EAE小鼠星形胶质细胞自噬水平研究及小檗碱对其自噬水平的影响
发布时间：2018-09-07 18:24
【摘要】：目的:探讨EAE小鼠星形胶质细胞的自噬水平变化以及小檗碱对在体和离体的星形胶质细胞自噬水平的影响。方法:使用MOG35-55多肽免疫C57BL/6小鼠构建EAE小鼠模型,将小鼠分为CFA组、EAE组、EAE+BBR(100mg/kg·d)组、EAE+RAPA(1mg/kg·d)组,观察各组小鼠神经系统功能评分。在EAE模型诱导成功后的3d、7d、15d、30d心脏灌注取脑和脊髓,制作石蜡切片。免疫荧光双标定位检测小鼠脊髓白质星形胶质细胞上自噬相关蛋白Beclin1、LC3B的表达水平;第30d的切片行HE染色和LFB染色观察发病30d时各组小鼠脑白质炎症浸润情况和脱髓鞘情况。从健康新生的C57BL/6小鼠大脑皮质中分离纯化出原代星形胶质细胞,经两次传代后得到稳定的星形胶质细胞,使用星形胶质细胞特异性标记GFAP抗体进行细胞鉴定,得到纯化率95%的可用于实验的星形胶质细胞。将细胞样品分为三组进行干预:LPS组、LPS+BBR组、LPS+RAPA组,提取未干预时、干预后0.75h、1.5h、3h、6h、12h的细胞蛋白样品并采用BCA法进行蛋白定量,Western Blot法检测各组的自噬相关蛋白Beclin1、LC3B的表达水平。结果:(1)EAE小鼠最早于免疫后8d发病,大多数发病时间集中在13-15d,并于15-19d达到疾病最高峰。与EAE组的最高分和总分相比,EAE+BBR组(P0.05)和EAE+RAPA组(P0.05)小鼠神经功能评分显著降低,疾病严重程度下降。(2)免疫后第30d,HE染色结果显示EAE组小鼠脑白质中炎性细胞浸润较CFA组(P0.01)、EAE+BBR组(P0.01)及EAE+RAPA组(P0.01)均更为严重,表现呈典型“血管袖套样”改变:大量淋巴细胞、中性粒细沿包膜和小血管由外向内延伸。LFB染色结果显示EAE组小鼠脑白质炎症性脱髓鞘较CFA组(P0.01)、EAE+BBR组(P0.01)及EAE+RAPA组(P0.01)均更为严重,在炎症浸润区域,有大小不等的片状脱髓鞘区,髓鞘纤维疏松,大量空泡形成。(3)通过星形胶质细胞特异性标记物GFAP与自噬相关蛋白Beclin1荧光双标共定位发现,EAE小鼠免疫后7d、15d、30d时,EAE组小鼠脊髓星形胶质细胞上Beclin1表达明显低于EAE+BBR组(P0.05)和EAE+RAPA组(P0.05)。各时间点CFA组表达Beclin1和EAE组表达Beclin1无统计学意义(P0.05)。(4)通过星形胶质细胞特异性标记物GFAP与自噬相关蛋白LC3B荧光双标共定位发现,EAE小鼠免疫后7d、15d、30d时,EAE组小鼠脊髓星形胶质细胞上LC3B表达明显低于EAE+BBR组(P0.05)和EAE+RAPA组(P0.01)。各时间点CFA组表达LC3B和EAE组表达LC3B无统计学意义(P0.05)。(5)WB结果示,在干预后第0.75h,1.5h,3h,6h,12h,LPS组星形胶质细胞上自噬相关蛋白Beclin1的相对表达量较干预以前无明显变化(P0.05),LPS+BBR组(P0.01)和LPS+RAPA组(P0.01)星形胶质细胞上自噬相关蛋白Beclin1的相对表达量较干预以前明显增高。(6)WB结果示,在干预后第0.75h,1.5h,3h,6h,12h,LPS组星形胶质细胞上自噬相关蛋白LC3-II/I相对表达量的比值较干预以前无明显变化(P0.05),LPS+BBR组(P0.01)和LPS+RAPA组(P0.01)星形胶质细胞上自噬相关蛋白LC3-II/I相对表达量的比值较干预以前明显增高。结论:(1)EAE小鼠免疫后星形胶质细胞上自噬持续处于低水平表达,离体培养的原代星形胶质细胞通过LPS刺激后自噬也持续处于低水平表达。(2)雷帕霉素和小檗碱可改善EAE小鼠神经功能缺损,减轻炎症浸润和脱髓鞘情况。(3)雷帕霉素和小檗碱可诱导EAE小鼠和离体星形胶质细胞上的自噬,这可能是雷帕霉素和小檗碱改善EAE症状的机制之一。
[Abstract]:AIM: To investigate the changes of autophagy level of astrocytes in EAE mice and the effects of berberine on autophagy level of astrocytes in vivo and in vitro. Methods: The mice were immunized with MOG35-55 polypeptide to construct an EAE mouse model. The mice were divided into CFA group, EAE group, EAE+BBR group (100mg/kg.d), EAE+RAPA group (1mg/kg.d). The expression of autophagy-associated proteins Beclin 1 and LC3B on mouse spinal cord white matter astrocytes was detected by immunofluorescence double labeling localization. The expression of autophagy-related proteins Beclin 1 and LC3B on mouse spinal cord white matter astrocytes was observed by HE staining and LFB staining on the 30th day after the induction of EAE model. Inflammatory infiltration and demyelination of white matter. Primary astrocytes were isolated and purified from the cerebral cortex of healthy newborn C57BL/6 mice. After two passages, stable astrocytes were obtained. The astrocytes were identified by using GFAP antibody, and the purified astrocytes with 95% purity were obtained. Glial cells were divided into three groups: LPS group, LPS + BBR group, LPS + RAPA group. Cell protein samples were extracted at 0.75h, 1.5h, 3h, 6h, 12h after intervention and quantified by BCA. The expression of autophagy-related proteins Beclin1 and LC3B was detected by Western Blot. On the 8th day, most of the onset time was concentrated in 13-15 days, and reached the peak of the disease on the 15th-19th day. Compared with the highest and total scores of EAE group, the neurological function scores of EAE+BBR group (P 0.05) and EAE+RAPA group (P 0.05) were significantly decreased, and the severity of the disease was decreased. (2) On the 30th day after immunization, the results of HE staining showed the infiltration of inflammatory