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作者黄小会（2019）在《MYSM1对星形胶质细胞活化的影响及其机制研究》一文中研究指出：星形胶质细胞在哺乳动物中枢神经系统（CNS）胶质细胞中约占90%。它们是大脑和脊髓的组成部分。在正常生理情况下,星形胶质细胞在神经发育、脑血流量调节,离子平衡,突触形成与功能,突触修剪,能量代谢中发挥重要作用。病理状态下,星形胶质细胞活化,在许多疾病和几乎所有形式的神经性疾病中发挥重要作用,包括重型脑损伤如中风和创伤性脑损伤（TBI）,神经退行性疾病如阿尔茨海默氏病（AD）、帕金森氏病（PD）、肌萎缩侧索硬化症（ALS）等。然而,在应对免疫攻击、慢性神经退行性疾病或急性损伤时,星形胶质的功能将如何改变,我们仍知之甚少。2012年Zamanian等人发现皮下注射LPS诱导神经炎症与脑缺血诱发大脑中动脉闭塞这两种不同形式的脑损伤产生了至少两种类型的“活化型”星形胶质细胞,并提供新的标志以划定不同状态的活化型星形胶质细胞。2017年Liddelow等人将这两种不同类型的活化型星形胶质细胞分别命名为"A1"和"A2"。这个术语与巨噬细胞和小胶质细胞被命名"M1"和"M2"相似。巨噬细胞和小胶质表现出不只两个极化状态,同样,活化型星形胶质细胞也很可能有两个以上的极化状态,这将是未来研究的重要领域。MYSM是一种去泛素化酶,全称是MYb-1ike,SWRIM and MPN domain-containing protein-1。研究发现,MYSM1缺陷小鼠造血全系细胞减少,影响T细胞、B细胞、DC细胞的发育;影响巨噬细胞的极化以及MSCs的分化。同时,MYSM1能通过去除TRAF3和TRAF6上的K63泛素链进而调控巨噬细胞的活化和功能。那么MYSM1是否会调控中枢神经系统内具有免疫调节能力的星形胶质细胞的功能,目前尚未有报道,所以我们从以下三个部分进行MYSM1对星形胶质细胞活化的影响及其机制的研究。第一部分:活化型星形胶质细胞的生物学特性实验方法:1.分离野生型（WT）小鼠星形胶质细胞,通过星形胶质细胞GFAP免疫荧光和qRT-PCR检测星形胶质细胞标志（Aldh1l1、GFAP、Vim、Aqp4）、小胶质细胞标志（Cx3cr1、Aif1）以及神经元标志（Snap25、Syt1）鉴定星形胶质细胞的纯度。2.qRT-PCR检测不同因子刺激后星形胶质细胞A1转录因子（H2-T23、H2-D1、Serping1、Gbp2）/A2转录因子（Tgm1、Ptx3、S100a10）的水平。3.qRT-PCR检测LPS刺激后星形胶质细胞iNOS、IL-1β、TNF-α、IFN-γ、IL-6、IL-10、FGF-2、BDNF、TGF-β、VEGF水平。Western Blot检测LPS刺激后星形胶质细胞iNOS的表达水平。Griess法检测LPS刺激后星形胶质细胞上清NO含量。4.ACM与神经元共培养实验检测星形胶质细胞对神经元的影响,qRT-PCR检测星形胶质细胞促突触形成因子（Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2）的表达。5.星形胶质细胞与红色荧光标记的Aβ42共培养检测星形胶质细胞的吞噬功能,qRT-PCR检测星形胶质细胞吞噬受体（megf10、mertk、Gas6、Axl）的表达。6.分离WT和IL10低表达(IL-10tm1/tm1)小鼠星形胶质细胞,qRT-PCR检测IL-10tm1/tm1星形胶质细胞A1转录因子（H2-T23、H2-D1、Gbp2）/A2转录因子（Tgm1、S100a10）水平。7.qRT-PCR检测IL-10tm1/tm1星形胶质细胞iNOS、IL-1β、TNF-α、IFN-γ、IL-6、FGF-2、BDNF、TGF-β的表达。8.ACM与神经元共培养检测IL-10tm1/tm1星形胶质对神经元的影响,qRT-PCR检测IL-10tm1/tm1星形胶质细胞促突触形成因子（Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2）的表达。9.星形胶质细胞与荧光标记的Aβ42共培养检测IL-10tm1/tm1星形胶质细胞的吞噬功能,qRT-PCR检测IL-10tm1/tm1星形胶质细胞吞噬受体（megf10、mertk、Gas6、Axl）的表达。实验结果:1.星形胶质细胞GFAP阳性率在95%以上,星形胶质细胞细胞标志（Aldh1l1、GFAP、Vim、Aqp4）的表达水平明显高于小胶质细胞标志（Cxcr3、Aif1）和神经元标志（Snap25、Syt1）的表达水平,纯度满足后续实验需求。2.LPS（100ng/mL）刺激的星形胶质细胞A1转录因子（H2-T23、H2-D1、Serping1、Gbp2）水平显著增加。3.LPS能够显著促进星形胶质细胞iNOS、IL-1β,TNF-α、IFN-γ、IL-6的合成分泌。Western Blot结果表明,LPS刺激后星形胶质细胞iNOS水平增加。Griess实验结果表明,LPS刺激后星形胶质细胞上清NO含量显著增加。4.ACM与神经元共培养24h后,与WT control ACM组相比,WT LPS ACM组神经元数量减少,胞体萎缩,突触明显减少或消失。qRT-PCR检测结果表明,LPS刺激后,星形胶质细胞Gpc4、Gpc6、SPARCL1、Thbs1和Thbs2表达均下降。5.Aβ42与星形胶质细胞共培养24h后,LPS刺激后,在星形胶质细胞周围聚集的Aβ42蛋白减少,不能得到有效的清除。qRT-PCR检测结果表明,LPS刺激后,星形胶质细胞吞噬受体（megf10、mertk、Gas6、Axl）表达减少。6.qRT-PCR结果表明,IL-10tm1/tm1星形胶质细胞A1转录因子（H2-T23、H2-D1、Gbp2）表达水平升高。