Establishment Of Orthotopic Colon Tumor Model By Coloclysis With MNU In Rat And The Suppression Of Colon Cancer By CD133siRNA Gene Therapy

ObjectiveTo establish an orthotopic colon tumor and metastasis model by the coloclysis with MNU in SD Rat, observe the carcinogenesis development and the metastasis process. To investigate the expression of CD44 and CD 133 proteins in this colon carcinoma model, analysis the relationship of the cancer pathology stage and the expression of CD44,CD133, to apply for immunocytochemical staining, real-time-PCR,Western Blotting assay CD44,CD133 gene expression of protein distribution in colon carcinoma respectively.For Targeting CD 133 inhibition in vivo effect of colon carcinoma cells to do a further test, in order to explore the targeting of gene therapy in colon gene treatment of the feasibility and preliminary effects in vivo. For clinical new methods of the treatment, to explore the apoptosis and invasion of HT-29 cells induced by siRNA-mediated CD 133 silencing. To construct an expression plasmid of siRNA against CD133 gene and to investigate its biological behavioral effect on human colon carcinoma cell line HT-29.MethodsSD Rats were given the MNU clyster, check the clinical symptom of the Rats, observe tumor growth, metastasis and the clinical symptom.To respectively sacrifice 10 SD Rats every time at 8,12,16 weeks later, observe the tumor in situ and the metastasis. Evaluate the effect of this colon cancer model through pathological methods. Immunohistochemical technique was used to detect the expression of the CD44 and CD133 gene protein in 6 cases of the colon carcinoma model with paraffin embedded tissue samples, we inhibited the CD 133 expression using the siRNA method. Expression levels of CD 133 mRNA and protein from HT—29 after transfection were examined by RT-PCR and Western blot respectively. Changes of the cell growth activity in response to transfected plasmid were evaluated by MTT assay.The migration ability of transfected HT-29 cells were measured quantitatively by Transwell migration assays. Intratumoral gene therapy with a LipofectamineTM 2000/siRNA CD 133 plasmid DNA complex resulted in tumor growth suppression in nude mice. After siRNA CD 133 intratumoral gene therapy, Immunohistochemical technique was used to detect the expression of the CD 133 expression in the tumors compared to the control vector group.ResultsNone of the SD Rats died,8-12 weeks later, clinical symptom show hematochezia and lymphadenectasis,16 weeks later,90% Rat formed colon neoplasma and many lymphadenectasis, pathological examination confirm the colon adencarcinoma with lymph node metastasis. Using immunohistochemistry, we confirmed elevated expression of CD44 and CD133 on the colon tumors. After CD133 siRNA transfeetion into HT-29 cells, compared with that in control groups, the level mRNA of CD133 in cancer cells transfected with CD133 siRNA was inhibited in a concentration and time dependent manner and the CD133 protein concentration decreased significandy. The growth of HT-29 cells was significantly inhibited than those in control groups(P<0.05). CD133 siRNA significandy inhibited invasion of HT—29 cells to matrigel (P<0.05). Intratumoral gene therapy with siRNA CD 133 into the xenografted of nude mice generated tumors with a reduced tumor volume and wet weight, as compared to control group.ConclusionThe successful establishment of orthotopic colon tumor model by the coloclysis of MNU in SD Rats. The induction process is convenience and utility, model the colon carcinoma autogenesis development successfully. The orthotopic tumor and metastasis model provide useful tools for the study of mechanism of metastasis and its treatment of colon cancer. The expression level of CD44 and CD 133 is closely related to the occurrence and development of colon carcinoma. CD133 positive rate increases with the colon carcinoma. Silencing CD133 gene by the siRNA technology can actively suppress the expression of CD133 gene, and then inhibit the growth and invasion of HT-29 cell. Based on the mouse xenograft model, siRNA targeting a discrete sequence of CD133 may provide a potential therapeutic option for colon cancer.