| Objective:This study aims to improve the model colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum,which has higher hepatic metastasis rate.we establish the model of the colorectal recessive liver metastases(CRLM)in order to provide the basis of exploring the mechanism of colorectal liver metastases further.First,using a transwell adhesion assay,we can collect the higher migratory CT26 colorectal cancer cells from the ordinary CT26 colorectal cancer cells,and using the monoclonal cultivation to replicate the higher migratory CT26 colorectal cancer cells.We use the higher migratory CT26 cells to establish a model of colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum,and increase the higher hepatic metastasis rate of the model.Using the ordinary,lower migratory and higher migratory CT26 cells to establish the model,and compear to each model to confirm using the higher magritory can increase the higher hepatic metastasis rate of the model or not. Immunohistochemistry was used to verify the accuracy of this method, a larger sample size is needed to verify the stability of the model.We observe the tumor growth characteristics of this model mice to filter the CRLM,and confirm CRLM through the pathological examination.At last,we use the 2D Difference Gel electrophoresis(2D DIGE) method to distinguish the differential proteins form the CRLM group,CDLM group and the normal control group,that we can confirm the theory of the CRLM preliminary.Methods:Part 1:The cultivation and screencing of high migratory CT26 colorectal cancer cells Select the CT26 colorectal cancer cells, were cultivated by the complete medium,which Containing 10% foetal bovine serum(FBS) and 90% DMEM.We using trypsin enzyme digesting cells,after cultivating enough cells,and inject these cells into the centrifuge tube.To seed some cells in to ordinary 6 well cell cultrue cluster after adjusting the concentration, and adding appropriate DMEM complete medium into the 6well cell cultrue cluster(this kind of cell labeling for Group A).And we seed equal the number of cells in to apical chamber of the Transwell and adding appropriate High glucose DMEM medium.We add appropriate complete medium in to the bottom membrane of the Transwell(marking within Group B).Every 12 hours dynamic observing the cells’ growth and morphology,which in the apical chanber and the bottom chamber of the Transwell.After about 72 hours cells in to the apical chamber of the Transwell were digestived and removed to normal petri dish(marking within Group Bup).The cells in to the bottom membrane of the Transwell were continue to cultured.(marking Group Bdown).Part 2:Establishment of the improved mice model and verifying the stability of the model Digested CT26 colorectal cancer cells of Group A, Group Bup and Group Bdowm from each culture dish and resuspended with 1×PBS.Injucted the cells’ suspension with with a syring in to the nape of three male mice(SPF,4-5week,i H.) respectively and marked Group A, Bup and Bdown. After 13 days’ normal diet, each mice grew a tumor of a size about 1.5cm * 1.5 cm in the nape. Take out the tumor of the three mice and cut them into the size of about 2 mm* 2 mm respectively. Inoculated those pieces of the tumor to the corresponding 6 mice named Group A, Group Bup and Group Bdown. We established hepatic metastasis of colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum.Mice had normal diet after established the model and were observed dynamically of the sistuation of tumor growth and the weight.All of them were sacrificed after 5weeks.Observed the livers in gross to check whether they had nodules on the surface. Livers were embeded in parafin and cut into thin slices for pathologic examination. Determind whether there is a dominant liver metastasis.Compared those groups who had dominant liver metastases and verifed whether the CT26 colorectal cancer cells of Group Bdown had highed migration ability.Compared the expression level of S100A8,MMP2,Vimentin between the liver and the carcinoma in site among among each group. Using Group Bdown cells to establih the model with 36 mices(labeling Group C).Compear Group C’ liver metastses rate to Group Bdown’,and confirmed the improved model has well stability.Part 3:Confirming the theory of the CRLM Using the CT26 colorectal cancer cells of Group Bdown to established hepatic metastasis of colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum. Mice were sacrificed after 5 weeks. Classified those who had nodules on the liver surface and pathologicaly hepatic metatase of colorectal cancer cells as Group