cells in the white matter of EAE group mice. Compared with CFA group (P 0.01), EAE+BBR group (P 0.01) and EAE+RAPA group (P 0.01), the changes were more serious, showing a typical "vascular cuff-like" change: a large number of lymphocytes, neutrophils along the capsule and small blood vessels extending from outside to inside. LFB staining showed that inflammatory demyelination of white matter in EAE group was more serious than that in CFA group (P 0.01), EAE+BBR group (P 0.01) and EAE+RAPA group (P 0.01). (3) Astrocyte specific marker GFAP and autophagy-associated protein Beclin1 fluorescence double labeling were used to localize Beclin1 on spinal astrocytes of EAE mice 7, 15 and 30 days after immunization. The expression of Beclin1 in CFA group and EAE + RAPA group was significantly lower than that in EAE + BBR group (P 0.05) and EAE + RAPA group (P 0.05). There was no significant difference in the expression of Beclin1 and EAE group at each time point (P 0.05). (4) By co-localization of astrocyte specific marker GFAP and autophagy-associated protein LC3B fluorescence double labeling, it was found that the spinal astrocytes of EAE mice were co-localized at 7, 15 and 30 days after immunization. The expression of LC3B in plasma cells was significantly lower than that in EAE+BBR group (P 0.05) and EAE+RAPA group (P 0.01). There was no significant difference between CFA group and EAE group (P 0.05). (5) WB results showed that the relative expression of autophagy-related protein Beclin 1 in astrocytes of LPS group had no significant change at 0.75 h, 1.5 h, 3 h, 6 h and 12 h after intervention (P The relative expression of autophagy-associated protein Beclin 1 on astrocytes in LPS+BBR group (P 0.01), LPS+RAPA group (P 0.01) and LPS+RAPA group (P 0.01) was significantly higher than that before intervention. (6) WB results showed that the ratio of autophagy-associated protein LC3-II/I on astrocytes in LPS group had no significant change at 0.75 h, 1.5 h, 3 h, 6 h and 12 h after intervention (P 0.0). 5) The ratio of autophagy-associated protein LC3-II/I on astrocytes in LPS+BBR group (P 0.01) and LPS+RAPA group (P 0.01) was significantly higher than that before the intervention. Conclusion: (1) Autophagy on astrocytes of EAE mice remained at a low level after immunization, and autophagy on primary astrocytes cultured in vitro persisted after LPS stimulation. (3) Rapamycin and berberine can induce autophagy on EAE mice and astrocytes in vitro, which may be one of the mechanisms of rapamycin and berberine improving EAE symptoms.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51
本文编号：2229064
[Abstract]:AIM: To investigate the changes of autophagy level of astrocytes in EAE mice and the effects of berberine on autophagy level of astrocytes in vivo and in vitro. Methods: The mice were immunized with MOG35-55 polypeptide to construct an EAE mouse model. The mice were divided into CFA group, EAE group, EAE+BBR group (100mg/kg.d), EAE+RAPA group (1mg/kg.d). The expression of autophagy-associated proteins Beclin 1 and LC3B on mouse spinal cord white matter astrocytes was detected by immunofluorescence double labeling localization. The expression of autophagy-related proteins Beclin 1 and LC3B on mouse spinal cord white matter astrocytes was observed by HE staining and LFB staining on the 30th day after the induction of EAE model. Inflammatory infiltration and demyelination of white matter. Primary astrocytes were isolated and purified from the cerebral cortex of healthy newborn C57BL/6 mice. After two passages, stable