【作者】 杨蕴天；

【导师】 江新梅；

【作者基本信息】 吉林大学 ， 神经病学， 2008， 博士

【摘要】 朊蛋白病(Prion diseases)即可传播性海绵状脑病(Transmissible spongiform encephalopathies,TSEs),是一类侵袭人类及多种动物中枢神经系统的退行性脑病,潜伏期长,致死率100%。克雅氏病(Creutzfeldt-Jakob disease,CJD)是人类最常见的朊蛋白病之一。朊蛋白病的病因主要是在人或动物脑组织内有大量异常的朊蛋白(PrP)沉积而致神经细胞死亡。其发病机制尚不清楚,目前认为由于脑内异常PrP的沉积导致胶质细胞活化是神经细胞死亡的主要原因,其中活化的小胶质细胞释放的细胞因子在神经元变性、死亡的过程中起着重要作用。本文通过对CJD患者脑脊液及PrP105-132对体外小胶质细胞和体外大脑皮质细胞作用的研究发现:CJD患者脑脊液中白细胞介素-6(IL-6)、白细胞介素-8(IL-8)含量明显升高;并且PrP能够诱导体外小胶质细胞分泌IL-6、IL-8,且具有剂量依赖关系,PrP诱导体外小胶质细胞分泌IL-6可能通过NF-κB和NFAT途径,PrP诱导体外小胶质细胞分泌IL-8可能通过NF-κB途径;PrP对体外大脑皮质细胞有毒性作用、可引起神经细胞凋亡,当与IL-6、IL-8共同作用时,毒性作用增强、神经细胞凋亡增多。这些结果说明小胶质细胞分泌的IL-6和IL-8在朊蛋白病的致病过程中起着重要的作用。对小胶质细胞及细胞因子的深入研究将有利于揭示朊蛋白病的致病机制,有助于疾病的预防及治疗。更多还原

【Abstract】 Prion diseases (Transmissible spongiform encephalopathies, TSEs) are invariably fatal neurodegenerative disorders involved in human and animals. Creutzfeldt-Jakob disease (CJD) is the most common form of human prion protein diseases. The neuropathology of prion disease is characterized by spongiform degeneration of the brain, neuronal loss, reactive increased number of glial and glial activitation. A key event in prion disease pathogenesis is the accumulation of conformationally changed prion protein (PrPSc) in the brain, resulting in neuronal loss. The mechanisms of neuronal loss during the prion disease are not fully understood. Studies show that gial activation precedes neuronal loss. And the cytokines secreted by activated microglia play an important role in the neurodegeneration and neuronal loss. This study investigated the effect of cytokines interleukin (IL) -6 and IL-8 in the prion disease used with CJD patients cerebrospinal fluid (CSF), rat microglia culture and primary cortical neuron culture exposed to PrP105-132 in vitro.Firstly, cytokines (IL-6, IL-8) levels in CSF were measured in commercial enzyme immunoassay (ELISA). The results showed that IL-6 level (37.11±6.86 pg/mL) in CJD patients CSF was significantly higher than it (26.27±3.83 pg/ mL) in normal people. But IL-6 level in CJD patients CSF was significantly lower than it (43.70±14.75 pg/mL) in encephalitis patients CSF. IL-8 level (174.13±80.99 pg/mL) in CJD patients CSF was significantly higher than it (62.80±38.34 pg/mL) in normal people. But IL-8 level in CJD patients CSF was significantly lower than it (264.79±126.24 pg/mL) in encephalitis patients CSF. These suggested that IL-6, IL-8 play important roles in the prion disease.Secondly, we used rat microglia culture exposed to PrP105-132 in vitro to investigate the resource of IL-6 and IL-8. The results showed that the microglia exposed to PrP105-132 in vitro had some changes: cell body became bigger, protoplasmic processes became shorter or disappeared, cell became round, rod and amebiform. A dose-dependent increase in IL-6 as well as IL-8 secretion by the PrP105-132 exposed rat microglia was obtained. At a concentration of 80 M the secretion of IL-6 (109.64±7.40 pg/mL), IL-8 (23.82±2.25 pg/mL) were the most. The NF-κB mRNA expression and NF-κB protein synthesis increased in the group treated with PrP105-132. At the same time the secretion of IL-6 and IL-8 increased. In the group treated with NF-κB inhibitor MG132 the NF-κB mRNA expression, NF-κB protein synthesis and the secretion of IL-6, IL-8 decreased. The NFAT mRNA expression and NFAT protein synthesis increased in the group treated with PrP105- 132. At the same time the secretion of IL-6 and IL-8 increased. In the group treated with NFAT inhibitor CsA the NFAT mRNA expression, NFAT protein synthesis and the secretion of IL-6 decreased, but the secretion of IL-8 didn’t decreased significantly. These suggest that PrP may induce microglia secret IL-6 and IL-8. The activation of NF-κB and NFAT pathway are involved in the IL-6 secretion, but the activation of NF-κB pathway is involved in the IL-8 secretion.Lastly, we used the rat primary cortical neuron culture exposed to PrP105-132 in vitro to investigate the effects of prion protein and IL-6, IL-8 on neuron. The cell viability or death was determined by using the MTT assay. Flow cytometry was used for detecting the apoptosis of cells. The results showed that the neuronal viability decreased 13.57% and the percent of apoptotic cell was 5.50±0.56% in the group treated with PrP105-132. The cell apoptosis rate (15.52±1.58%) and motality (20.70%) in the group treated with PrP105-132+IL-6, IL-8 were both significantly higher than them in the group treated with PrP105-132. But the cell apoptosis rate and mortality in the group treated with IL-6, IL-8 had no differences with the control group significantly. These suggest that PrP105-132 has the toxic effects on primary cortical neuron culture, can result in cell apoptosis. IL-6, IL-8 may enchance this neurotoxic effects of PrP105-132.更多还原