LPS刺激后,IL-10tm1/tm1星形胶质细胞的A1转录因子（H2-T23、H2-D1、Gbp2）也显著高于WT组。7.qRT-PCR结果表明,相对于WT组,IL-10tm1/tm1星形胶质细胞iNOS、IL-1β、TNF-α、IL-6这些炎性因子水平都没有显著变化,IFN-γ水平显著升高;LPS刺激后,IL-10tm1/tm1星形胶质细胞iNOS、IL-1β、IL-6表达水平明显高于WT组。8.ACM与神经元共培养24h后,IL-10tm1/tm1control组神经元胞体萎缩,突触断裂,神经元死亡。LPS刺激后,IL-10tm1/tm1LPS组神经元胞体裂解,突触消失,神经元死亡。qRT-PCR结果也显示,LPS刺激与否,IL-10tm1/tm1星形胶质细胞Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2减少。9.Aβ42与IL-10tm1/tm1星形胶质细胞共培养24h。在相同时间点,IL-10tm1/tm1星形胶质细胞胞内聚集的Aβ42蛋白明显少于WT组。LPS刺激后,相对于WT组,IL-10tm1/tm1星形胶质细胞对Aβ42的吸收明显下降,Aβ42蛋白得不到有效的清除。qRT-PCR实验也表明,与WT组相比,IL-10tm1/tm1星形胶质细胞吞噬受体megf10、Mertk、Gas6、Axl表达均下降。LPS刺激后,相比WT组,IL-10tm1/tm1星形胶质细胞吞噬受体megf10、Gas6、Axl表达也下降第二部分:MYSM1对星形胶质细胞活化的影响实验方法:1.分离WT小鼠星形胶质细胞,qRT-PCR和Western Blot检测LPS刺激后星形胶质细胞MYSM1的表达水平。2.分离WT和MYSM1敲除(MYSM1-/-)小鼠星形胶质细胞,qRT-PCR检测MYSM1的敲除效率。3.qRT-PCR检测MYSM1-/-星形胶质细胞A1转录因子（H2-T23、H2-D1、Gbp2）/A2转录因子（Tgm1、Ptx3、S100a10）转录因子的水平。4.qRT-PCR检测MYSM1-/-星形胶质细胞iNOS、IL-1β、TNF-α、IL-10水平。5.ACM与神经元共培养检测MYSM1-/-星形胶质细胞对神经元的影响,qRT-PCR检测MYSM1-/-星形胶质细胞促突触形成因子Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2的表达。6.星形胶质细胞与荧光标记的Aβ42共培养检测MYSM1-/-星形胶质细胞的吞噬功能,qRT-PCR检测MYSM1-/-星形胶质细胞吞噬受体megf10、mertk、Gas6、Axl的表达。实验结果:1.qRT-PCR结果和Western Blot结果表明,LPS刺激后星形胶质细胞MYSM1转录水平和蛋白表达水平降低。2.qRT-PCR结果表明,MYSM1-/-小鼠星形胶质细胞MYSM1显著降低。3.qRT-PCR结果表明,相比WT组,MYSM1-/-星形胶质细胞A1转录因子（H2-T23、H2-D1、Gbp2）水平增加;LPS刺激后,相比WT组,MYSM1-/-星形胶质细胞H2-D1、Gbp2转录因子水平增加,但其它因子变化不显著。4.qRT-PCR结果表明,相比WT组,MYSM1-/-星形胶质细胞iNOS、IL-1β水平升高,TNF-α、IL-10差异不显著。LPS刺激后后,相比WT组,MYSM1-/-星形胶质细胞iNOS、IL-1β表达显著增加,TNF-α、IL-10变化不显著。5.ACM与神经元共培养24h。相对于WT组,MYSM1-/-星形胶质上清与神经元共培养组的神经元,胞体萎缩,突触断裂,神经元死亡。LPS刺激后,MYSM1-/-星形胶质细胞上清组,神经元胞体裂解,突触消失,神经元死亡。qRT-PCR结果也显示,相比WT组,LPS刺激与否,MYSM1-/-星形胶质细胞促突触形成蛋白Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2减少。6.将Aβ42与MYSM1-/-星形胶质细胞共培养24h。在相同时间点,MYSM-/-星形胶质细胞胞内聚集的Aβ42蛋白明显少于WT组。但随着时间的延长,胞体内的荧光强度也逐渐增加,呈现时间依赖性。LPS刺激后,相对于WT组,MYSM-/-星形胶质细胞对Aβ42的吸收明显下降。qRT-PCR实验表明,与WT组相比,MYSM-/-星形胶质细胞吞噬受体megf10、Mertk、Gas6、Axl表达均下降。LPS刺激后,相比WT组,MYSM-/-星形胶质细胞吞噬受体megf10、Mertk、Gas6、Axl表达均下降。第三部分:MYSM1对星形胶质细胞活化机制的研究实验方法:1.分离WT小鼠星形胶质细胞,qRT-PCR检测LPS刺激后miR129的表达水平。2.分离WT和MYSM1-/-小鼠星形胶质细胞,qRT-PCR检测miR129表达水平。3.分离WT小鼠星形胶质细胞,体外慢病毒转染干扰miR129（shmiR129）,qRT-PCR检测shmiR129星形胶质细胞A1转录因子（H2-T23、H2-D1、Serping1、Gbp2）/A2转录因子（Tgm1、Ptx3、S100a10）水平。4.qRT-PCR检测shmiR129星形胶质细胞iNOS、IL-1β、TNF-α、IL-6、IL-10、BDNF、FGF-2的转录水平。Griess法检测shmiR129星形胶质细胞上清NO含量。5.ACM与神经元共培养检测shmiR129星形胶质对神经元的影响,qRT-PCR检测shmiR129星形胶质细胞促突触形成因子Gpc4、Gpc6、Thbs1、SPARCL1、Thbs2的表达。实验结果:1.qRT-PCR结果表明,LPS刺激后星形胶质细胞miR129表达降低。2.MYSM1敲除后,星形胶质细胞miR129表达降低。3.qRT-PCR结果表明,shmiR129星形胶质细胞A1转录因子（H2-T23、H2-D1、Serping1、Gbp2）水平增加,A2转录因子（Tgm1、Ptx3、S100a10）水平降低。