| Hepatocellular carcinoma (HCC) is one of the common malignant tumor in world-wide. High incidence, poor prognosis, high mortality, and short survival time are its features. Surgical excision is the best treatment, However, the majority of tumors (75%) are in intermediate or advanced stage when found. The volume of tumor is so big that it can not be completely resected. The traditional chemotherapy and radiotherapy are ineffective in the unresectable primary liver cancer. Radioimmunoassay treatment is respected as a better treatment of advanced hepatocellular carcinoma in recent years. It uses monoclonal antibodies as carrier which has specific oriented ability, and it can kill tumor cell when coupling with radionuclide. a variety of monoclonal antibodies are applied to radioimmunoassay experimental study as carrier at home and abroad. But as for some factors such as characteristics of Monoclonal Antibody, the expressive heterogeneity of tumor antigen,the antigen modulation, the special barriers function of tumor angiogenesis, the route of administration, the effects of radioimmunotherapy has been greatly affected. Some means such as applying cytokines, increasing vascular permeability are used to strengthen the binding capacity of labeled antibody with tumor cell antigen, then the effect of radioimmunotherapy can be enhanced. Recently, some methods such as combining two or more different targeting antibodies, enhancing the concentration of radionuclide in the tumor tissue has become the new hot spots of RIA treatment.Anti HBsAg Fab is a complete humorized Fab fragment which targets for hepatitis B surface antigen(HBsAg), and it can bind with the HBsAg excreted from cell membrane specificly. The epidemiology shows that HBsAg has a close correlation with the occurrence of liver cancer in China, and HBsAg is called "HCC associated antigen" in a sense. Chimeric Tumor Necrosis Treatment (chTNT) is a human/mouse chimeric monoclonal antibody which can bind with nucleoprotein that exposes when tumor cell necrosis (DNA histone H1 complex). Study shows that chTNT labeled with 131I has a suppressive function to a variety of solid tumors, and it is licensed to treat advanced lung cancer by Chinese Food and Drug Administration (SFDA).Sorafenib is a multi-target small molecule oral anticancer drug. Preclinical study and clinical trial predicts that sorafenib has a wide anti-tumor function, for it can not only inhibit the formation of vessel but also suppress proliferation of tumor cell directly, and it is licensed to treat advanced renal carcinoma by Food and Drug Administration (FDA). It also achieves encouraging results in II /III stage reseaches on other solid tumor. Our aim is to study the inhibitory effect of combining 131I-chTNT with 131I- anti-HBsAg Fab to the subcutaneous humor liver caner xenografts of HepG2.2.15 and the effect of distribution in vivo of 131I-chTNT by applying sorafenib to suppress the Tumor micrangiogenesis, to provide experiment statistics for clinic, and to lay the foundation for combining multi-antibodies target therapy and Small molecule drugs.Chapter 1. Animal experiment studies on the radioimmunotherapy of the hepatoma with intratumoral injection combining 131I-humanrized anti-HBsAgFab with 131I-chTNTMethods:1. A model of human liver cancer was establishes by subcutaneous transplantation of HepG2.2.15 cells in nude mice: 0.2ml suspension containing 1×107 HepG2.2.15 hepatoma cells is inject into the hypo of armpit. Then divided the tumor tissue into small pieces of l-2cm3 when the tumor has big enough, and implantate them into the hypo of armpit of other nude mice.2. Measure the marking rate, constancy and the immunobinding rate of the anti-HBsAg Fab, chTNT and IgG was labeled by 131I.3. 25 mice are randomly divided into 5 groups: 131I-anti-HBsAg Fab group; 131I-chTNT group; combination routine treatment group; positive control group; negative control group.4. Treatments is designed as follows: the mice in 131I-anti-HBsAg Fab group (A group) receives 14.8MBq/40ul 131I-anti-HBsAg Fab; the mice in 131I-chTNT group(B group) receives 14.8MBq/40ul 131I-chTNT; the combination routine treatment group(C group) receives 7.4MBq/20ul 131I-chTNT + 7.4MBq/20ul 131I- anti-HBsAg Fab; the mice in positive control group(D group) receives 14.8MBq/40ul 131I-IgG; the mice in negative control group(E group) receives 40ul normal saline. Treatment is given by intratumorally injection.5.After treatment, the situation of growth and