摘要
目的
通过MNU灌肠法诱导大鼠原位结肠癌模型,探讨MNU肿瘤诱导过程中对于肿瘤干细胞标记物CD44、CD133表达的影响,建立研究肿瘤发展过程肿瘤干细胞标记物变化趋势的模型,尝试通过肠镜下对该模型进行干预研究。通过应用RNAi干扰技术,沉默大肠癌细胞株HT-29的CD133表达,研究其对HT-29细胞增殖和侵袭力的影响,为抑制肿瘤细胞体内生长实验做进一步验证,选择通过裸鼠肿瘤模型的治疗对照研究,对靶向CD133抑制大肠癌细胞的体内效果做了进一步验证,探讨靶向基因治疗在大肠癌基因治疗中的可行性、效应及初步作用机制,为研究RNAi体内干扰对大肠癌临床效果作出初步探索。
方法
应用MNU灌肠方法诱导大鼠结肠癌模型,并且通过肠镜实时监测肿瘤的发生发展,尝试在肠镜下对肿瘤进行注射治疗,应用免疫组织化学染色法、RT—PCR(实时定量PCR)、Western Blotting分别检测模型中诱导成功的结肠癌与正常结肠组织中肿瘤干细胞标记物CD44、CD133的基因及蛋白表达对比情况,分析其表达与肿瘤发生的关系。体外实验中,应用siRNA干扰大肠癌细胞株HT-29的CD133的表达,以RT-PCR及Western Blotting分析并优化转染的效率及效果,以MTT法检测细胞的增殖活性,以Transwell小孔实验方法检测转染对肿瘤细胞侵袭力的影响。体内试验中,通过建立裸鼠结肠癌模型,分组对照进行瘤体内多点注射,探讨CD133siRNA体内治疗的方法及效果,探讨siRNA在大肠癌临床治疗的可行性,并且进一步在MNU诱导大鼠结肠模型中进行原位肿瘤的干扰治疗打下基础。
结果
通过MNU灌肠成功诱导出大鼠结肠癌模型,病理分析证明该模型中肿瘤生长为腺瘤—不典型增生—腺癌的渐进过程,通过肠镜下检查成功做到了对该模型的实时监测,并成功尝试了对该模型中原位肿瘤治疗的干预。CD133siRNA成功转染了HT-29细胞,通过RT-PCR及Western Blotting检测,分析并优化了siRNA转染的浓度及时间,证明了转染过程伴有时间及浓度依存关系,MTT法检测证实,转染后细胞的增殖活性被抑制,Transwell实验表明转染后肿瘤细胞的侵袭力有所下降。动物体内实验表明,瘤内注射CD133siRNA能有效抑制裸鼠移植瘤生长,免疫组化分析了各组裸鼠肿瘤中CD133蛋白的表达,实验组与对照组有统计学意义(P<0.05),提示该基因治疗在临床应用上有良好的前景。
结论
MNU灌肠可以成功诱导大鼠原位结肠癌,可以通过肠镜下实时监测及实验干预此过程,是研究大肠癌发生、发展过程及临床治疗的良好模型,利于大肠隐窝干细胞在肿瘤发展过程中的变化研究,利于研究肿瘤干细胞在肿瘤发展过程中所扮演的角色及作用,CD133siRNA可以有效的抑制CD133基因在肿瘤细胞HT—29中的表达,可以有效抑制肿瘤细胞的增殖及侵袭力。裸鼠肿瘤模型中,该基因治疗能够有效的抑制移植瘤的生长,诱导肿瘤细胞的凋亡,今后可以进一步尝试通过肠镜直接对原位结肠肿瘤进行干预治疗,CD133与大肠癌密切相关,可能是靶向基因治疗潜在的分子标靶,可以尝试将CD133siRNA进一步应用于成功建立的大鼠结肠癌模型,为大肠癌的基因干扰治疗探索新的方法。
Objective
To 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.
Methods
SD 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.
Results
None 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.
Conclusion
The 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.
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