4.qRT-PCR结果表明,shmiR129星形胶质细胞分泌的细胞因子iNOS、IL-1β、TNF-α、IL-6、IL-10、BDNF、FGF-2没有显著变化。Griess实验结果表明,WT和shmiR129星形胶质细胞上清NO含量也没明显差异。5.ACM与神经元共培养24h后,shmiR129组神经元突触断裂或消失。qRT-PCR实验结果表明,与对照组相比,shmiR129星形胶质细胞促突触形成受体Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2的表达无明显变化。结论:1.不同因子对星形胶质细胞的活化的影响不同。100ng/mL LPS为活化星形胶质细胞最为合适的因子。经LPS刺激后,星形胶质细胞A1特异性转录因子（H2-T23/H2-D1/Serping1/Gbp2）表达水平升高,炎性因子表达水平升高,对神经元产生细胞毒性促突触形成蛋白表达水平降低,吞噬Aβ42的能力降低,吞噬受体的表达水平也降低,这提示LPS刺激后星形胶质细胞易于向A1型活化。2.IL-10低表达的星形胶质细胞验证了以上结果。3.MYSM1敲除后,星形胶质细胞A1特异性转录因子（H2-T23/H2-D1/Gbp2）表达水平升高,炎性因子表达水平升高,对神经元营养支持作用减弱,促突触形成蛋白表达水平降低,吞噬能力降低,吞噬受体的表达降低,提示MYSM1敲除后星形胶质细胞易于向A1型活化。4.miR129干扰后,星形胶质细胞A1（H2-T23/H2-D1/Serping1/Gbp2）特异性转录因子表达升高,A2特异性转录因子（Tgm1/Ptx3/S100a10）表达水平升高,促进神经元神经元存活的能力降低,吞噬能力降低;MYSM1敲除后miR129表达水平下降,提示MYSM1可能是通过miR129调控星形胶质细胞活化,但具体的调控机制还需要进一步深入研究。
Abstract
xing xing jiao zhi xi bao zai bu ru dong wu zhong shu shen jing ji tong （CNS）jiao zhi xi bao zhong yao zhan 90%。ta men shi da nao he ji sui de zu cheng bu fen 。zai zheng chang sheng li qing kuang xia ,xing xing jiao zhi xi bao zai shen jing fa yo 、nao xie liu liang diao jie ,li zi ping heng ,tu chu xing cheng yu gong neng ,tu chu xiu jian ,neng liang dai xie zhong fa hui chong yao zuo yong 。bing li zhuang tai xia ,xing xing jiao zhi xi bao huo hua ,zai hu duo ji bing he ji hu suo you xing shi de shen jing xing ji bing zhong fa hui chong yao zuo yong ,bao gua chong xing nao sun shang ru zhong feng he chuang shang xing nao sun shang （TBI）,shen jing tui hang xing ji bing ru a er ci hai mo shi bing （AD）、pa jin sen shi bing （PD）、ji wei su ce suo ying hua zheng （ALS）deng 。ran er ,zai ying dui mian yi gong ji 、man xing shen jing tui hang xing ji bing huo ji xing sun shang shi ,xing xing jiao zhi de gong neng jiang ru he gai bian ,wo men reng zhi zhi shen shao 。2012nian Zamaniandeng ren fa xian pi xia zhu she LPSyou dao shen jing yan zheng yu nao que xie you fa da nao zhong dong mai bi sai zhe liang chong bu tong xing shi de nao sun shang chan sheng le zhi shao liang chong lei xing de “huo hua xing ”xing xing jiao zhi xi bao ,bing di gong xin de biao zhi yi hua ding bu tong zhuang tai de huo hua xing xing xing jiao zhi xi bao 。2017nian Liddelowdeng ren jiang zhe liang chong bu tong lei xing de huo hua xing xing xing jiao zhi xi bao fen bie ming ming wei "A1"he "A2"。zhe ge shu yu yu ju shi xi bao he xiao jiao zhi xi bao bei ming ming "M1"he "M2"xiang shi 。