living of nude mice is observed everyday. Measure the long-diameter (a) and the short diameter (b). Calculate the volume of transplanted tumor using the formula: volume =0.5*a*b*b.6.On the first, forth and seventh day, anesthetize the nude mice for SPECT imaging, and calculate the ratio of radioactivity in tumor/non-tumor tissue, then observe the distribution and metabolism of antibody labeled with 131I in vivo. On the 28th day, all mice are killed by anesthetic overdose. The tumors are obtained from xenografts, then calculate the inhibition ratio of transplanted tumor and observe the pathological situation in microscope.7. The sample data are analyzed statistically by using SPSS 11.5 software. The quantity data are represented by the way of "Mean±SD"; As for the immunobinding rate of antibody labeled with radionuclide, Independent-Sample T Test is used ; As the ratio of T/NT of antibody in nude mice and the comparison of the transplant tumor volume among groups before and after taking the medicine, Repeated Measure ANOVA is used. among it, the comparison between two groups use LSD Test while Welch or Tambane's T2 Test with equal variances not assumed.Results:1.The average marking ratio of anti-HBsAg Fab, chTNT and IgG measured by TCA is 60%, 50% and 58.3% separately. The radiochemical purity of 131I-anti-HBsAg Fab,131I-chTNT and 131I-IgG after purification is 100%,99 %和99.3% separately, and the mean activity ratio of them is 22.2MBq/mg,30.8MBq/mg和19.5MBq/mg individually. The immunobinding rate of 131I-anti-HBsAg Fab with HepG2.2.15 cell is 68.3% while that of 131I-anti-HBsAg Fab with HepG2 cell is 10.9% ( P<0.001).2. The ratios of T/NT in 131I-chTNT group (B group), 131I-anti-HBsAg Fab (A group) group and combination group (C group) arise gradually, Form the point of view of time, there is no significant difference in T / NT ratio among groups on the first day after the treatment (P >0.05).On the seventh day, there is significantly difference in ratio of T/NT between C group and A or B group, the P value is 0.005 and 0.029 separately, nor is the ratio between A group and B group(P=0.386). image is visible in positive control group on the first day, but on the seventh day, the concentration of radiation decreased significantly. Radioactivity can not be found in negative control group all the time. 3. As for the volume of tumor, there is significant difference among groups at the different time(F= 14.287,P<0.001), so is the difference in the volume of tumor among groups (F= 14.287,P<0.001). In the forth week, there is significantly difference in volume of tumor between C group and B group(P<0.003), so is the difference between C group and A group(P<0.001), and nor is the difference between A group and B group(P=0.325). The growth inhibitory rates of C group is 84.32% when the observation is over, which is significant higher than 63.86% of B group, 51.49% of A group and 14.87% of D group.4. After staining with HE, the necrosis tissue in the there groups of drug treatment are more than that in control group, the nucleus of tumor cells are pycnosis significantly, and the combination group is most obviously.Conclusion:1. The 131I-anti-HBsAg Fab and 131I-chTNT have good constancy and immunocompetence.2. Intratumoral injection can increase the concentration of antibody labeled with radionuclide in the tumor tissue to improve the effect of targeted therapy, because it can induce the antibody to enter into tumor tissue directly and need not circulate through the blood.3. Combining 131I-anti-HBsAg Fab with 131I-chTNT, radionuclides can be more durable in the tumor tissue and have a stronger capacity of killing tumor cells than using 131I-anti-HBsAg Fab or 131I-chTNT alone.Chapter 2 The study of how sorafenib effect the distribution of 131I-chTNT innude mice and the ability to inhibit tumor growth of 131I-chTNTMethods1. A model of human liver cancer was establishes by subcutaneous transplantation of HepG2 cells in nude mice: 0.2ml suspension containing 2×106 HepG2 hepatoma cells is inject into the hypo of armpit. Then divided the tumor tissue into small pieces of 1-2cm3 when the tumor has big enough, and implantate them into the hypo of armpit of other nude mice.2. Labeling chTNT by 131I.3. 