【作者】 刘颖；

【导师】 汪锡金；

【作者基本信息】 上海交通大学 ， 神经病学， 2020， 硕士

【摘要】 第一部分Smad3缺陷对大鼠行为学和黑质纹状体多巴胺能系统的影响及其机制研究目的:通过建立Smad3抑制剂大鼠模型,观察Smad3抑制剂对大鼠行为学和黑质纹状体多巴胺能系统影响,并探讨其相关作用机制。方法:我们将200-250g SD大鼠随机分成四个组,分别为对照组(Vehicle)、Smad3抑制剂组(SIS3)、脂多糖组(LPS)、联合处理组(SIS3+LPS)。4ug Smad3抑制剂溶于5ul DMSO制备SIS3溶液,使用立体定位仪在SD大鼠双侧黑质区分别给予5ul SIS3处理,一天后腹腔注射溶于生理盐水的1mg/kg脂多糖。在注射脂多糖之后的第7、14、21天进行行为学测试,采用矿场实验和平衡木实验检测大鼠的行为学。在最后一次行为学检测后,采用蛋白免疫印迹法检测黑质和纹状体中的酪氨酸羟化酶(tyrosine hydroxylase,TH)蛋白表达水平,采用免疫组化方法检测大鼠黑质中的TH、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和离子钙接头分子(ionized calcium binding adapter molecule 1,IBA-1),采用蛋白免疫印迹法检测大鼠黑质区白介素-1β(interleukin-1 beta,IL-1β)、白介素-6(interleukin 6,IL-6)和诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)蛋白表达,采用氧化还原试剂盒检测大鼠黑质中活性氧(reactive oxygen species,ROS)生成。结果:本研究中观察到与对照组相比,Smad3抑制剂和脂多糖处理均引起大鼠行为缺陷,黑质纹状体区TH表达明显下降。并且Smad3抑制剂和脂多糖联合处理组相比于对照组和单独处理组,大鼠的行为缺陷增加和黑质和纹状体区TH表达明显下降。同时,与对照组相比,Smad3抑制剂和脂多糖均能诱导大鼠黑质区小胶质细胞活化和促炎因子IL-1β,IL-6,iNOS和ROS的产生。与其他三组相比,Smad3抑制剂和脂多糖联合处理组的大鼠黑质区小胶质细胞活化和促炎因子IL-1β,IL-6,iNOS和ROS的产生显着增强。结论:Smad3抑制剂处理诱导大鼠行为缺陷和黑质纹状体多巴胺能系统紊乱,可能通过促进小胶质细胞炎症反应引起损害作用。第二部分Smad3缺陷对小胶质细胞的影响及其机制研究目的:通过建立Smad3抑制剂小胶质细胞模型,观察Smad3抑制剂对小胶质细胞炎症反应的影响,并探讨其相关作用机制。方法:我们在体外培养新生SD乳鼠(2-3天)小胶质细胞,分为对照组(Vehicle)、Smad3抑制剂组(SIS3)、脂多糖组(LPS)、联合处理组(SIS3+LPS),每个组均添加TGF-β。在小胶质细胞体外培养的第10天,加入Smad3抑制剂SIS3(10μM)。在1小时后加入LPS(300 ng/ml),在1小时后给予TGF-β(10 ng/ml)孵育24小时。采用蛋白免疫印迹法检测小胶质细胞中的白介素-1β(interleukin-1 beta,IL-1β)、白介素-6(interleukin 6,IL-6)和诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)蛋白表达,采用氧化还原试剂盒检测小胶质细胞中活性氧(reactive oxygen species,ROS)生成。采用蛋白免疫印迹法检测小胶质细胞中MAPK通路蛋白表达情况。进一步使用ERK抑制剂和p38抑制剂处理小胶质细胞,观察促炎因子IL-1β、IL-6、iNOS和ROS生成有无变化。结果:与对照组相比,Smad3抑制剂和脂多糖均能显著诱导小胶质细胞促炎因子IL-1β,IL-6,iNOS和ROS的产生。与对照组和单独处理组相比,Smad3抑制剂和脂多糖联合处理使促炎因子IL-1β、IL-6、iNOS和ROS生成显著增加。同时,与对照组相比,Smad3抑制剂和脂多糖均提高ERK磷酸化和p38磷酸化水平。与对照组和单独处理组相比,联合处理组的ERK磷酸化和p38磷酸化水平显著增加。进一步给予ERK抑制剂后,发现Smad3抑制剂和脂多糖联合处理对促炎因子IL-1β、IL-6、iNOS和ROS生成的诱导作用得以抑制。给予p38抑制剂也有类似的结果。结论:Smad3抑制剂和脂多糖联合处理可增强小胶质细胞炎症反应,这可能是通过ERK/MAPK和p38/MAPK通路起作用。第三部分老化相关的黑质区Smad3缺陷对小鼠的影响及其相关性研究目的:观察不同年龄组小鼠黑质区Smad3和pSmad3的表达以及促炎因子的产生水平,探究老化相关的黑质区Smad3表达量与神经炎症的相关性。方法:我们选取C57BL/6小鼠,按不同年龄分为青年组(2-3个月)和老年组(18-20个月),采用蛋白免疫印迹法检测小鼠黑质区和小脑区中Smad3和pSmad3蛋白表达情况。采用蛋白免疫印迹法和Real Time PCR法检测小鼠黑质区中的白介素-1β(interleukin-1 beta,IL-1β)、白介素-6(interleukin 6,IL-6)和诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)表达,采用氧化还原试剂盒检测小鼠黑质区活性氧(reactive oxygen species,ROS)生成。应用相关性分析计算黑质区Smad3和pSmad3表达与促炎因子(IL-1β、IL-6、iNOS和ROS)表达是否具有相关性。结果:本研究发现,与青年组相比,老年组黑质区Smad3和pSmad3蛋白表达显著下降。而青年组和老年组小脑区的Smad3和pSmad3蛋白表达未见明显变化。同时,与青年组相比,老年组黑质区促炎因子IL-1β、IL-6、iNOS(蛋白和m RNA)和ROS生成增加,炎症反应增强。通过相关性分析,Smad3与促炎因子(IL-1β、IL-6、iNOS和ROS)之间存在显着的负相关。同样地,pSmad3与炎症因子(IL-1β、IL-6、iNOS和ROS)之间也有相似的结果。结论:我们的结果表明,老化相关的黑质区Smad3蛋白表达和磷酸化水平的损害与促炎因子增多有关。更多还原

