过表达IL-8对三阴乳腺癌发展的影响及其机制的初步探究

发布时间：2018-03-21 17:49

本文选题：IL-8　切入点：TNBCs　出处：《重庆医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的构建含有白细胞介素8(IL-8)基因的重组腺病毒,观察三阴乳腺癌细胞(Triple-negative breast cancer cells,TNBCs)过表达IL-8后,细胞增殖、细胞周期、迁移能力以及体内成瘤能力的变化,并初步探究其相关机制。方法划痕愈合实验检测5株乳腺癌细胞的迁移能力,RT-PCR和ELISA法检测细胞中内源性IL-8 mRNA和蛋白表达水平。以人骨肉瘤143B细胞c DNA为模版,PCR扩增IL-8基因,与穿梭质粒pAdTrack-TO4连接,电转入AdEasier细菌中,获得重组腺病毒质粒pAdIL-8,酶切后转入HEK293细胞包装扩增并检测滴度;AdIL-8感染Hs578T、BT549和MDA-MB-231细胞后,RT-PCR法检测各株细胞中IL-8 mRNA水平,ELISA法检测细胞上清液中IL-8蛋白水平;流式细胞术(flow cytometer,FCM)检测细胞凋亡以及细胞周期情况;MTT法检测细胞增殖活性;划痕愈合实验及Transwell实验检测细胞迁移能力。Western blot检测过表达IL-8后对PI3K-Akt和MAPK信号通路的作用以及对CXCR1、CXCR2、E-cadherin、MMP2、Cyclin B1、Bcl-2和Caspase 3表达的影响;裸鼠皮下成瘤实验观察过表达IL-8对MDA-MB-231细胞成瘤能力的影响。结果5株乳腺癌细胞中Hs578T迁移最快,BT549较快,MCF7和SKBr3迁移较慢,MDA-MB-231最慢;Hs578T中IL-8表达水平最高,BT549次之,MDA-MB-231、MCF7和SKBr3 IL-8表达较低;PCR以及测序证实pAdTrack-TO4-IL-8构建成功;pAd IL-8经酶切证实重组正确;重组腺病毒Ad IL-8感染Hs578T、BT549和MDA-MB-231细胞后,可高表达IL-8;过表达IL-8的细胞迁移能力增强,且可将细胞周期阻滞于S期,但其增殖活性和凋亡情况无明显变化。AdIL-8感染MDA-MB-231细胞后可激活PI3K-Akt和MAPK信号通路,其特异性受体CXCR1和CXCR2表达增加,E-cadherin表达有所减少,Caspase 3和Bcl-2表达无明显变化,而Cyclin B1表达增加。体内实验表明过表达IL-8可促进肿瘤的生长。结论过表达IL-8可促进TNBCs的迁移能力及肿瘤形成能力。
[Abstract]:Objective to construct a recombinant adenovirus containing interleukin-8 (IL-8) gene and observe the changes of cell proliferation, cell cycle, migration ability and tumorigenic ability of triple-negative breast cancer cell line triple-negative breast cancer cells (IL-8) after overexpression of IL-8. Methods the migration ability of 5 breast cancer cells was detected by scratch healing assay and the expression of endogenous IL-8 mRNA and protein were detected by RT-PCR and ELISA. IL-8 gene was amplified by using c DNA of human osteosarcoma 143B cell as template. Ligated with shuttle plasmid pAdTrack-TO4 and electrotransferred into AdEasier bacteria, The recombinant adenovirus plasmid pAdIL-8 was obtained. The recombinant adenovirus plasmid pAdIL-8 was digested into HEK293 cells for packaging and amplification. The titer of Hs578TnBT-BT549 and MDA-MB-231 cells were detected by RT-PCR. The levels of IL-8 mRNA in the supernatants were detected by Elisa and the levels of IL-8 protein in supernatants were detected by Elisa. Flow cytometerus (FCM) was used to detect cell apoptosis and cell cycle. MTT assay was used to detect cell proliferation activity. The effect of IL-8 overexpression on PI3K-Akt and MAPK signaling pathway and the expression of Bcl-2 and Caspase 3 in CXCR1CXCR2CXCR2CXCR2CXCR2E-cadherin B1C1CXCR2CXCR2 and CXCR1CXCR2CXCR2CXCR2CXCR2CXCR2CXCR1CXCR1CXCR2CXCR2CXCR2C@@. The effect of overexpression of IL-8 on the tumorigenic ability of MDA-MB-231 cells was observed in nude mice. Results among the 5 breast cancer cell lines, the fastest migration of Hs578T was the fastest MCF7 and the slowest migration of SKBr3 was MDA-MB-231. The highest level of IL-8 expression in Hs578T was MDA-MB-231MCF7 and SKBr3 IL-8, followed by BT-549, MDA-MB-231MCF7 and SKBr3 IL-8. Low expression PCR and sequencing confirmed that pAdTrack-TO4-IL-8 was successfully constructed and the recombinant pAd IL-8 was confirmed by restriction endonuclease digestion. After the recombinant adenovirus Ad IL-8 was infected with Hs578TfBT549 and MDA-MB-231 cells, IL-8 was overexpressed, and the migration ability of the cells was enhanced, and the cell cycle was blocked in S phase. However, there was no significant change in proliferation activity and apoptosis. AdIL-8 infected MDA-MB-231 cells could activate PI3K-Akt and MAPK signaling pathway. The expression of CXCR1 and CXCR2 increased and the expression of E-cadherin decreased. There was no significant change in the expression of Caspase-3 and Bcl-2. In vivo, overexpression of IL-8 can promote the growth of tumor. Conclusion overexpression of IL-8 can promote the ability of TNBCs migration and tumor formation.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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