ju shi xi bao he xiao jiao zhi biao xian chu bu zhi liang ge ji hua zhuang tai ,tong yang ,huo hua xing xing xing jiao zhi xi bao ye hen ke neng you liang ge yi shang de ji hua zhuang tai ,zhe jiang shi wei lai yan jiu de chong yao ling yu 。MYSMshi yi chong qu fan su hua mei ,quan chen shi MYb-1ike,SWRIM and MPN domain-containing protein-1。yan jiu fa xian ,MYSM1que xian xiao shu zao xie quan ji xi bao jian shao ,ying xiang Txi bao 、Bxi bao 、DCxi bao de fa yo ;ying xiang ju shi xi bao de ji hua yi ji MSCsde fen hua 。tong shi ,MYSM1neng tong guo qu chu TRAF3he TRAF6shang de K63fan su lian jin er diao kong ju shi xi bao de huo hua he gong neng 。na me MYSM1shi fou hui diao kong zhong shu shen jing ji tong nei ju you mian yi diao jie neng li de xing xing jiao zhi xi bao de gong neng ,mu qian shang wei you bao dao ,suo yi wo men cong yi xia san ge bu fen jin hang MYSM1dui xing xing jiao zhi xi bao huo hua de ying xiang ji ji ji zhi de yan jiu 。di yi bu fen :huo hua xing xing xing jiao zhi xi bao de sheng wu xue te xing shi yan fang fa :1.fen li ye sheng xing （WT）xiao shu xing xing jiao zhi xi bao ,tong guo xing xing jiao zhi xi bao GFAPmian yi ying guang he qRT-PCRjian ce xing xing jiao zhi xi bao biao zhi （Aldh1l1、GFAP、Vim、Aqp4）、xiao jiao zhi xi bao biao zhi （Cx3cr1、Aif1）yi ji shen jing yuan biao zhi （Snap25、Syt1）jian ding xing xing jiao zhi xi bao de chun du 。2.qRT-PCRjian ce bu tong yin zi ci ji hou xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Serping1、Gbp2）/A2zhuai lu yin zi （Tgm1、Ptx3、S100a10）de shui ping 。3.qRT-PCRjian ce LPSci ji hou xing xing jiao zhi xi bao iNOS、IL-1β、TNF-α、IFN-γ、IL-6、IL-10、FGF-2、BDNF、TGF-β、VEGFshui ping 。Western Blotjian ce LPSci ji hou xing xing jiao zhi xi bao iNOSde biao da shui ping 。Griessfa jian ce LPSci ji hou xing xing jiao zhi xi bao shang qing NOhan liang 。4.ACMyu shen jing yuan gong pei yang shi yan jian ce xing xing jiao zhi xi bao dui shen jing yuan de ying xiang ,qRT-PCRjian ce xing xing jiao zhi xi bao cu tu chu xing cheng yin zi （Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2）de biao da 。5.xing xing jiao zhi xi bao yu gong se ying guang biao ji de Aβ42gong pei yang jian ce xing xing jiao zhi xi bao de tun shi gong neng ,qRT-PCRjian ce xing xing jiao zhi xi bao tun shi shou ti （megf10、mertk、Gas6、Axl）de biao da 。6.fen li WThe IL10di biao da (IL-10tm1/tm1)xiao shu xing xing jiao zhi xi bao ,qRT-PCRjian ce IL-10tm1/tm1xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Gbp2）/A2zhuai lu yin zi （Tgm1、S100a10）shui ping 。7.qRT-PCRjian ce IL-10tm1/tm1xing xing jiao zhi xi bao iNOS、IL-1β、TNF-α、IFN-γ、IL-6、FGF-2、BDNF、TGF-βde biao da 。