【Abstract】 Part Ⅰ Effect of Smad3 deficiency and mechanisms of action on behavior and nigrostriatal dopaminergic neurons in ratsObjective:To investigate the effect of Smad3 deficiency on behavior and nigrostriatal dopaminergic neurons of rats,and to further investigate its potential mechanisms.Methods:Sprague-Dawley rats(250-300 g)were randomly divided into four groups:Vehicle group,Smad3 inhibitor(SIS3)group,lipopolysaccharide(LPS)group,SIS3+LPS group.Smad3 inhibitor(4 ug)was dissolved in dimethyl sulfoxide(DMSO)and injected to the substantia nigra of SD rats by using a stereotactic device.Same way to inject 4 ug SIS3 on the other side of rats SN,and each rat received total of 8 ug of SIS3 administration.One day later,LPS was dissolved in saline at 1mg/kg and intraperitoneally injected to rats.On the 7th,14th and 21st day after LPS treatment,the behavior of rats were measured by Open field and Beam walk tests.Western blotting was used to examine tyrosine hydroxylase(TH)expression in the substantia nigra and striatum of rats.TH,ionized calcium binding adapter molecule 1(IBA-1)and glial fibrillary acidic protein(GFAP)in the substantia nigra of rats were examined by immunohistochemistry analysis.Western blotting was used to evaluate interleukin-1 beta(IL-1β),interleukin 6(IL-6)and inducible nitric oxide synthase(iNOS)expression in the substantia nigra of rats.The levels of reactive oxygen species(ROS)in the substantia nigra of rats were examined by commercially available kits.Results:In this study,both SIS3 and LPS induced significant behavior deficits in the rats compared with the rats treated with vehicle.Both SIS3and LPS significantly decreased TH expression in the substantia nigra and striatum of rats compared with the rats treated with vehicle.Significant behavior deficits and nigrostriatal dopaminergic neurodegeneration were observed in the rats co-treated with SIS3 and LPS compared with the rats treated with vehicle,SIS3 or LPS.Furthermore,both SIS3 and LPS induced significant microglia activation and pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the rats compared with the rats treated with vehicle.SIS3 and LPS co-treatment induced significant microglia activation and pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the rats compared with the rats treated with vehicle,SIS3 or LPS.Conclusion:Our results indicate that Smad3 inhibitor may induce behavior deficits and nigrostriatal dopaminergic neurodegeneration by inducing microglia-mediated neuroinflammatory responses in rats.Part Ⅱ Effect of Smad3 deficiency and mechanisms of action on inflammation in microgliaObjective:To investigate the effect of Smad3 deficiency on inflammation in microglia,and to further investigate its potential mechanisms.Methods:Primary microglia cultures were separated from the cortical tissues of 2-3 days SD rat pups.They were randomly divided into four groups:Vehicle group,SIS3 group,LPS group,SIS3+LPS group.It was particularly important to add TGF-βto all group.SIS3(10μM)was added on day 10 prior 1 hour after the enriched microglia were cultured.The microglia were exposed to LPS(300ng/ml)for 1 h.Then TGF-β(10ng/ml)was added to the microglial cultures,which were incubated for 24 h.Western blotting was used to examine interleukin-1 beta(IL-1β),interleukin 6(IL-6)and inducible nitric oxide synthase(iNOS)expression in the microglia.The levels of reactive oxygen species(ROS)in microglia were examined by commercially available kits.Western blotting was used to evaluate ERK,p38 and JNK expression in the microglia.We analyzed the