【作者】 李学忠；

【导师】 刘春风；

【作者基本信息】 苏州大学 ， 神经病学， 2008， 博士

【摘要】 第一部分脂多糖通过上调Toll样受体表达、激活MAPK通路和NF-κB通路,介导星形胶质细胞的生长双重性目的:研究不同浓度脂多糖作用于星形胶质细胞不同时间后星形胶质细胞的生长特性,脂多糖受体表达情况,星形胶质细胞内部信号通路激活情况。方法:在原代培养的星形胶质细胞中加入不同浓度的脂多糖分别作用不同时间,观察星形胶质细胞生长的情况,MAPK通路抑制剂和NF-κB通路抑制剂对星形胶质细胞生长的影响。同时以不同浓度的LPS作用于星形胶质细胞24h后,观察随后8d星形胶质细胞生长的变化。通过RT-PCR、Western-blot、免疫细胞化学的方法观察星形胶质细胞Toll样受体的基因水平、蛋白水平和细胞水平的表达。Western-blot检测星形胶质细胞细胞内转录因子p65和P-p38的表达情况,观察MAPK通路抑制剂对P-p38表达及NF-κB通路抑制剂对p65表达的影响。结果:只有在作用24h后,低剂量的脂多糖才可使星形胶质细胞的生长加快,高剂量的脂多糖则可抑制星形胶质细胞的增殖。以≤10mg/L的脂多糖作用于星形胶质细胞24h后,短期内都可促进星形胶质细胞的生长,长期则抑制星形胶质细胞生长,脂多糖浓度越大,抑制细胞生长的能力越明显。NF-κB通路抑制剂、MAPK通路抑制剂皆可阻断脂多糖对星形胶质细胞生长的影响。在正常状况下,星形胶质细胞在胞浆和胞膜表达大量的TLR3受体,很少的TLR4受体。在脂多糖的刺激下,星形胶质细胞的TLR3受体表达保持不变,TLR4受体的表达随予以脂多糖的浓度增加而增高。予以10mg/L脂多糖作用于星形胶质细胞不同时间,我们发现星形胶质细胞内各组皆有P-p38表达,基础水平为内参GAPDH的0.176±0.021倍,30min后明显升高,24h后达到高峰,达GAPDH的0.435±0.044倍,随后逐渐减弱,48h组和72h组仅为GAPDH的0.287±0.028倍。预先加用MAPK通路抑制剂PD98059可下调P-p38的表达。同样,p65蛋白表达高峰出现在24h,随后下降, NF-κB通路抑制剂可阻断p65蛋白的升高。结论:低剂量的脂多糖可促进星形胶质细胞生长,高剂量时则抑制星形胶质细胞生长,而炎症刺激对星形胶质细胞的长期影响是对细胞的生长增殖起抑制作用。星形胶质细胞Toll样受体的表达是不同源的,TLR4随环境的变化而改变。其分子机制可能与NF-κB通路、MAPK通路的激活有关。第二部分星形胶质细胞在脂多糖诱导的多巴胺能神经元损伤中起双重作用目的:(1)建立含有高比例多巴胺能神经元的神经元培养体系。(2)建立星形胶质细胞和神经元的共培养体系。(3)研究星形胶质细胞在不同浓度脂多糖诱导的多巴胺能神经元损伤中的作用。方法:利用L-抗坏血酸-2-磷酸酯倍半镁盐和人成纤维生长因子促进孕12d的SD大鼠中脑前体细胞扩增、分化为多巴胺能神经元,通过阿糖胞苷抑制胶质细胞的增殖,建立高比例多巴胺能神经元的神经元培养体系。原代培养纯化的星形胶质细胞,使用终浓度为0、0.1、10mg/L脂多糖分别作用于星形胶质细胞24h,收集细胞上清液,高速离心,去除杂质,获得星形胶质细胞条件培养液(ACM)。在此基础上,利用transwell双室培养系统建立星形胶质细胞和神经元共培养体系,观察脂多糖、星形胶质细胞条件培养液和星形胶质细胞对神经元培养体系中多巴胺能神经元存活及酪氨酸羟化酶mRNA表达的影响。结果:脂多糖对神经元体系中的多巴胺能神经元有损伤作用,并呈剂量依赖性。