CD(Colorectal dominant liver metastase).Classified those which had not dules on the liver surface but pathologicaly micro-hepatic metastases of colorectal cancer cells as Group CR(Colorectal recessive liver metastases).Classifiedthe 10 mice who only had the same operational procedures without transplanting the tumor as group NC(non-Colorectal).Ramd only selected 7 fresh livers respectively from Group CD,CR and NC,made relevant pretreatment and then detected with Two-Dimensional Difference Gel Electrophoresis method. Seperated the liver protein of each group and took statistical analysis of differential proteins to verify the theory of colorectal cancer recessive liver metastases.Results:Part 1:The cultivation and screencing of high migratory CT26 colorectal cancer cells After 12 hours the bottom membrane of the Transwell in which has not CT26cells(Group Bdowm).24 hours later some CT26 cells appear in the bottom,compared to the Group A cells and Group Bup cells,we can find that Group Bdowm cells’ morphology was round,the synapses was shorter,the number of the synapses and the stellate cells was less.72 hours later we can find the more number of the Group Bdowm cells.And this cells’ synapses prolong after adherencing.Compared to the Group A cells,the number Group Bdowm cells’ synapses was less,but the length of the Group Bdowm cells’ synapses was longer.Part 2:Establishment of the improved mice model and verifying the stability of the model Modeled mice recovered well after the operation, and there were no wound infection and fistula.NO.5 of Group Bup did not form tumor.The overall tumor rate was94.4%.The weight of Group Bdowm were lower than those of the rest of the 2 groups,and it had a greater difference than Group Bup(P < 0.05).The tumour volume of the Group Bdown was significantly greater than the rest of the two groups(P < 0.05).Mice were scrificed after 5 weeks.Group A and Group Bup had no liver metastases in the gross,while there were dominant metastases in the two mice of the Group Bdowm.Pathologic results suggested the mice of the former two groups only had minor metastases, and the two mice from Group Bdowm indeed had hepatic metastases from colorectal cancer. The expression level of S100A8 in Group Bdowm’ carcinoma insitu was significantly higher than the other two groups(P < 0.05);The expression level of S100A8 in Group Bup’ liver metastases was higher than their carcinoma insitu, but without statistical significance.The expression level of MMP-2 in Group Bdown’carcinoma insitu was higher than the rest of the two groups(P < 0.05), and Group Bup’was significantly higher than Group Bup’(P < 0.05);the difference of expression level of MMP-2 had no statistical significance between the liver metastases and the carcinoma insitu.The expression level of Vimentin in Group Bdowm’ carcinoma in situ was significantly higher than the other two groups(P< 0.05);the expression level of Vimentin in Group Bdowm’ liver metastases was higher than their carcinoma insitu.Expanding the model number, wich liver metastases rate is 27.7%,compared with the previous cmodel’ liver metastasis rate with no statistically significant(P> 0.05), and confirmed the improved model has well stability. Part 3:Confirming the theory of the CRLM Pathologic results suggested there were dominant metastases in the seven mices of the Group CD.The mices had minor metastases labeled as Group CR.Through statistical analysis,the dominant liver metastases rate was 25%,which approaching to the Group Bdown’ rate.We use Two-Dimensional Difference Gel Electrophoresis method.to seperated the liver protein from each group and using Decyder TM2 D 6.5 to statistical analysis,compare Group CD to Group CR we can find that there were 23 differential proteins.Compare Group CD to Group NC we can find that there were 132 differential proteins.And we can verify the theory of colorectal cncer recessive liver metastases preliminary.Conclusion:1. Using a transwell adhesion assay,we can collect the higher migratory CT26 colorectal cancer cells2. Using these higher migratory CT26 cells to establish a model of colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum,wich had preferably stability.3. Simulating the recessive liver metastasis model,according to the model of colorectal cancer in mice by orthotopic transplantation with fresh tumor tissues on herniated cecum. Through the methods of pathologys and proteomics, we can preliminary prove the feasibility of theory of this "colorectal cancer recessive liver metastasis". |
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