EAE小鼠星形膠質(zhì)細(xì)胞自噬水平研究及小檗堿對(duì)其自噬水平的影響
發(fā)布時(shí)間：2018-09-07 18:24
【摘要】：目的:探討EAE小鼠星形膠質(zhì)細(xì)胞的自噬水平變化以及小檗堿對(duì)在體和離體的星形膠質(zhì)細(xì)胞自噬水平的影響。方法:使用MOG35-55多肽免疫C57BL/6小鼠構(gòu)建EAE小鼠模型,將小鼠分為CFA組、EAE組、EAE+BBR(100mg/kg·d)組、EAE+RAPA(1mg/kg·d)組,觀察各組小鼠神經(jīng)系統(tǒng)功能評(píng)分。在EAE模型誘導(dǎo)成功后的3d、7d、15d、30d心臟灌注取腦和脊髓,制作石蠟切片。免疫熒光雙標(biāo)定位檢測(cè)小鼠脊髓白質(zhì)星形膠質(zhì)細(xì)胞上自噬相關(guān)蛋白Beclin1、LC3B的表達(dá)水平;第30d的切片行HE染色和LFB染色觀察發(fā)病30d時(shí)各組小鼠腦白質(zhì)炎癥浸潤(rùn)情況和脫髓鞘情況。從健康新生的C57BL/6小鼠大腦皮質(zhì)中分離純化出原代星形膠質(zhì)細(xì)胞,經(jīng)兩次傳代后得到穩(wěn)定的星形膠質(zhì)細(xì)胞,使用星形膠質(zhì)細(xì)胞特異性標(biāo)記GFAP抗體進(jìn)行細(xì)胞鑒定,得到純化率95%的可用于實(shí)驗(yàn)的星形膠質(zhì)細(xì)胞。將細(xì)胞樣品分為三組進(jìn)行干預(yù):LPS組、LPS+BBR組、LPS+RAPA組,提取未干預(yù)時(shí)、干預(yù)后0.75h、1.5h、3h、6h、12h的細(xì)胞蛋白樣品并采用BCA法進(jìn)行蛋白定量,Western Blot法檢測(cè)各組的自噬相關(guān)蛋白Beclin1、LC3B的表達(dá)水平。結(jié)果:(1)EAE小鼠最早于免疫后8d發(fā)病,大多數(shù)發(fā)病時(shí)間集中在13-15d,并于15-19d達(dá)到疾病最高峰。與EAE組的最高分和總分相比,EAE+BBR組(P0.05)和EAE+RAPA組(P0.05)小鼠神經(jīng)功能評(píng)分顯著降低,疾病嚴(yán)重程度下降。(2)免疫后第30d,HE染色結(jié)果顯示EAE組小鼠腦白質(zhì)中炎性細(xì)胞浸潤(rùn)較CFA組(P0.01)、EAE+BBR組(P0.01)及EAE+RAPA組(P0.01)均更為嚴(yán)重,表現(xiàn)呈典型“血管袖套樣”改變:大量淋巴細(xì)胞、中性粒細(xì)沿包膜和小血管由外向內(nèi)延伸。LFB染色結(jié)果顯示EAE組小鼠腦白質(zhì)炎癥性脫髓鞘較CFA組(P0.01)、EAE+BBR組(P0.01)及EAE+RAPA組(P0.01)均更為嚴(yán)重,在炎癥浸潤(rùn)區(qū)域,有大小不等的片狀脫髓鞘區(qū),髓鞘纖維疏松,大量空泡形成。(3)通過星形膠質(zhì)細(xì)胞特異性標(biāo)記物GFAP與自噬相關(guān)蛋白Beclin1熒光雙標(biāo)共定位發(fā)現(xiàn),EAE小鼠免疫后7d、15d、30d時(shí),EAE組小鼠脊髓星形膠質(zhì)細(xì)胞上Beclin1表達(dá)明顯低于EAE+BBR組(P0.05)和EAE+RAPA組(P0.05)。各時(shí)間點(diǎn)CFA組表達(dá)Beclin1和EAE組表達(dá)Beclin1無統(tǒng)計(jì)學(xué)意義(P0.05)。(4)通過星形膠質(zhì)細(xì)胞特異性標(biāo)記物GFAP與自噬相關(guān)蛋白LC3B熒光雙標(biāo)共定位發(fā)現(xiàn),EAE小鼠免疫后7d、15d、30d時(shí),EAE組小鼠脊髓星形膠質(zhì)細(xì)胞上LC3B表達(dá)明顯低于EAE+BBR組(P0.05)和EAE+RAPA組(P0.01)。各時(shí)間點(diǎn)CFA組表達(dá)LC3B和EAE組表達(dá)LC3B無統(tǒng)計(jì)學(xué)意義(P0.05)。(5)WB結(jié)果示,在干預(yù)后第0.75h,1.5h,3h,6h,12h,LPS組星形膠質(zhì)細(xì)胞上自噬相關(guān)蛋白Beclin1的相對(duì)表達(dá)量較干預(yù)以前無明顯變化(P0.05),LPS+BBR組(P0.01)和LPS+RAPA組(P0.01)星形膠質(zhì)細(xì)胞上自噬相關(guān)蛋白Beclin1的相對(duì)表達(dá)量較干預(yù)以前明顯增高。(6)WB結(jié)果示,在干預(yù)后第0.75h,1.5h,3h,6h,12h,LPS組星形膠質(zhì)細(xì)胞上自噬相關(guān)蛋白LC3-II/I相對(duì)表達(dá)量的比值較干預(yù)以前無明顯變化(P0.05),LPS+BBR組(P0.01)和LPS+RAPA組(P0.01)星形膠質(zhì)細(xì)胞上自噬相關(guān)蛋白LC3-II/I相對(duì)表達(dá)量的比值較干預(yù)以前明顯增高。結(jié)論:(1)EAE小鼠免疫后星形膠質(zhì)細(xì)胞上自噬持續(xù)處于低水平表達(dá),離體培養(yǎng)的原代星形膠質(zhì)細(xì)胞通過LPS刺激后自噬也持續(xù)處于低水平表達(dá)。(2)雷帕霉素和小檗堿可改善EAE小鼠神經(jīng)功能缺損,減輕炎癥浸潤(rùn)和脫髓鞘情況。(3)雷帕霉素和小檗堿可誘導(dǎo)EAE小鼠和離體星形膠質(zhì)細(xì)胞上的自噬,這可能是雷帕霉素和小檗堿改善EAE癥狀的機(jī)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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R744.51
本文編號(hào)：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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R744.51
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