弓形虫通过极化星形胶质细胞诱导宿主神经细胞死亡的分子机制研究

Toxoplasmic Encephalitis (TE) is a common clinical manifestation in immunosuppressive individuals and newborn babies infected with Toxoplasma gondii, along with a large quantity of neuron death in brain. Astrocyte polarization is a noval celluar differentiation phenomenon recently found in neurodegenerative diseases. A1 astrocytes are neurotoxic, and in LPS-induced CNS inflammation model astrocyte polarization to A1 type is induced by activated microglia. In mice TE model, we found A1 astrocytes in mice brain. However, it is still unclear how resting astrocytes polarize to A1 type and how these A1 astrocytes trigger neuron death in mice TE. In our study, we will perform the following in vitro experiments:1. Incubate mice primary astrocytes with cell medium containing Toxoplasma MICs，Wh6 tachyzoites pretreated by Cyto-D, Wh6 parasitic lysate, or freshly collected Wh6 tachyzoites for certain time periods, and detect A1 astrocyte-specific gene transcription and expression profiles using qRT-PCR and western blotting; 2. Infect microglia cells (BV2) for certain time, collect cell medium to prepare microglial conditioned medium (MCM) for astrocyte culture, and detect A1 astrocyte-specific gene transcription and expression profiles using qRT-PCR and western blotting; 3. Collect astrocyte cell medium in above two experiments to prepare astrocyte conditioned medium (ACM) for neuron cell (CATH.a) culture, at different time points detect cellular apoptosis level using FACS and key molecules in apoptosis pathway and necroptosis pathway using western blotting. NFkB pathway activation will be detected as well. Also, we will establish mice TE model in vivo. Microglia will be depleted by Pexidartinib; microglial activation will be inhibited by minocycline. The in vivo relationship between T. gondii infection, microglia activation and A1 type astrocyte will be evaluated using IFA. In vivo neuron death will be analysed using western blotting and IFA. The concentration of neurotoxic elements (NO and glutamate) and neuronutrients (Gpc, Hevin and THBS) in astrocyte cell medium and mice brain will be detected using ELISA kit or other commercial kit in vitro and in vivo. Our study will provide new direction to investigate the machenisms of neuron death in TE, combining astrocyte, microglia and neuron together, which may be helpful for development of novel anti-toxoplasmosis therapy.

脑炎是弓形虫感染免疫抑制患者和婴幼儿常见的临床症状，伴有神经细胞的死亡。在多种神经退行性疾病中，星形胶质细胞发生极化。其中A1型具神经毒性，其极化依赖于小胶质细胞活化。我们在小鼠弓形虫脑炎模型中发现A1型星形胶质细胞的存在，但其极化机制、诱导神经细胞死亡的机制尚未明确。本申请将通过⑴经不同处理的虫体与星形胶质细胞共孵育，⑵虫体感染小胶质细胞，而后配制小胶质细胞条件培养基培养星形胶质细胞，⑶用星形胶质细胞培养上清配制条件培养基培养神经细胞，⑷免疫抑制建立小鼠弓形虫脑炎模型等方式，运用免疫印迹、qRT-PCR、间接免疫荧光、ELISA、流式细胞术等检测细胞或组织中的星形胶质细胞极化标志分子和主要信号通路、神经毒性因子和神经营养物质、不同细胞死亡途径的关键信号分子，旨在阐明弓形虫通过极化星形胶质细胞引发神经细胞死亡的分子机制，为寻找治疗弓形虫脑炎药物新靶点提供科学依据。此项研究国内外未见报道。

脑炎是弓形虫感染免疫抑制患者和婴幼儿常见的临床症状，伴有神经细胞的死亡。在多种神经退行性疾病中，星形胶质细胞发生极化。其中A1型具神经毒性，其极化依赖于小胶质细胞活化。我们在小鼠弓形虫脑炎模型中发现A1型星形胶质细胞的存在，但其极化机制、诱导神经细胞死亡的机制仍未明确。我们通过建立小鼠弓形虫脑炎模型观测到星形胶质细胞极化现象，但星形胶质细胞极化的特异性基因表达水平和先前报道的不一致。体外实验的初步结果显示，星形胶质细胞极化可能和弓形虫分泌性蛋白有关，但是，弓形虫是一种细胞内寄生原虫。模拟弓形虫对细胞的真实感染能更好的解释其致病性。鉴于此，培养性能稳定的原代星形胶质细胞是未来解析弓形虫引起星形胶质细胞极化的关键。我们还在检测弓形虫急性感染到慢性感染阶段小鼠脑组织星形胶质细胞极化的动态变化。.除此之外，我们检测了弓形虫入侵巨噬细胞的过程，鉴定出一种新的弓形虫入侵宿主细胞的模式PAI。这个过程和弓形虫微线蛋白的释放有关，为研究弓形虫效应蛋白和宿主蛋白的相互作用提供了新的模型。