豚鼠诱导性多能干细胞系的建立及其诱导分化为功能性树突状细胞的研究

Dendritic cells (DCs) have become the focus of many academic-based and industry-based research and development programs, because they are highly effective in overcoming immune tolerance by priming potent T-cell immunity in models for cancer or viral disease. The primary drawback of using DCs in clinical trials is that the amount of DCs in the body is very few. Studies have shown that pluripotent embryonic stem cells can be differentiated into DCs with either immunostimulatory or tolerogenic function. The issue of histoincompatibility between patients to be treated and the ethical concerns related to the use of ES cells are anticipated to be serious obstacles, which will hinder the realization of the medical use of ES-DCs. It was recently revealed that ES cell-like pluripotent stem cells, designated as iPS cells, can be directed along multiple lineage pathways in vitro. Guinea pig is an ideal model for the pathogenesis of tuberculosis and drug screening, due to the highly sensitive to Mycobacterium tuberculosis. In this proposal, we will establish the guinea pig iPS cell lines, and differentiate the guinea pig iPS cells to dendritic cells in vitro. The properties of the guinea pig iPS derived DCs will be characterized. Furthermore, to explore the immunological response and mechanism against Mycobacterium tuberculosis, the DCs derived from guinea pig iPS will be stimulated with attenuated Bacillus Calmette-Guerin (BCG). The aims of this proposal are to provide a scalable resource for broadly applicable active GiPS-DCs to explore the immunological mechanism of DCs against mycobacteria infection and screen anti-tuberculosis drugs and vaccines using a guinea pig model, as well as establish a reliable cell culture model in vitro for investigating the functions of DCs in the pathogenesis of tuberculosis. This propsal may also provide an insight into the new application of iPS to other immune system diseases.

树突状细胞在多种免疫性疾病中发挥着重要的作用，以其为靶点的治疗策略成为多种免疫相关疾病防治的新途径。机体内树突状细胞数量极少，已成为制约其在细胞治疗应用中的瓶颈之一。研究证实胚胎干细胞可以诱导分化为树突状细胞，但胚胎干细胞有免疫排斥反应和伦理道德两大缺陷。诱导性多能干细胞(iPS)与胚胎干细胞类似，具有多向分化的潜能，在体外特定的培养环境中能被诱导向特定谱系的细胞分化。豚鼠由于其对结核分枝杆菌高度敏感，使其成为研究结核病致病机理与药物筛选的理想动物模型。为此我们拟建立豚鼠iPS细胞系，并于体外诱导分化为树突状细胞，鉴定由豚鼠iPS细胞分化的树突状细胞的生物学特性和免疫学功能；并进一步应用卡介苗刺激豚鼠iPS细胞分化的树突状细胞，探索其对结核分支杆菌的免疫学反应及机制，试图建立一种能够从豚鼠iPS获得大量、具有抗原提呈功能的树突状细胞的方法体系，为iPS应用于免疫性疾病的防治提供新思路。

iPS细胞在疾病模型、药物筛选以及疾病治疗中具有重要的应用价值。但iPS真正应用于人类疾病治疗之前，尚需要建立适宜的动物模型对其进行系统而深入的研究。基于此，本项目以豚鼠为研究对象，首先通过携带鼠源Oct4、Sox2、Klf4和c-Myc(OSKM)四个转录因子基因的逆转录病毒和慢病毒分别感染原代分离培养的豚鼠成纤维细胞；利用干细胞培养条件培养感染后的细胞，并进行形态学、表面抗原、干性相关基因mRNA和蛋白质水平表达分析、动物畸胎瘤分化实验等鉴定，结果表明，所获得豚鼠iPS细胞(GiPS)碱性磷酸酶染色呈阳性，高表达Sox2, Oct4和Nanog基因，具有分化为内、中、外三个胚层不同组织细胞的能力和正常的染色体核型，证实获得了完全重编程的豚鼠GiPS。在此基础上，体外诱导GiPS细胞分化为豚鼠树突状细胞 (GiPS-DCs)；同时，也探讨了将GiPS细胞在体外诱导分化为豚鼠巨噬细胞(GiPS-Mø)，并以牛结核分枝杆菌弱毒卡介苗(BCG)感染刺激GiPS-Mø，研究GiPS-Mø对结核菌的免疫反应。结果表明，GiPS-DCs表达具有典型的树突状细胞的形态特征，表达表面特征性标志分子CD11c；GiPS-Mø具有典型的巨噬细胞形态特征和较强的吞噬能力，表达巨噬细胞特异性的表面抗原CD163和CD68。BCG感染24 h后，与未感染的对照组相比，BCG感染组巨噬细胞产生的NO显著增加(P<0.01)，细胞的凋亡率分别为凋亡率为73.56±2.97%，显著高于对照组的32.43±4.28% (P<0.01)。充分说明，诱导获得的GiPS-Mø细胞在抗结核免疫中起重要的作用。本项目的实施，为利用基于豚鼠iPS细胞技术研究免疫相关疾病提供细胞来源；也为进一步阐明Mø在结核免疫中的作用机制和药物的筛选建立一种体外细胞模型。