33 mice are randomly divided into 3 groups. Treatments is designed as follows: twelve mice in the first group receive sorafenib ,40mg.kg-1; twelve mice in the second group and nine mice in the third group receive normal saline at the same dose, Treatment is given by intragastric administration and lasts for 14 days; In the 15th day, take 3 mice from the first and the second group separately, kill them by anesthetic overdose and dislodge the transplanted tumor, and tumor microvessel density(MVD) is measured by inmunohistochemistry. The remaining mice are given intratumorally injection with 14.8MBq/20ul 131I-chTNT, then the mice in second group receives sorafenib, 40mg.kg-1. The other mice receives normal saline at the same dose. On the first, forth and seventh day after being treated with 131I-chTNT, take 3 mice separately in every group to make SPECT imaging, Then kill the mice, take out and weigh(g) the tumor, heart, blood, liver, lung, kidney, spleen of each mouse. Next measure the radiocounting (D) by well-type calibrating machine. Calculate the proportionality of D/g and the ratio of tumor/non-tumor tissue (T/NT).4. 25 mice and divide them into 5 groups stochastically. Treatments is designed as follows: the mice in sorafenib group receives sorafenib 40mg.kg-1+ normal saline 40ul by intratumorally injection; the mice in 131I-chTNT group receives 14.8MBq/40ul 131I-chTNT + normal saline 1ml, with intragastric administration lasting for 14 days; the sequent combination group receives 14.8MBq/40ul 131I-chTNT after sorafenib 40mg.kg-1 is given 14 days; the mice in simultaneous combined group receives 14.8MBq/40ul 131I-chTNT + sorafenib 40mg.kg-1 at the same time; the mice in control group receives 40ul normal saline intratumoral injection + normal saline 1ml intragastric administration, lasting 14 days. sorafenib is given by intragastric administration and continues for 14 days, and 131I-chTNT is given by intratumoral injection. After treatment, observe the situation of growth and living of nude mice. All mice are killed after 4 weeks, then calculate the volume of transplanted tumor and the inhibition ratio, then observe the pathological situation of transplanted tumor in microscope.5. The sample data are analyzed statistically by using SPSS 11.5 software. The quantity data are represented by the way of "Mean±SD"; As for MVD in tumor tissue of two groups, Independent-Sample T Test is used ; As the ratio of T/NT of antibody in nude mice and the comparison of the transplant tumor volume among groups before and after taking the medicine,, the Repeated Measure ANOVA is used, among it,, and the comparison between two groups use LSD Test while Welch or Tambane's T2 Test with equal variances not assumed.Results1. The MVD of tumor tissue in sorafenib group is significantly lower than that of in control group (P=0.004).2. The radionuclide concentration level of tumor tissue in sequent combination group is higher than that of in other groups obviously (F=74.258, P<0.001). From the first day to the seventh day after intratumoral injection, comparing with the simultaneous combination group and the 131I-chTNT group, the ratio of T/NT in sequent combination group is obviously higher than that of in them after intratumorally injecting 131I-chTNT, and it has statistical difference among there groups , the value of P is all less than 0.01. At the same time, there is no statistical difference between simultaneous combination group with the 131I-chTNT group, and the P value in the first and seventh day is 0.187 and 0.933 respectively. 3. In the transplantat tumor suppressive experiment, tumor growth in sequent combination group and simultaneous combination group is suppressed significantly, and there is no statistical difference between them, but there is statistical difference between groups mentioned above and other groups, the P values are less than 0.05. At the end of experimental period, the growth inhibitory rates in sequent combination group and simultaneous combination group is 85.02% and 89.05%, while that of 131I-chTNT group and sorafenib group is 57.64% and 56%.4.the necrosis tissue in the groups of drug treatment are more than that in control group, the nucleus of tumor cells are pycnosis significantly, and the simultaneous combination group and sequent combination group is most obviously.Conclusion:1. Sorafenib can significantly inhibit the angiogenesis of transplant tumor.2. The decrease MVD transplant tumor can extend the metabolism time of radionuclides and enhance concentration of 131I-chTNT in tumor tissue.3. Combining sorafenib with 131I-chTNT have a stronger anti-tumor effect than using sorafenib or 131I-chTNT alone. |
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