8.ACMyu shen jing yuan gong pei yang jian ce IL-10tm1/tm1xing xing jiao zhi dui shen jing yuan de ying xiang ,qRT-PCRjian ce IL-10tm1/tm1xing xing jiao zhi xi bao cu tu chu xing cheng yin zi （Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2）de biao da 。9.xing xing jiao zhi xi bao yu ying guang biao ji de Aβ42gong pei yang jian ce IL-10tm1/tm1xing xing jiao zhi xi bao de tun shi gong neng ,qRT-PCRjian ce IL-10tm1/tm1xing xing jiao zhi xi bao tun shi shou ti （megf10、mertk、Gas6、Axl）de biao da 。shi yan jie guo :1.xing xing jiao zhi xi bao GFAPyang xing lv zai 95%yi shang ,xing xing jiao zhi xi bao xi bao biao zhi （Aldh1l1、GFAP、Vim、Aqp4）de biao da shui ping ming xian gao yu xiao jiao zhi xi bao biao zhi （Cxcr3、Aif1）he shen jing yuan biao zhi （Snap25、Syt1）de biao da shui ping ,chun du man zu hou xu shi yan xu qiu 。2.LPS（100ng/mL）ci ji de xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Serping1、Gbp2）shui ping xian zhe zeng jia 。3.LPSneng gou xian zhe cu jin xing xing jiao zhi xi bao iNOS、IL-1β,TNF-α、IFN-γ、IL-6de ge cheng fen bi 。Western Blotjie guo biao ming ,LPSci ji hou xing xing jiao zhi xi bao iNOSshui ping zeng jia 。Griessshi yan jie guo biao ming ,LPSci ji hou xing xing jiao zhi xi bao shang qing NOhan liang xian zhe zeng jia 。4.ACMyu shen jing yuan gong pei yang 24hhou ,yu WT control ACMzu xiang bi ,WT LPS ACMzu shen jing yuan shu liang jian shao ,bao ti wei su ,tu chu ming xian jian shao huo xiao shi 。qRT-PCRjian ce jie guo biao ming ,LPSci ji hou ,xing xing jiao zhi xi bao Gpc4、Gpc6、SPARCL1、Thbs1he Thbs2biao da jun xia jiang 。5.Aβ42yu xing xing jiao zhi xi bao gong pei yang 24hhou ,LPSci ji hou ,zai xing xing jiao zhi xi bao zhou wei ju ji de Aβ42dan bai jian shao ,bu neng de dao you xiao de qing chu 。qRT-PCRjian ce jie guo biao ming ,LPSci ji hou ,xing xing jiao zhi xi bao tun shi shou ti （megf10、mertk、Gas6、Axl）biao da jian shao 。6.qRT-PCRjie guo biao ming ,IL-10tm1/tm1xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Gbp2）biao da shui ping sheng gao 。LPSci ji hou ,IL-10tm1/tm1xing xing jiao zhi xi bao de A1zhuai lu yin zi （H2-T23、H2-D1、Gbp2）ye xian zhe gao yu WTzu 。7.qRT-PCRjie guo biao ming ,xiang dui yu WTzu ,IL-10tm1/tm1xing xing jiao zhi xi bao iNOS、IL-1β、TNF-α、IL-6zhe xie yan xing yin zi shui ping dou mei you xian zhe bian hua ,IFN-γshui ping xian zhe sheng gao ;LPSci ji hou ,IL-10tm1/tm1xing xing jiao zhi xi bao iNOS、IL-1β、IL-6biao da shui ping ming xian gao yu WTzu 。