以基因功能喪失策略研究人類巨噬細胞在肺結核致病機轉的角色

Identification of the genes involved in human macrophage THP-1 during Mycobacterium tuberculosis infection by loss-of-function screening

林思瑋(Shih-Wei Lin)

指導教授 ： 顏伯勳

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10693

摘要

結核分枝桿菌 (Mycobacterium tuberculosis, Mtb) 是造成人類肺結核的致病菌，估計全球每年約有200萬人死於結核病。 雖然結核分枝桿菌自從被發現以來已經有一百多年，但是該菌與宿主細胞間的互動機制卻尚未完全了解。現今隨著多重抗藥性菌株 (MDR-TB) 及廣泛抗藥性菌株 (XDR-TB) 的出現，結核分枝桿菌儼然成為公共衛生的重要議題。因此，研究及了解結核分枝桿菌複雜的致病機轉是唯一可以提供我們治療以及消滅該菌的方法。結核分枝桿菌是經由患者將含有病源菌的氣膠呼出傳播。 當細菌被吸入人體肺部，便會被肺泡上的巨噬細胞吞噬，並在胞內形成吞噬體，然後利用吞噬體當做一個成長繁殖的場所。因此，巨噬細胞被視為結核分枝桿菌感染人類的主要目標。 巨噬細胞亦會使用多種殺菌機制，包括產生活性氧、吞噬體-溶小體融合、自體吞噬及細胞凋亡機制來抑制細菌生長。我們的實驗利用了82,000個 shRNA 病毒匯集文庫對人類單核球細胞株進行RNA干擾分析掃描，希望可以找出參與在調控結核分枝桿菌感染後人類單核球細胞株細胞凋亡過程之基因。我們先將 shRNA 病毒匯集文庫感染人類單核球細胞株，感染後24小時，以嘌呤黴素(puromycin)篩選出有成功嵌入shRNA的細胞。接著用不同感染複數 (MOI) 結核分枝桿菌菌株H37Rv感染這些細胞。細菌感染後分離出存活細胞，抽取其基因組去氧核醣核酸，再以客製化的晶片做微陣列分析。藉此找出結核分枝桿菌感染後參與在細胞凋亡機制有關的基因。我們從分析的結果篩選出一百零六個與細胞凋亡、訊息傳遞、免疫反應相關的候選基因，接著再以這一百零六基因個別剔除的細胞株感染結核分枝桿菌並觀察是否有表現型的差異。

關鍵字

肺結核 ； 結核分枝桿菌 ； 全基因組核醣核酸干擾篩選技術

並列摘要

Mycobacterium tuberculosis (Mtb) is the pathogen which causes human lung tuberculosis. It is estimated that two million people die from tuberculosis world-wide annually. Mtb has been discovered for over a century; however, its exact interaction with the host cell still remains largely unclear. With the emergence of multi-drug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB), tuberculosis is becoming a significant public health issue. Thereby, investigating and understanding the pathogenesis of Mtb may be the only way for its eradication. Mtb is spread through aerosol cough out by the patients. When Mtb is inhaled into the lung, it is engulfed by alveolar macrophage forming a phagosome. Mtb will then reproduce intracellularly within the phagosome. Alveolar macrophage is the major host cell of Mtb and can activate various bactericidal mechanisms to digest intracellular Mtb. The mechanisms include the production of oxygen radical, phagosome-lysosome fusion and apoptosis. Our studies used an 82,000 pooled lentiviral-shRNA library for RNA interference screening to identify genes that are involved in regulation of host cell death upon Mtb infection. THP-1 cells were transduced with the lentiviral-shRNA library for loss-of-function screening. The cells were first transduced with lentivirus and treated with puromycin to select cells that were successfully incorporated with shRNA by lentivirus 24 hours post transduction. The cells were then infected with Mtb strain H37Rv at various MOI for 24 hours, and genomic DNA was extracted from the surviving cells. The shRNA bar-codes were amplified through a series of PCR and analyzed by customized microarray for gene identification. Our result identified 106 candidate genes associated with cell death, signal transduction, and immune responses. Then we infected the clone with each of these 106 genes stably knockdown with Mtb to validate for phenotypic changes.

並列關鍵字

Tuberculosis ； Mycobacterium tuberculosis ； Genome-wide RNA interference screen

參考文獻

McManus, M. T., and Sharp, P. A. (2002) Gene silencing in mammals by small interfering RNAs. Nat. Rev. 3, 737-747.

Zumla, A., Raviglione, M., Hafner, R., and von Reyn, C. F. (2013). Tuberculosis. N. Engl. J. Med. 368, 745-755.

De Backer, A. I., Mortelé, K. J., De Keulenaer, B. L., and Parizel, P. M. (2006). Tuberculosis: Epidemiology, manifestations and the value of medical imaging in diagnosis. J. Belg. Radiol. 89, 243-250.

van Crevel, R., Ottenhoff, T. H. M., and van der Meer, J. W. M. (2002). Innate immunity to Mycobacterium tuberculosis. Clin. Microbiol. Rev. 15, 294-309.

Kleinnijenhuis, J., Oosting, M., Joosten, L. A. B., Netea, M. G., and van Crevel, R. (2011). Innate immune recognition of Mycobacterium tuberculosis. Clin. Dev. Immunol. 2011, 1-12.