高通量免疫单细胞温度介导细胞因子动态分析

Human immunoregulation is closely associated with core body temperature, and fever is a cardinal response when immunological function of immune system is activated. However, the effect of temperature on immunological mechanisms with respect to whether pro-inflammatory cytokine level of innate immune cell population be upregulated under physiological range hyperthermia during infection is still not clear. Cytokine production from individual cells varies because of single cell heterogeneity. Therefore, revealing the dynamic regulation of cytokine secretion from a number of live single immune cells in response to temperature changes is of great importance. Herein, we develop a high throughput, multiplexed single cell secretion analysis temperature-controllable platform assembled with transparent microelectrode array, a newly designed microchannels array to immobilize high density space-resolved antibodies for multiplexed protein secretion detection and a corresponding microwell array for single cell capture. Ten kinds of immune effector cytokine (or proteins) secreted from 5000 single macrophage cells can be in situ detected simultaneously at precisely controlled temperature. This platform will be used to investigate the regulating correlation and diversity of cytokine releasing from human single macrophage cells challenged with lipopolysaccharide with/without human breast cancer coculture under different physiological rang hyperthermia. Therefore, immunological mechanisms from the same single macrophage cells in response to temperature would be uncovered.

人体免疫功能调控与体内温度密切相关，发热是免疫系统启动免疫机制的主要表现形式，由发热引起的免疫调节机制尚不清楚，严重阻碍了细胞免疫学的发展。细菌感染条件下，发热对天然免疫细胞的细胞因子分泌调节方式的研究结果存在争议，而免疫细胞异质性可能是争议存在的关键原因。因此单细胞水平，深入研究发热如何调节免疫细胞的免疫应答及其动态过程有望成为解决争议的突破点。本项目拟设计一种集透明温控微电极阵列、高通量单细胞捕获池及高密度微流体通道抗体阵列于一体的微流控芯片用于单细胞蛋白检测，实现原位精确温控下，5000个巨噬细胞十余种细胞因子（或分泌蛋白）的动态分析，并模拟研究人体天然免疫系统在细菌感染和人乳腺肿瘤条件下，发热对单个巨噬细胞十余种细胞因子调节变化的影响，分析多种细胞因子免疫调节的相关性、个体差异性，探索细菌感染和肿瘤条件下发热对免疫调节的规律，为细胞免疫学发展和热疗临床应用提供重要参考。

本项目在单细胞水平上，发展了一种原位精确温控的单细胞蛋白分泌分析平台用于模拟细菌感染情况下不同发热对免疫细胞免疫功能影响研究。通过ITO透明电极实现了区域上的精密温控和在线模拟不同发热情况；与高通量单细胞捕获池及抗体玻片集成，监测免疫细胞的促炎症因子，研究免疫细胞在细胞感染及肿瘤微环境下，不同程度发热 (37 ℃、40 ℃)调节的免疫响应，并在ITO温控平台上初步实现了单细胞水平上， U937分化的巨噬细胞TNF-a和IL-6的同时检测。以PDMS微柱为三维基底，以肿瘤细胞共培养模拟细胞通讯，研究处于细菌感染及三维肿瘤微环境中单个巨噬细胞的免疫响应，发现三维肿瘤微环境抑制巨噬细胞的TNF-a分泌，却增加了IL-6分泌；U937巨噬细胞的表型鉴定表明肿瘤微环境诱导巨噬细胞从M1型向M2型的极化，可能是导致TNF-a和IL-6的分泌调节不同的原因。为进一步模拟体内三维肿瘤微环境，利用表面活性剂F-68促进肿瘤微球生成，可同时完成10800个肿瘤微球的成球及长达12天的连续培养，以SCC25肿瘤微球为例，考察了U937分化的巨噬细胞的动态免疫响应。我们发现在细菌感染条件下，SCC25肿瘤微球将进一步抑制U937巨噬细胞的TNF-a和IL-6的分泌，而对IL-8分泌无明显影响。