8.ACMyu shen jing yuan gong pei yang 24hhou ,IL-10tm1/tm1controlzu shen jing yuan bao ti wei su ,tu chu duan lie ,shen jing yuan si wang 。LPSci ji hou ,IL-10tm1/tm1LPSzu shen jing yuan bao ti lie jie ,tu chu xiao shi ,shen jing yuan si wang 。qRT-PCRjie guo ye xian shi ,LPSci ji yu fou ,IL-10tm1/tm1xing xing jiao zhi xi bao Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2jian shao 。9.Aβ42yu IL-10tm1/tm1xing xing jiao zhi xi bao gong pei yang 24h。zai xiang tong shi jian dian ,IL-10tm1/tm1xing xing jiao zhi xi bao bao nei ju ji de Aβ42dan bai ming xian shao yu WTzu 。LPSci ji hou ,xiang dui yu WTzu ,IL-10tm1/tm1xing xing jiao zhi xi bao dui Aβ42de xi shou ming xian xia jiang ,Aβ42dan bai de bu dao you xiao de qing chu 。qRT-PCRshi yan ye biao ming ,yu WTzu xiang bi ,IL-10tm1/tm1xing xing jiao zhi xi bao tun shi shou ti megf10、Mertk、Gas6、Axlbiao da jun xia jiang 。LPSci ji hou ,xiang bi WTzu ,IL-10tm1/tm1xing xing jiao zhi xi bao tun shi shou ti megf10、Gas6、Axlbiao da ye xia jiang di er bu fen :MYSM1dui xing xing jiao zhi xi bao huo hua de ying xiang shi yan fang fa :1.fen li WTxiao shu xing xing jiao zhi xi bao ,qRT-PCRhe Western Blotjian ce LPSci ji hou xing xing jiao zhi xi bao MYSM1de biao da shui ping 。2.fen li WThe MYSM1qiao chu (MYSM1-/-)xiao shu xing xing jiao zhi xi bao ,qRT-PCRjian ce MYSM1de qiao chu xiao lv 。3.qRT-PCRjian ce MYSM1-/-xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Gbp2）/A2zhuai lu yin zi （Tgm1、Ptx3、S100a10）zhuai lu yin zi de shui ping 。4.qRT-PCRjian ce MYSM1-/-xing xing jiao zhi xi bao iNOS、IL-1β、TNF-α、IL-10shui ping 。5.ACMyu shen jing yuan gong pei yang jian ce MYSM1-/-xing xing jiao zhi xi bao dui shen jing yuan de ying xiang ,qRT-PCRjian ce MYSM1-/-xing xing jiao zhi xi bao cu tu chu xing cheng yin zi Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2de biao da 。6.xing xing jiao zhi xi bao yu ying guang biao ji de Aβ42gong pei yang jian ce MYSM1-/-xing xing jiao zhi xi bao de tun shi gong neng ,qRT-PCRjian ce MYSM1-/-xing xing jiao zhi xi bao tun shi shou ti megf10、mertk、Gas6、Axlde biao da 。shi yan jie guo :1.qRT-PCRjie guo he Western Blotjie guo biao ming ,LPSci ji hou xing xing jiao zhi xi bao MYSM1zhuai lu shui ping he dan bai biao da shui ping jiang di 。2.qRT-PCRjie guo biao ming ,MYSM1-/-xiao shu xing xing jiao zhi xi bao MYSM1xian zhe jiang di 。3.qRT-PCRjie guo biao ming ,xiang bi WTzu ,MYSM1-/-xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Gbp2）shui ping zeng jia ;LPSci ji hou ,xiang bi WTzu ,MYSM1-/-xing xing jiao zhi xi bao H2-D1、Gbp2zhuai lu yin zi shui ping zeng jia ,dan ji ta yin zi bian hua bu xian zhe 。