effect of ERK inhibitor PD98059 or p38 inhibitor SB203580 on the pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the cultures co-administered with SIS3 and LPS.Results:In this study,both SIS3 and LPS induced significant pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the cultures compared with the cultures treated with vehicle.Co-treatment of SIS3 and LPS significantly increased pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the cultures compared with the cultures treated with vehicle,SIS3 or LPS.Both SIS3 and LPS induced significant increase in the phosphorylation of ERK and p38 in the cultures compared with the cultures treated with vehicle.Co-treatment of SIS3 and LPS significantly increased the phosphorylation of ERK and p38 in the cultures compared with the cultures treated with vehicle,SIS3 or LPS.Both ERK inhibitor PD98059 and p38 inhibitor SB203580 inhibited significant pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production in the cultures co-treated with SIS3 and LPS.Conclusion:Our results indicate that Smad3 inhibitor may enhance inflammation response by through the ERK/MAPK and p38/MAPK pathways in microglia.Part Ⅲ Effect of aging-related Smad3 deficiency in the substantia nigra on mice and their correlationObjective:To investigate the effect of Smad3 and pSmad3 expression in mice SN on inflammation at different ages,and to further investigate its correlation.Methods:Mice were divided into young and aged groups who were aged to 2-3 months and 18-20 months,respectively.We investigated the Smad3and pSmad3 expression in the substantia nigra and cerebellum of mice by western blot analysis.We investigated the expression of interleukin-1 beta(IL-1β),interleukin 6(IL-6)and inducible nitric oxide synthase(iNOS)in the substantia nigra of mice by using western blot and PCR analysis.The levels of reactive oxygen species(ROS)in the substantia nigra of mice were examined by commercially available kits.We investigated the correlation between Smad3 expression and pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production by correlation analysis.We investigated the correlation between pSmad3 expression and pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production by correlation analysis.Results:Our results showed that Smad3 and pSmad3 expression were significantly decreased in the SN of aged mice compared with the young mice.However,no significant difference in the Smad3 and pSmad3expression were observed in cerebellum between the aged mice and young mice.In contrast with the decreased expression of Smad3,the levels of pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production were significantly increased in the SN of aged mice compared with the young mice.There was a significantly inverse correlation between Smad3 and pro-inflammatory factors(IL-1β,IL-6,iNOS and ROS)production by correlation analysis.Similarly,there was a similar result between pSmad3and pro-inflammatory factors production.Conclusion:Our results shown that the amount and phosphorylation of Smad3 impairment were related with pro-inflammatory factors production enhancement.更多还原