結核分枝桿菌(M. tb)是一種侵入呼吸系統的病原菌，所引起的肺結核(tuberculosis) ，每年造成全球超過200萬人的死亡。結核菌在藉由噬菌作用(phagocytosis)進入肺泡內巨噬細胞(alveolar macrophage)後，利用其作用分子(effector molecule)阻礙成熟噬菌小體(phagosomal maturation)的形成，於是結核菌便以此為侵入人體的前線基地，並在細胞內生長(intracellular growth)，最後導致了肺部功能的永久性損壞，甚至致患者於死亡。截至目前為止，用以治療結核患者的藥物都是屬於抗生素(antibiotics)，但以提升自體免疫力，來對抗結核感染的概念，卻從未被利用在結核用藥的開發上。另一方面，肺泡表面蛋白D(surfactant protein D, SPD)，一種由肺泡第二型細胞(alveolar type II cells)所分泌的C型辨醣蛋白(C-type lectin)，參與著各種抵禦呼吸道病原體侵犯肺部的免疫反應，目前已知肺泡表面蛋白D基因(SPD gene)具有單一核甘酸多型性(single nucleotide polymorphism, SNP)，反映在其蛋白質結構上的第11個胺基酸殘基(residue 11)變化，將決定吾人是否易受特定感染性疾病侵擾；尤甚者，流行病學的研究亦指出，肺泡表面蛋白D第11個胺基酸殘基若為羥丁氨酸(threonine)，將較易受結核菌之感染。至於不同單一核苷酸多型性下的肺泡表面蛋白D，是否會對結核菌侵入肺泡內巨噬細胞有不盡相同的抑制能力，未曾有人做此方面的研究；我們假設將可藉著遞予編碼肺泡表面蛋白D第11個特定殘基的基因，來發展治療肺結核之終極目的。在本篇研究中，我們將以幾丁聚醣(Chitosan)包覆肺泡表面蛋白D基因所形成的奈米微粒(nanoparticle)，來進行基因遞送，利用幾丁聚醣包覆，除了可有效避免雙股核糖核酸酶(DNase)破壞基因，亦可有效提升肺泡第二型細胞攝入遞送基因；同時, 在此我們的結果也證明了，以幾丁聚醣遞送基因可在毫無細胞毒性(cytotoxicity)的安全性前提下，大幅提高轉染效率(transfection efficiency)。之後行有餘力，我們也將用小鼠動物實驗來證實，給予幾丁聚醣包覆肺泡表面蛋白D基因所形成的奈米微粒，可達成治療肺結核之目的。總結來說，遞送肺泡表面蛋白D基因不僅可提供肺部長久性的保護效果而具有預防感染性疾病之功能外，同時也可用在治療結核患者身上，以提升人體免疫機制的方式，與利用滅菌活性的抗生素雙管齊下，來更快速有效殲滅肺結核!

Mycobacterium tuberculosis (M. tb), a respiratory pathogen, could cause tuberculosis which is responsible for more than 2 million deaths in the world each year. After been phagocytosised by human alveolar macrophages, M. tb uses effector molecules to arrest phagosomal maturation at an early stage, resulting in its intracellular growth, and finally leading to permanent lung destruction and deadly respiratory failure. So far, all drugs applied to clinical therapy for tuberculosis belonged to antibiotics, but designed molecule for enhancing host innate immunity against M. tb infection is still not available. On the other hand, surfactant protein D (SPD), a C-type lectin that is produced in alveolar type II cells of lung, has important functions with respect to the respiratory innate immunity toward various pathogens. Up to now, four single nucleotide polymorphisms (SNP) of SPD gene have been found, and SNP of SPD at residues 11 has been confirmed to associate with infectious diseases in lung. The Thr11 homologous allele has linked with susceptibility to M. tb infection in the epidemiological study. Interestingly, our data indicate entirely different results from the previous study. Here we showed different SPD gene of SNP had diverse potential to prevent alveolar macrophages from M. tb infection, thus the delivery of SPD gene encoded specific residue 11 had possible potential in the application of clinical therapy for tuberculosis. In this study, SPD gene delivery was designed in the form of chitosan-DNA nanoparticles, so that SPD gene could be protected from DNase degradation and then be ingested easily by alveolar type II cells. Our results also showed chitosan nanoparticle could be a safe vector of gene delivery with high transfection efficiency; In the future, we will demonstrate the animal model with M. tb infection do be improved by SPD gene delivery via Chitosan nanoparticle. In conclusion, SPD gene delivery not only may support persistent lung protection, but also can be combined collocate with antibiotics to cure tuberculosis involving in both innate immunity and bactericidal activity.

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