肺癌转移动物模型的建立及活体成像观察

发布时间：2018-01-07 06:17

本文关键词：肺癌转移动物模型的建立及活体成像观察　出处：《南京农业大学》2011年博士论文　论文类型：学位论文

【摘要】：目的：建立了肺癌转移动物模型,采用分子病理学方法检测肿瘤转移相关基因表达,验证部分关键microRNA对人肺癌高转移细胞系SPC-A-lsci侵袭和迁移能力的影响；应用小动物活体成像技术,以肺癌动物模型为主要对象,建立荧光报告基因的细胞转染方法,活体观察肺癌动物模型的发生、发展及转移,为小动物活体成像技术在肿瘤动物模型中应用提供参考依据。方法：采用小鼠尾静脉注射方法观察长期传代的高、低转移细胞系的转移特性；通过Western blot、FICC、IHC等分子病理方法检测已知转移转移促进基因CD44,OPN, MMP-9,EGFR在高、低转移细胞系间的差异表达；采用siRNA干扰技术,筛选对人肺癌高转移细胞系SPC-A-lsci侵袭和迁移能力有影响的microRNA.通过慢病毒转染技术建立肺癌荧光报告基因稳转细胞,进而建立相应的肺癌动物模型,采用小动物活体成像技术观察模型的成像效果,观察指标包括标记率、灵敏度、光子信号面积,光子信号强度等。结果：动物体内实验表明8周时高转移细胞系的肺转移率为100%(12/12),而低转移细胞系的肺转移率仅为16.67%(2/12)。与低转移细胞系相比,转移促进基因CD44, OPN,MMP-9,EGFR在高转移细胞中的表达均显著上调。选择23个差异显著的microRNA进行细胞水平的转移潜能筛选,发现过表达microRNA148a和microRNA200c能显著降低人肺癌高转移细胞系SPC-A-lsci的侵袭和迁移能力,而对microRNA148a和microRNA200c进行siRNA干扰则能显著提高人肺癌低转移细胞系SPC-A-1的侵袭和迁移能力。建立了GFP和GFP/Luc双标记报告基因的细胞转染方法,标记了SMMC-7721,SPC-A-1, SPC-A-lsci,NCI-H460, MDA-MB-231sci等5个细胞系,标记率达90%以上。小动物活体成像系统的灵敏度,体外水平为100个GFP细胞,体内水平为1×105个GFP细胞或100个Luc细胞。建立了小鼠皮下移植瘤模型和原位移植瘤模型的小动物活体成像实验技术,观察了肿瘤生长、转移等生物学特性。结论：建立了肺癌转移动物模型,采用分子病理方法观察肺癌动物模型转移相关的生物学特性,应用小动物活体成像应用技术并活体观察肺癌动物模型的生长、转移,为肿瘤动物模型的推广应用和临床肺癌防治研究提供了参考依据。
[Abstract]:Objective: to establish an animal model of lung cancer metastasis and to detect the expression of tumor metastasis related genes by molecular pathology. To verify the effect of some key microRNA on the invasion and migration of high metastatic human lung cancer cell line SPC-A-lsci. Using small animal in vivo imaging technique and taking lung cancer animal model as the main object, the cell transfection method of fluorescent reporter gene was established, and the occurrence, development and metastasis of lung cancer animal model were observed in vivo. It provides a reference for the application of small animal in vivo imaging in tumor animal model. Methods: the metastatic characteristics of high and low metastatic cell lines were observed by tail vein injection in mice for a long time. The known metastasis promoter gene CD44 / OPN and MMP-9 / EGFR were detected by Western blottir FICC- IHC and other molecular pathological methods. Differential expression among low metastatic cell lines; SiRNA jamming technology is adopted. MicroRNAs, which have an effect on the invasion and migration of human lung cancer cell lines with high metastasis, were screened. The stable transfer cells of lung cancer fluorescent reporter gene were established by lentivirus transfection technique. Then the corresponding animal model of lung cancer was established, and the imaging effect of the model was observed by the small animal in vivo imaging technique. The observation indexes included labeling rate, sensitivity, photon signal area, photon signal intensity and so on. Results: in vivo animal experiments showed that the lung metastasis rate of high metastatic cell line was 100% 12 / 12 at 8 weeks. The lung metastasis rate of low metastasis cell line was only 16.67 / 12. Compared with low metastasis cell line, CD44, OPNmMP-9 were the metastasis promoter genes. The expression of EGFR was significantly up-regulated in high metastatic cells. Twenty-three highly differentiated microRNA were selected for cell level metastasis potential screening. It was found that overexpression of microRNA148a and microRNA200c could significantly reduce the ability of invasion and migration of human lung cancer cell line SPC-A-lsci with high metastasis. SiRNA interference with microRNA148a and microRNA200c could significantly improve the invasion and migration ability of human lung cancer cell line SPC-A-1 with low metastasis. GFP and GFP/Luc double labeling reporter gene transfection method. Five cell lines, SMMC-7721, SPC-A-1, SPC-A-lscin NCI-H460 and MDA-MB-231sci, were labeled. The labeling rate was more than 90%. The sensitivity of small animal imaging system was 100 GFP cells in vitro. In vivo, 1 脳 105 GFP cells or 100 Luc cells were used to establish mouse subcutaneous transplanted tumor model and orthotopic transplantation tumor model. Biological characteristics such as transfer. Conclusion: the animal model of lung cancer metastasis was established and the biological characteristics related to metastasis of lung cancer animal model were observed by molecular pathological method. The growth and metastasis of lung cancer animal model were observed in vivo by using small animal imaging technique, which provided a reference for the popularization and application of tumor animal model and the study of clinical prevention and treatment of lung cancer.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R734.2;R-332

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