Xia Di, Sun Wen-Kui, Tan Ming-Ming, Ding Yuan, Liu Zhi-Cheng, Li Pei, Qian Qian, Su Xin, Shi Yi
Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, People's Republic of China.
Lung. 2015 Aug;193(4):549-57. doi: 10.1007/s00408-015-9740-8. Epub 2015 May 6.
The incidence of invasive pulmonary aspergillosis (IPA) has increased significantly over the last two decades. Alveolar macrophages (AMs) represent the first line of pulmonary host response to Aspergillus conidia. Recognition of conidia by AMs involves Dectin-1 (CLEC7A), which is a conserved structure to combine β-glucans. The deficiency of Dectin-1 results in impaired fungal killing and uncontrolled growth of Aspergillus fumigatus. Thus, we hypothesized that high expression of Dectin-1 would enhance the host recognition and fungal killing.
We set out to develop an adenoviral vector encoding full-length Dectin-1 (Ad-Dectin-1-EGFP) and then transfect it to MH-S cells. Transfect cell model was verified by using real-time RT-PCR, Western blot, flow cytometric, and confocal microscopic assays. And also, the function of Dectin-1 was explored by measuring cytokine release and killing ability during the course of A. fumigatus infection.
We constructed a recombinant adenovirus which could upregulate the expression of Dectin-1 and verified that Dectin-1 was expressed on cell membrane. The function of Dectin-1 was also demonstrated by its ability in promoting the production of cytokines and increasing the killing ability during the course of A. fumigatus infection.
An adenoviral vector was successfully applied to the production of a recombinant adenovirus encoding full-length Dectin-1, and also, its function in Aspergillus-induced innate immune response was demonstrated.
在过去二十年中，侵袭性肺曲霉病（IPA）的发病率显著增加。肺泡巨噬细胞（AMs）是肺部对曲霉分生孢子宿主反应的第一道防线。AMs对分生孢子的识别涉及到识别β-葡聚糖的保守结构——Dectin-1（CLEC7A）。Dectin-1的缺乏会导致真菌杀伤受损和烟曲霉的生长失控。因此，我们假设Dectin-1的高表达会增强宿主识别和真菌杀伤能力。
我们着手构建一种编码全长Dectin-1的腺病毒载体（Ad-Dectin-1-EGFP），然后将其转染至MH-S细胞。通过实时RT-PCR、蛋白质印迹、流式细胞术和共聚焦显微镜检测来验证转染细胞模型。此外，在烟曲霉感染过程中，通过测量细胞因子释放和杀伤能力来探索Dectin-1的功能。
我们构建了一种能上调Dectin-1表达的重组腺病毒，并证实Dectin-1在细胞膜上表达。在烟曲霉感染过程中，Dectin-1促进细胞因子产生和增强杀伤能力的作用也得到了证实。
一种腺病毒载体成功应用于生产编码全长Dectin-1的重组腺病毒，并且其在曲霉诱导的固有免疫反应中的功能也得到了证实。