4.qRT-PCRjie guo biao ming ,xiang bi WTzu ,MYSM1-/-xing xing jiao zhi xi bao iNOS、IL-1βshui ping sheng gao ,TNF-α、IL-10cha yi bu xian zhe 。LPSci ji hou hou ,xiang bi WTzu ,MYSM1-/-xing xing jiao zhi xi bao iNOS、IL-1βbiao da xian zhe zeng jia ,TNF-α、IL-10bian hua bu xian zhe 。5.ACMyu shen jing yuan gong pei yang 24h。xiang dui yu WTzu ,MYSM1-/-xing xing jiao zhi shang qing yu shen jing yuan gong pei yang zu de shen jing yuan ,bao ti wei su ,tu chu duan lie ,shen jing yuan si wang 。LPSci ji hou ,MYSM1-/-xing xing jiao zhi xi bao shang qing zu ,shen jing yuan bao ti lie jie ,tu chu xiao shi ,shen jing yuan si wang 。qRT-PCRjie guo ye xian shi ,xiang bi WTzu ,LPSci ji yu fou ,MYSM1-/-xing xing jiao zhi xi bao cu tu chu xing cheng dan bai Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2jian shao 。6.jiang Aβ42yu MYSM1-/-xing xing jiao zhi xi bao gong pei yang 24h。zai xiang tong shi jian dian ,MYSM-/-xing xing jiao zhi xi bao bao nei ju ji de Aβ42dan bai ming xian shao yu WTzu 。dan sui zhao shi jian de yan chang ,bao ti nei de ying guang jiang du ye zhu jian zeng jia ,cheng xian shi jian yi lai xing 。LPSci ji hou ,xiang dui yu WTzu ,MYSM-/-xing xing jiao zhi xi bao dui Aβ42de xi shou ming xian xia jiang 。qRT-PCRshi yan biao ming ,yu WTzu xiang bi ,MYSM-/-xing xing jiao zhi xi bao tun shi shou ti megf10、Mertk、Gas6、Axlbiao da jun xia jiang 。LPSci ji hou ,xiang bi WTzu ,MYSM-/-xing xing jiao zhi xi bao tun shi shou ti megf10、Mertk、Gas6、Axlbiao da jun xia jiang 。di san bu fen :MYSM1dui xing xing jiao zhi xi bao huo hua ji zhi de yan jiu shi yan fang fa :1.fen li WTxiao shu xing xing jiao zhi xi bao ,qRT-PCRjian ce LPSci ji hou miR129de biao da shui ping 。2.fen li WThe MYSM1-/-xiao shu xing xing jiao zhi xi bao ,qRT-PCRjian ce miR129biao da shui ping 。3.fen li WTxiao shu xing xing jiao zhi xi bao ,ti wai man bing du zhuai ran gan rao miR129（shmiR129）,qRT-PCRjian ce shmiR129xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Serping1、Gbp2）/A2zhuai lu yin zi （Tgm1、Ptx3、S100a10）shui ping 。4.qRT-PCRjian ce shmiR129xing xing jiao zhi xi bao iNOS、IL-1β、TNF-α、IL-6、IL-10、BDNF、FGF-2de zhuai lu shui ping 。Griessfa jian ce shmiR129xing xing jiao zhi xi bao shang qing NOhan liang 。5.ACMyu shen jing yuan gong pei yang jian ce shmiR129xing xing jiao zhi dui shen jing yuan de ying xiang ,qRT-PCRjian ce shmiR129xing xing jiao zhi xi bao cu tu chu xing cheng yin zi Gpc4、Gpc6、Thbs1、SPARCL1、Thbs2de biao da 。shi yan jie guo :1.qRT-PCRjie guo biao ming ,LPSci ji hou xing xing jiao zhi xi bao miR129biao da jiang di 。