To investigate the effect and potential mechanism of ethyl acetate extracts from Inonotus obliquus in inhibiting the replicaton of prion, multiple methods were utilized. Using CCK-8 assay to evaluate the cytotoxicity of dichloromethane extracts, ethyl acetate extracts, n-butanol extracts and obtain the maximal concentrations of various extracts. Prion-infected cell model SMB-S15 were treated with three extracts at safe concentrations (0.1×2−14 mg/mL~0.1×2−8 mg/mL) and the inhibitory effects of different extracts on prion replication were analyzed by PrP-specific Western blots. The expression of reactive oxygen species, hydrogen peroxide and related antioxidant factors were measured in SMB-S15 cells treated with ethyl acetate extracts. The results showed that the maximum safe concentrations of dichloromethane extracts, ethyl acetate extracts and n-butanol extracts was 0.1×2−8 mg/mL. Prion replication was significantly inhibited in SMB-S15 cells treated with ethyl acetate extracts at this concentration of 0.1×2−8 mg/mL for 3 days (P<0.01). While there were no inhibitory effects on prion replication in SMB-S15 cells treated with dichloromethane extracts and n-butanol extracts. In addition, ethyl acetate extracts decreased the levels of reactive oxygen species and hydrogen peroxide, increased the expression level of HO-1, GCLC, total glutathione and enhanced the activity of SOD, with statistical difference. These data suggested that the ethyl acetate extracts from Inonotus obliquus could inhibit prion replication in vitro which might be related to the activation of Nrf2 signaling pathway.

优化桦褐孔菌乙醇粗提物提取工艺并初步探究其对朊病毒复制的影响.采用醇提方法,利用正交试验方法,确定最佳提取工艺.使用CCK-8试剂盒对桦褐孔菌乙醇粗提物处理细胞的安全性进行评价,确定其安全质量浓度;根据安全性评价结果,使用蛋白免疫印记(Western blot)方法检测桦褐孔菌乙醇粗提物对朊病毒复制的影响;通过测定过氧化氢及抗氧化因子等对桦褐孔菌乙醇粗提物的抗氧化活性进行研究.桦褐孔菌乙醇粗提物的最佳提取工艺为:料液比(桦褐孔菌生粉:乙醇)1:10(g/mL),乙醇体积分数75％,提取时间2 h,提取温度80℃,在此条件下,得率为6.32％.桦褐孔菌乙醇粗提物的最大安全质量浓度为6.25×10-4 mg/mL;在此浓度下,处理细胞24 h后,桦褐孔菌乙醇粗提物对朊病毒的复制有抑制作用(p<0.01),可减少H2O2的含量,增加SOD和抗氧化因子GCLM、GCLC的含量,且具有统计学意义.本试验表明桦褐孔菌乙醇粗提物对朊病毒的复制具有抑制作用,且这种作用可能与其抗氧化活性有关.

目前由于缺失特异性的商品化抗体,不同tau异构体在中枢神经系统疾病中,尤其在朊病毒病中的变化鲜有报道.本研究利用杂交瘤技术制备了抗tau蛋白外显子2和10的单克隆抗体,同时对制备抗体的灵敏度、特异性、免疫反应亲和力、种属反应性等方面进行了评价,随后对制备的抗体应用范围进行了摸索.结果 显示,制备的四株单抗特异性高、灵敏度好,尤其可以识别小鼠脑组织中的tau蛋白.制备的四株单抗可用于间接细胞免疫荧光实验和多种感染神经细胞和感染小鼠模型脑组织的蛋白免疫印迹和免疫组织化学实验.综上,本研究制备了四株tau外显子特异性单抗,上述单抗具有较广的应用范围,将成为神经退行性疾病中tau蛋白检测的有力工具.