| Tumor necrosis therapy was firstly proposed by Epstein, Professor of University of Southern California. It was a radioimmunotherapy for solid tumors, and the binding sites were nuclear antigens, which exposed after tumor cell degeneration and necrosis. There were spontaneous necrosis region in tumor tissue;131I-chTNT could be combined with the nuclear antigen in the necrosis region. There were not necrotic areas in normal tissues due to the monitoring of the reticuloendothelial system, so TNT had a characteristic of broad-spectrum and specificity. The experiments confirmed that TNT can bind to tumor cell nuclear antigen of mammals including the human, mouse and so on.131I-chimeric tumor necrosis therapy monoclonal antibody(131I-chTNT) was a drug of RIT which was firstly used in solid tumors in the world.131I-chTNT was a standard radioimmunotherapy drug, which was consituted of specifical antibodies to identify tumor and radionuclide to anti-tumor. But spontaneous necrosis in tumor tissue was limited; the expansion of tumor necrosis could increase131I-chTNT binding sites, increasing the distribution of drug in the tumor to increase its efficacy. There were thermal ablation and cryablation in clinical temperature ablation, microwave ablation and argon-helium refrigeration were the representative. Microwave ablation and argon-helium refrigeration could increase the necrosis in the tumor and combining with131I-chTNT could increase efficacy theoretically. It had been reported microwave ablation followed by intratumoral injection of131I-chTNT could increase the131I-chTNT uptaking in tumor tissue. Between argon-helium refrigeration, microwave ablation followed by intratumoral injection of131I-chTNT the differences had not been reported and we could study.We designed this experiment and combined the argon-helium refrigeration, microwave ablation with131I-chTNT. Firstly we simulated argon-helium refrigeration, microwave ablation to treat of Lewis lung carcinoma cells and combined with131I-chTNT.Inspection of the differences between argon-helium refrigeration, microwave ablation with131I-chTNT in the cellular level, which provided a reference for animal experiments.Chapter1Control study of simulating the argon-helium refrigeration/microwave ablation followed by131I-chTNT to the Lewis lung carcinoma cellsPurposeThe Lewis lung carcinoma cells were heat-inactivated/frozen inactivated followed by131I-chTNT and investigated the differences after the tumor cells treated by two ablation necrosis with131I-chTNT.MethodTaking to garithmic phase cells were divided into groups A, B, and C. Group A was the control group, which was not treated. Group B was simulating microwave ablation group. The cells in the group B were put into the37℃water to heat. Until the water was boiled and maintained for10mins. Group C was simulating argon-helium refrigeration. The cells in the group C was put into the liquid nitrogen for5min and melted in the room temperature for two cycles. The two sets of trypan blue detecting necrosis should reach100%necrosis.0.001mci131I-chTNT was put into the each group in each tube and incubated30mins under37℃conditions and centrifuge5mins under low temperature and high-speed of12000rpm. Supernatant was removed with washing with PBS and the above process was repeated for three times. The radioactivity was measured by y counter and compared the differences of combination between cells in the three groups with131I-chTNT.Statistical analysis. The sample data were analyzed statistically by SPSS13.0software. The uantity dates were represented by the way of "Mean±SD". The differences in each group of radioactive count were analyzed in one-way analysis of variance (One-way ANOVO).The comparison between groups using LSD Test when the variances were equal. When the variances were not equal, Welch was used in the overall comparison and Dunnett’3Test was used in the two groups. P<0.05was defined statistically significant.ResultsThe radioactive counts in the group B and group C was higher than group A; the radioactive counts in the group C was higher than in the group B (P<0.05)ConclusionsArgon-helium refrigerating and microwave ablating the Lewis cells could improve the combination of131I-chTNT with the tumor necrosis; compared the argon-helium refrigeration to the microwave ablation, the former was beneficial for uptake131I-chTNTChapter2Control study on percutaneous argon-helium refrigeration/microwave ablation followed by intratumoral injection of131I-chTNT to the Lewis lung carcinomaPurposeThe Lewis lung carcinoma was percutaneously refrigerated/microwave ablated followed by intratumoral injection of131I-chTNT and investigated the differences after the tumor treated by two ablation necrosis with131I-chTNT.Method1.A mouse tumor model was established by subcutaneous injection with Lewis lung cancer cells. Lewis cells in logarithmic growth phase was injected in the right side of the back skin in906-week-old C57BL/6mice. About three weeks,when the maximum tumor diameter reach15mm,they could be used in the experiment.2.Group and treatment.75tumor-bearing mice were selected to used in experiments in accordance with the principles of tumor size. The75mice were divided into group A, group B and group C in accordance with the principle of completely randomization. Group A was single-drug and group B was microwave ablation group and group C was argon-helium refrigeration group. There were25mice in each group,20mice in each group were used to measure the drug distribution in tumor tissues and the remaining5mice in each group were used for SPECT imaging. Group A,single group, only with the injection of131I-chTNT drugs,18.5MBq/0.05ml.Group B, microwave ablation were implemented5days prior drug injection then the drugs were injected into the center of the tumor. The methods and dose of drug injection were the same as group A. Group C, argon-helium refrigeration were implemented5days prior drug injection then the drugs were injected into the center of the tumor. The methods and dose of drug injection were the same as group A.3. Drug distribution in the tumor of mice.1,3,5,7day after drug injection five mice were killed in each group in each time, get the tumor organization and use y counter to weight determination of radioactivity uptake per gram of tissue(%ID/g).4. Radionuclide scanning and imaging of tumor-bearing mice.1,3,5,7day after injection,five mice in group A, group B, group C were implemented SPECT