2.MYSM1qiao chu hou ,xing xing jiao zhi xi bao miR129biao da jiang di 。3.qRT-PCRjie guo biao ming ,shmiR129xing xing jiao zhi xi bao A1zhuai lu yin zi （H2-T23、H2-D1、Serping1、Gbp2）shui ping zeng jia ,A2zhuai lu yin zi （Tgm1、Ptx3、S100a10）shui ping jiang di 。4.qRT-PCRjie guo biao ming ,shmiR129xing xing jiao zhi xi bao fen bi de xi bao yin zi iNOS、IL-1β、TNF-α、IL-6、IL-10、BDNF、FGF-2mei you xian zhe bian hua 。Griessshi yan jie guo biao ming ,WThe shmiR129xing xing jiao zhi xi bao shang qing NOhan liang ye mei ming xian cha yi 。5.ACMyu shen jing yuan gong pei yang 24hhou ,shmiR129zu shen jing yuan tu chu duan lie huo xiao shi 。qRT-PCRshi yan jie guo biao ming ,yu dui zhao zu xiang bi ,shmiR129xing xing jiao zhi xi bao cu tu chu xing cheng shou ti Gpc4、Gpc6、SPARCL1、Thbs1、Thbs2de biao da mo ming xian bian hua 。jie lun :1.bu tong yin zi dui xing xing jiao zhi xi bao de huo hua de ying xiang bu tong 。100ng/mL LPSwei huo hua xing xing jiao zhi xi bao zui wei ge kuo de yin zi 。jing LPSci ji hou ,xing xing jiao zhi xi bao A1te yi xing zhuai lu yin zi （H2-T23/H2-D1/Serping1/Gbp2）biao da shui ping sheng gao ,yan xing yin zi biao da shui ping sheng gao ,dui shen jing yuan chan sheng xi bao du xing cu tu chu xing cheng dan bai biao da shui ping jiang di ,tun shi Aβ42de neng li jiang di ,tun shi shou ti de biao da shui ping ye jiang di ,zhe di shi LPSci ji hou xing xing jiao zhi xi bao yi yu xiang A1xing huo hua 。2.IL-10di biao da de xing xing jiao zhi xi bao yan zheng le yi shang jie guo 。3.MYSM1qiao chu hou ,xing xing jiao zhi xi bao A1te yi xing zhuai lu yin zi （H2-T23/H2-D1/Gbp2）biao da shui ping sheng gao ,yan xing yin zi biao da shui ping sheng gao ,dui shen jing yuan ying yang zhi chi zuo yong jian ruo ,cu tu chu xing cheng dan bai biao da shui ping jiang di ,tun shi neng li jiang di ,tun shi shou ti de biao da jiang di ,di shi MYSM1qiao chu hou xing xing jiao zhi xi bao yi yu xiang A1xing huo hua 。4.miR129gan rao hou ,xing xing jiao zhi xi bao A1（H2-T23/H2-D1/Serping1/Gbp2）te yi xing zhuai lu yin zi biao da sheng gao ,A2te yi xing zhuai lu yin zi （Tgm1/Ptx3/S100a10）biao da shui ping sheng gao ,cu jin shen jing yuan shen jing yuan cun huo de neng li jiang di ,tun shi neng li jiang di ;MYSM1qiao chu hou miR129biao da shui ping xia jiang ,di shi MYSM1ke neng shi tong guo miR129diao kong xing xing jiao zhi xi bao huo hua ,dan ju ti de diao kong ji zhi hai xu yao jin yi bu shen ru yan jiu 。
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