astrocytes were obtained. The astrocytes were identified by using GFAP antibody, and the purified astrocytes with 95% purity were obtained. Glial cells were divided into three groups: LPS group, LPS + BBR group, LPS + RAPA group. Cell protein samples were extracted at 0.75h, 1.5h, 3h, 6h, 12h after intervention and quantified by BCA. The expression of autophagy-related proteins Beclin1 and LC3B was detected by Western Blot. On the 8th day, most of the onset time was concentrated in 13-15 days, and reached the peak of the disease on the 15th-19th day. Compared with the highest and total scores of EAE group, the neurological function scores of EAE+BBR group (P 0.05) and EAE+RAPA group (P 0.05) were significantly decreased, and the severity of the disease was decreased. (2) On the 30th day after immunization, the results of HE staining showed the infiltration of inflammatory cells in the white matter of EAE group mice. Compared with CFA group (P 0.01), EAE+BBR group (P 0.01) and EAE+RAPA group (P 0.01), the changes were more serious, showing a typical "vascular cuff-like" change: a large number of lymphocytes, neutrophils along the capsule and small blood vessels extending from outside to inside. LFB staining showed that inflammatory demyelination of white matter in EAE group was more serious than that in CFA group (P 0.01), EAE+BBR group (P 0.01) and EAE+RAPA group (P 0.01). (3) Astrocyte specific marker GFAP and autophagy-associated protein Beclin1 fluorescence double labeling were used to localize Beclin1 on spinal astrocytes of EAE mice 7, 15 and 30 days after immunization. The expression of Beclin1 in CFA group and EAE + RAPA group was significantly lower than that in EAE + BBR group (P 0.05) and EAE + RAPA group (P 0.05). There was no significant difference in the expression of Beclin1 and EAE group at each time point (P 0.05). (4) By co-localization of astrocyte specific marker GFAP and autophagy-associated protein LC3B fluorescence double labeling, it was found that the spinal astrocytes of EAE mice were co-localized at 7, 15 and 30 days after immunization. The expression of LC3B in plasma cells was significantly lower than that in EAE+BBR group (P 0.05) and EAE+RAPA group (P 0.01). There was no significant difference between CFA group and EAE group (P 0.05). (5) WB results showed that the relative expression of autophagy-related protein Beclin 1 in astrocytes of LPS group had no significant change at 0.75 h, 1.5 h, 3 h, 6 h and 12 h after intervention (P The relative expression of autophagy-associated protein Beclin 1 on astrocytes in LPS+BBR group (P 0.01), LPS+RAPA group (P 0.01) and LPS+RAPA group (P 0.01) was significantly higher than that before intervention. (6) WB results showed that the ratio of autophagy-associated protein LC3-II/I on astrocytes in LPS group had no significant change at 0.75 h, 1.5 h, 3 h, 6 h and 12 h after intervention (P 0.0). 5) The ratio of autophagy-associated protein LC3-II/I on astrocytes in LPS+BBR group (P 0.01) and LPS+RAPA group (P 0.01) was significantly higher than that before the intervention. Conclusion: (1) Autophagy on astrocytes of EAE mice remained at a low level after immunization, and autophagy on primary astrocytes cultured in vitro persisted after LPS stimulation. (3) Rapamycin and berberine can induce autophagy on EAE mice and astrocytes in vitro, which may be one of the mechanisms of rapamycin and berberine improving EAE symptoms.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51
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