imaging. ROI technology was used, the tumor tissue (T) and corresponding contralateral parts of the same size region (NT) were selected, radioactive counts was measured and T/NT was analyzed.5. Statistical analysis. The sample data were analyzed statistically by SPSS13.0software. The unantity dates were represented by the way of "Mean±SD". The difference of drug distribution in tumor tissue in mice were analyzed in factorial design information variance analysis.The ratio of T/NT was compared by repeated measures analysis of variance. The Greenhouse-Geisser calibration coefficient was used when the statistical was not meeting the spherical assumption. P<0.05was defined statistically significant.Results1. The drugs distribution in each group of tumors. The radioactivity uptake per gram of tissue(%ID/g) in group B and C was higher than in group A. The radioactivity uptake per gram of tissue(%ID/g) in group C was higher than in group B.2. Radionuclide scanning and imaging of tumor-bearing mice. There were obviously enrichment in tumor tissue Id after injection. The radioactive in tumor tissues was highest in5day in the three groups. The tumor imaging were still visible until7days. The tumor radioactivity in group B and C was higher than in group A. Compared with Group C and group B, the tumor radioactivity in former was higher.3.T/NT ratio. There was significant differences of drug distribution between tumor tissue and non-tumor tissue. T/NT in the group B and group C was significantly higher than in the group A. Compared group C with group B, the former of T/NT was higher.Conclusions131I-chTNT uptake in the tumors could be promoted by argon-helium refrigeration, and microwave ablation. Compared the argon-helium refrigeration with microwave ablation followed by intratumoral injection of131I-chTNT, the former was beneficial for uptake131I-chTNT.Chapter3The study of efficacy on percutaneous argon-helium refrigeration/microwave ablation followed by intratumoral injection of131I-chTNT to Lewis lung cancerPurposeTo study the drug distribution in tumor after intratumoral injection. The differences of efficacy were observed when the Lewis lung carcinoma treated by percutaneous argon-helium refrigeration/microwave ablation followed by intratumoral injection of131I-chTNT.Methods1.A mouse tumor model was established by subcutaneous injection with Lewis lung cancer cells. Lewis cells in logarithmic growth phase was injected in the right side of the back skin in606-week-old C57BL/6mice. About three weeks,when the maximum tumor diameter reach15mm,they can be used in the experiment.2. Group and treatment.45tumor-bearing mice were selected to used in experiments in accordance with the principles of tumor size. The45mice were divided into group A, group B, group C, group D, group E and group F in accordance with the principle of completely randomization. Group A was single-drug and there were10mice in the group, only with the injection of1311-chTNT drugs,18.5MBq/0.05ml. Group B, there were10mice in the group and microwave ablation were implemented5days prior drug injection then the drugs were injected into the center of the tumor. The methods and dose of drug injection were the same as group A. Group C, there were10mice in the group and argon-helium refrigeration were implemented5days prior drug injection then the drugs were injected into the center of the tumor. The methods and dose of drug injection were the same as group A. Group D was singal microwave group. There were5mice in the group and they were microwave ablated singally. Group E was singal argon-helium refrigeration group. There were5mice in the group and they were argon-helium refrigerated singally. Group F was control group and there were5mice in the group.0.05ml saline was injected in the center of the tumor.3.Autoradiography and observation for HE staining. Five mice were killed3days after131I-chTNT injection in group A、B、C. Tumor tissues were taken and done conventional biopsy, two consecutive slices of each tissue were performed autoradiography and HE staining. The images of HE staining and autoradiography were observed and the different tumor necrosis radioactive distribution in groups were compared.4. Observing the efficiency. Observe appetite,movement, coat and mental changes of mice every day after the Lewis lung cancer cells were injected. The long-diameter(a) and the short diameter(b) were measured every3days until28days. According to volume=(axb2)/2.The tumor volume were calculated and the tumor volume growth curves were drawn in mice.5. Statistical analysis. The sample data were analyzed statistically by SPSS13.0software. The unantity dates were represented by the way of "Mean±SD". The tumor volume changes were compared by repeated measures analysis of variance. The Greenhouse-Geisser calibration coefficient was used when the statistical was not meeting the spherical assumption. P<0.05was defined statistically significant.Results1. The distribution of the drug in the tumor. Both autoradiography and HE staining show that:group A had smaller tumor necrosis region and there were also less drug distribution in the tumor. The regions of tumor necrosis in group B and C were significantly greater than the group A, the distribution of drugs in the tumor were also more. The tumor necrosis region in group C were greater than the group B, the distribution of drugs in the tumor were also more.2. Drug-specific binding verification. Compare autoradiography and HE staining we could see that the drug combinated with necrotic regions.There was no distribution in non-necrotic regions.3. The observation of drug efficacy. Tumor growth in different groups were different.Group B and C significantly slowed in tumor growth. The tumor volume in group B and C were significantly smaller than in group A,D,E and F. Compared group B with group C, tumor growth of latter slowed than the former, the volume was less than the former.Conclusions131I-chTNT specifically bind with tumor necrosis area, non-necrotic area. There was no distribution in non-necrotic regions. Injection of131I-chTNT after giving the argon-helium refrigeration and microwave ablation could increase drug uptake in tumor tissue, thereby increasing efficacy. Compared the argon-helium refrigeration with microwave ablation followed by intratumoral injection of131I-chTNT,the former was beneficial for uptake131I-chTNT and thereby increasing efficacy... |
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