本文關鍵詞：肺癌轉(zhuǎn)移動物模型的建立及活體成像觀察　出處：《南京農(nóng)業(yè)大學》2011年博士論文　論文類型：學位論文

  更多相關文章： 肺癌 動物模型 活體成像 轉(zhuǎn)移相關基因 microRNA

【摘要】：目的：建立了肺癌轉(zhuǎn)移動物模型,采用分子病理學方法檢測腫瘤轉(zhuǎn)移相關基因表達,驗證部分關鍵microRNA對人肺癌高轉(zhuǎn)移細胞系SPC-A-lsci侵襲和遷移能力的影響；應用小動物活體成像技術,以肺癌動物模型為主要對象,建立熒光報告基因的細胞轉(zhuǎn)染方法,活體觀察肺癌動物模型的發(fā)生、發(fā)展及轉(zhuǎn)移,為小動物活體成像技術在腫瘤動物模型中應用提供參考依據(jù)。 方法：采用小鼠尾靜脈注射方法觀察長期傳代的高、低轉(zhuǎn)移細胞系的轉(zhuǎn)移特性；通過Western blot、FICC、IHC等分子病理方法檢測已知轉(zhuǎn)移轉(zhuǎn)移促進基因CD44,OPN, MMP-9,EGFR在高、低轉(zhuǎn)移細胞系間的差異表達；采用siRNA干擾技術,篩選對人肺癌高轉(zhuǎn)移細胞系SPC-A-lsci侵襲和遷移能力有影響的microRNA.通過慢病毒轉(zhuǎn)染技術建立肺癌熒光報告基因穩(wěn)轉(zhuǎn)細胞,進而建立相應的肺癌動物模型,采用小動物活體成像技術觀察模型的成像效果,觀察指標包括標記率、靈敏度、光子信號面積,光子信號強度等。 結果：動物體內(nèi)實驗表明8周時高轉(zhuǎn)移細胞系的肺轉(zhuǎn)移率為100%(12/12),而低轉(zhuǎn)移細胞系的肺轉(zhuǎn)移率僅為16.67%(2/12)。與低轉(zhuǎn)移細胞系相比,轉(zhuǎn)移促進基因CD44, OPN,MMP-9,EGFR在高轉(zhuǎn)移細胞中的表達均顯著上調(diào)。選擇23個差異顯著的microRNA進行細胞水平的轉(zhuǎn)移潛能篩選,發(fā)現(xiàn)過表達microRNA148a和microRNA200c能顯著降低人肺癌高轉(zhuǎn)移細胞系SPC-A-lsci的侵襲和遷移能力,而對microRNA148a和microRNA200c進行siRNA干擾則能顯著提高人肺癌低轉(zhuǎn)移細胞系SPC-A-1的侵襲和遷移能力。建立了GFP和GFP/Luc雙標記報告基因的細胞轉(zhuǎn)染方法,標記了SMMC-7721,SPC-A-1, SPC-A-lsci,NCI-H460, MDA-MB-231sci等5個細胞系,標記率達90%以上。小動物活體成像系統(tǒng)的靈敏度,體外水平為100個GFP細胞,體內(nèi)水平為1×105個GFP細胞或100個Luc細胞。建立了小鼠皮下移植瘤模型和原位移植瘤模型的小動物活體成像實驗技術,觀察了腫瘤生長、轉(zhuǎn)移等生物學特性。 結論：建立了肺癌轉(zhuǎn)移動物模型,采用分子病理方法觀察肺癌動物模型轉(zhuǎn)移相關的生物學特性,應用小動物活體成像應用技術并活體觀察肺癌動物模型的生長、轉(zhuǎn)移,為腫瘤動物模型的推廣應用和臨床肺癌防治研究提供了參考依據(jù)。
[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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R734.2;R-332

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