL-12 had synergism with ATV-NDV, and comparing with the effect of each alone, the difference was significant, P<0.01; The effect of IL-15, IL-12 and ATV-NDV on human peripheral blood lymphocytes had significant difference comparing with the controls, P<0.01; There was no significant difference between the effect of IL-15 and ATV-NDV, P=0.35; While the effect of IL-12 had significant difference comparing with the effect of IL-15 and ATV—NDV, P<0.01.2. The Inhibitory Effect of ATV-NDV, IL-15 and IL-12 on Tumor Cell: Theactivated lymphocytes could kill carcinoma cells if they were cultured together with target cells, and the effect was promoted as the time prolonged, P<0.05;The difference of cell growth inhibitory rate (GIR) among different cell strains had no significance, P>0.05; IL-15 and IL-12 had synergism with ATV-NDV on the effect of activating lymphocytes, and the difference was significant comparing with the effect of each alone, P<0.01; There was no difference between the effect of IL-15 and ATV-NDV, P>0.05; Lymphocytes did not work on the normal ovarian epithelial cells.3. Cell Modality Changing: The tumor cells spreaded finely in the control group, the cell nucleus was purplish red and circular, the chromatin was homogeneous and the cytoplasm was pink. As the acting time of lymphocyte prolonged, the 3AO cells fades, the bulk became little, chromatin agglutinated, block likely and near the nuclear membrane fringe, became the apoptosis cells (the cell displayed the modality of apoptosis cell when acted 48 hours); AT last the cell fell off, suspended, crumbled and died, presented the necrosis cell form(the necrosis cell form arose when acted 96 hours).Conclusion: ATV-NDV, IL-12 and IL-15 are proved to have effect on the proliferation of normal human peripheral blood lymphocyte, and the human peripheral blood lymphocyte can effectively inhibit the growth of the ovarian carcinoma cell strains 3AO, SKOV3 and OVCAR3 after cultured together with ATV-NDV, IL-12, IL-15, ATV-NDV + IL-12 and ATV-NDV + IL-15.The effect has the time and concentration dependence. Objective :To discuss the effect and the mechanism of autologous tumor cell vaccines- Newcastle disease virus-infected (ATV-NDV), interleukin-12 (IL-12) and interleukin-15(IL-15) on the tumor planted into nude mice subcutaneously with human ovarian carcinoma cell strain 3 AO.Metheds: The human ovarian carcinoma cell strain 3AO was cultured in vitro to logarithm growth phase; The BALB/C nude mice were 4⁶ weeks old, weighed 16-20 gram and were transplanted with the cultured human ovarian cancer cell strain 3AO 1 ×106 into the right crura subcutaneously; The largest (LD) and smallest diameters(SD)of each tumor were measured using vernier clipers. When the transplanted tumor grew to 1 centimeter3 (LD X SD2/2), the nude mice were injected with cisplatin (3mg/kg) intraperitoneally. The cisplatin were confected on the criterion of 0.2ml every nude mice. After one week, the nude mice were randomly assigned to 6 groups, every group had 10 nude mice. The nude mice in the first group were injected ATV-NDV 0.2ml(with 4⁸X 106cells) at the right crura subcutaneously; The second group were injected intraperitoneally with lOOug/kg IL-15;The third group were injected intraperitoneally with lOOng/kg IL-12;The forth group were injected intraperitoneally with 100 u g/kg IL-15 and injected at right crura subcutaneously with 0.2ml ATV-NDV at the same tine; The fifth group were injected intraperitoneally with lOOng/kg IL-12 and injected at right crura subcutaneously with 0.2ml ATV-NDV simultaneously; The sixth group were controls which were injectedintraperitoneally with normal saline 0.2ml; IL-15 and IL-12 were confected on the criterion of 0.2ml every nude mice. The nude mice were treated once every tow days, altogether 4 times. They were observed the rate of the outgrowth of the transplanted tumor, survival time, the prolonged survival rate and the tumor growth curve diagram. The apoptosis and cell cycle of the transplanted tumor cell were observed with flow cytometry (FCM). At the same time tumor NK cell cluster was taken account in double marker. Nude mice were executed after 3 days of twice and forth treatment. The nude mice spleen NK cell activity was determined with MTT assay and the spleens were weighed. The transplanted tumor, liver, spleen and kidney were checked by routine pathological examination.Results: 1. Establishing of Nude Mice Transplant Tumor Model with Human Ovarian Carcinoma Cell Strain 3AO: The nude mice model could be established with human ovarian carcinoma cell strain 3AO.The outgrowth rate of the nude mice transplanted tumor was 100%.2. Survival Condition of Nude Mice Holding Tumor: The first group nude mice began to die at the 27th day, the second group at the 28th day, the third group at the 38th day, the forth group at 54th day, the fifth group at 76th day and the sixth group at 21th day. The survival rate of nude mice in the experimental groups was much longer than that in the control group, P<0.01; The survival time was not different between the first group and the second group, P=0.315.The survival time of nude mice in the third, the forth, and the fifth group were greatly longer than that in the first and the second group, P<0.01.And it was longer in the forth and fifth group than that in the first, second and third group, P<0.01.The survival rate of the nude mice in forth and fifth group were much longer than that in the first group, P<0.01.3. Apoptosis and Cell Cycle of Transplanted Tumor Cells: Subdiplont apoptosis peak of transplanted tumor were observed in Gl cytocycling of the experimental groups and there was significant difference compared between experimental and control group, P<0.01.The apoptosis rate was not different in the first and the second group, P>0.05.And there was much difference of apoptosis rate in the third, the forthand the fifth group, P<0.05.The apoptosis rate in the forth and the fifth group were much higher than that in the first group, P<0.01.4. Tumor NK Cell Cluster Counting: All the fresh tumor tissue of nude mice 3 days after treating 2 and 4 times had NK cluster cells and there was significant difference in the experimental group and the control group, P<0.05.The NK cell number wasn't different in the first and the second group, P>0.05,while it was different greatly in the third, forth and the fifth group, P<0.05.There was much difference of the NK cell number in the forth and fifth group than that in the first group, P<0.01.The NK cell number rose with the treating times increasing. The NK cluster cell increased distinctly when treated forth than treated twice in the forth and the fifth group, P<0.01.5. Changing of Nude Mice Spleen Weight: The nude mice spleen in the treatment group augmented distinctly than that in the controls, P<0.01; And it aggrandized with the treatment time prolonging. The weight of the nude mice spleen in the first and the second group was different after treated 2 times, P=0.024, while there was no difference after treated 4 times, P=0.131; The spleen weighed greater in the forth and fifth group than in the first group, P<0.01.6. NK Cell Activity of Nude Mice Spleen: The NK cell activity toned up with the treatment time prolonging and the different was significant, P<0.05. When the effect-target-ratio of the treatment groups was 5:1,10:1 and 20:1, the NK cell activity in the treatment groups had much difference than that in the control group, P<0.01. There was significant difference of the NK cell activity in the forth and fifth group than in the first group, f<0.01. All these showed that IL-15 and IL-12 had adjuvant effect on ATV-NDV and IL-15, IL-12 could be used as an effective adjuvant to tumor vaccine.7. Pathologic Examination of Nude Mice: The transplanted tumor cells of nude mice augmented duplicately, the size and the modality varied greatly, the frame of the tumor tissue was foul-up, took on flakes and strips. The karyon was bigger, the chromatin dyed greater, the form was abnormal, the atypia was distinct. All the...