Improvement Of Establishing An Animal Model For Thymic Lymphoma Induced By N-methyl-N-nitrosourea In Inbred Mice And Preliminary Study On The Treatment

【Objective】To improve the tumor rate and reduce the mortality rate of the animal model for thymic lymphoma in inbred mice induced by N-methyl-N-nitrosourea (MNU), we tried to change the program and experimental cycle. On this basis, we meant to establish a new cell line from the tumor we induced. To reveal the effect and mechanism of icaritin(ICT) on EL-4 cells in vitro and its anti-tumor effects on xenografts of thymic lymphoma induced by MNU in nude mice.【Methods】1. In the first part of our study, 80 male and female mice of the C57BL/6 strain at 6-8 weeks of age were randomly assigned to exposure and control groups, 66 and 14 animals respectively. Briefly, the exposure groups were divided into three groups (G1,G2,G3). They were injected by the intraperitoneal route with MNU solution shortly after dissolution at the dosage of 50,50,100mg/kg body weight separately. The control group received the same dosage of sterile 0.9% NaCl solution. The experimental animals were boosted 4 weeks later. The G1,G2 groups were injected at the dosage of 50,70mg/kg body weight separately. The G3 group and the control group received the same dosage of sterile 0.9% NaCl solution. Following injection , the mice were examined frequently. Seriously ill animals were killed and with mice found dead were completely autopsied. All mice were sacrificed by cervical dislocation for autopsy before the 25th experimental week. Complete gross examination was performed for detection of tumor masses. Thymic lymphomas as well as involved organs were fixed and routinely processed for light microscopy examination to analyze histopathologic features of the tumors. Sections were submitted to immunohistochemical staining with CD3, CD20, TdT and PCNA to identify the origin and subtype classification of the neoplasias.2. In the second part of our study, on the basis of the induced tumor model, utilize trypsin digestion method or mechanical dispersion method to obtain sing-cell mixing suspension. In vitro primary culture of the tumor cells.3. In the third part of our study, we try to investigate the effect and probable mechanism of icaritin(ICT) on EL-4 cells in vitro: MTT was used to examine the effect of ICT on the proliferation of EL-4 cells; transmission electron microscope (TEM),flow cytometry analysis were used to examine apoptotic cells;The expression of Bcl-2,P21 mRNA was determined by reverse transcript-polymerase chain reaction (RT-PCR) assay; Colorimetric method was used to measure Caspase-3 and Caspase-9 activities.4. In the fourth part of our study, we establish the xenografts of MNU induced tumor in nude mice , then evaluate the effect of ICT on xenografts growth. Mice bearing growing tumors were divided into five groups. There were sterile 0.9% NaCl solution negative control group and three treatment groups in which the dose of ICT in every animal was 0.5mg/kg,1.0mg/kg,1.5mg/kg.Reagents were administered via intraperitoneal injection once every day for ten times. There was Cyclophosphamide positive control group in which the dose was 0.2ml (10mg/ml ). Reagents were administered every two days for three times. All mice were sacrificed by cervical dislocation for autopsy 24h after the experiment. Weigh the tumors and detect the important organs by histology assay to evaluate the effect of ICT on xenografts growth .【Results】1. At the 25th week, the proportion of MNU-treated animals developing thymic lymphoma after injection of MNU was 83.3%( 55/66). The mortality rate was 7.6%. Histopathologic study revealed that 55 cases of thymus became totally replaced by sheets of cells of the lymphoid series. The thymic tumors were densely infiltrated with relatively uniform, mitotically active, medium-sized cells with round to ovoid and sometimes convoluted nuclei that were surrounded by small amounts of cytoplasm. The chromatin pattern was finely granular and prominent nucleoli were present in most of the cells which had a high mitotic rate. There may be a"starry sky"pattern in some cases. All the tumors coexpressed CD3 and TdT.2. Utilizing trypsin digestion method or mechanical dispersion method to obtain sing-cell mixing suspension. During the in vitro primary culture of the tumor cell, the rate of live cells decline from 98%±1.5% to 30%±3% which was impossible to continu culture.3. ICT could inhibit the proliferation of EL-4 cells significantly in a dose-and time-dependent manner. Apoptotic cells were observed by TEM and flow cytometry analysis in EL-4 cells treated with ICT . RT-PCR analysis showed that the expression of bcl-2,P21 were down-regulated. Besides ,Caspase-3 and Caspase-9 activity in EL-4 cells elevated remarkably after 40 ,80μmol/ L ICT treatment for 6h compared with the control group.4. Though, before and after the treatment of ICT, the average size of xenografts in nude mice between exposure groups and control group was nearly the same. There were more cell shrinkage and dead cells in 1.0mg/kg,1.5mg/kg treatment groups than incontrol group by light microscopy assay. .But only one case in each treatment group.【Conclusions】1. By changing the program and experimental cycle, we can improve the tumor rate of the mice thymic lymphoma model induced by MNU from 67.5% to 83.3%and reduce the mortality rate from 10% to 7.6%.2. We fail to culture the tumor cells in vitro primarily. The proper method needs to be researched.3. ICT could inhibit the proliferation of EL-4 cells and induce EL-4 cells apoptosis in vitro. The probable mechanism may be down-regulate the expression of bcl-2,P21and elevate Caspase-3 and Caspase-9 activity in EL-4 cells . 4. ICT may could induce EL-4 cells apoptosis in vivo. But needs further research.