同单用脂多糖相比,条件培养液显著促进多巴胺能神经元的存活,其中0.1mg/L脂多糖处理的ACM组多巴胺能神经元的存活能力最强,其次为10mg/L组,最后为0mg/L组。同条件培养液组相比,共培养体系中多巴胺能神经元存活能力更高,其中0.1mg/L脂多糖干预后多巴胺能神经元的存活能力最强,随后依次为10mg/L、0mg/L、20mg/L脂多糖。多巴胺能神经元表达酪氨酸羟化酶mRNA的变化类似多巴胺能神经元数量的变化。结论:脂多糖对多巴胺能神经元有损伤作用,并呈剂量依赖性,适度激活的星形胶质细胞对多巴胺能神经元有保护作用,过度激活则削弱了这种保护作用,星形胶质细胞和多巴胺能神经元之间的对话可进一步增加多巴胺能神经元的存活。第三部分脂多糖刺激的星形胶质细胞可能通过IL-6介导PC12细胞增殖双重性目的:研究脂多糖刺激的星形胶质细胞条件培养液对PC12细胞生长的影响及其可能的因子。方法:在原代培养的星形胶质细胞中加入不同浓度的脂多糖作用24h,收集条件培养液,用不同分子量的过滤单位(Ultrafree MC filter units)将培养液进行分层处理,观察不同分子量范围的星形胶质细胞条件培养液对PC12细胞生长的影响,同时对星形胶质细胞的条件培养液中的细胞因子进行分析,观察以不同浓度的脂多糖作用24h后,星形胶质细胞分泌的细胞因子的变化。观察重组IL-6和加用IL-6中和抗体后的星形胶质细胞条件培养液对PC12细胞生长的影响。最后在含高比例多巴胺能神经元的神经元培养体系中,加用添加IL-6中和抗体的星形胶质细胞条件培养液,观察他们对多巴胺能神经元存活及酪氨酸羟化酶mRNA表达的影响。结果:星形胶质细胞条件培养液可促进PC12细胞的增殖,低剂量的脂多糖刺激的星形胶质细胞条件培养液可增强星形胶质细胞对PC12细胞的增殖作用,高剂量的脂多糖则可削弱星形胶质细胞对PC12细胞的影响。5-30KDa分子量范围的星形胶质细胞条件培养液对PC12细胞的影响最大。以脂多糖作用星形胶质细胞24 h后,脂多糖可促进星形胶质细胞分泌IL-6,并呈剂量依赖性,GDNF、TNF-α、NO的分泌影响不大,未达统计性差异。低剂量脂多糖促进GSH合成、上调GPx活性,高剂量则起相反作用,重组IL-6可模拟星形胶质细胞条件培养液的作用,IL-6中和抗体可阻断星形胶质细胞条件培养液的这种效应。IL-6中和抗体加入到ACM中,消除ACM中IL-6的作用后,0、0.1mg/L脂多糖处理组多巴胺能神经元的数量下降,细胞突起变短,暗淡,但10mg/L脂多糖处理组多巴胺能神经元的数量没有下降,突起较未加IL-6中和抗体前延长、明亮,并且消除了星形胶质细胞对多巴胺能神经元的双重作用。细胞中酪氨酸羟化酶mRNA的变化类似于细胞数量的变化。结论:星形胶质细胞可促进PC12细胞的活存和增殖,星形胶质细胞的适度激活上调星形胶质细胞对PC12细胞的保护作用,而过度激活则削弱了星形胶质细胞的保护作用。不是通过调节GDNF的合成,而是诱导致炎因子的变化、抗氧化分子的多寡介导星形胶质细胞的双重性,IL-6可能是关键的因素之一。更多还原

【Abstract】 Part I Lipopolysaccharide exerts a dual effect on the growth of astrocytes by upregulating the expression of toll-like receptors and activating MAPK and NF-κB pathwayObjectives: The purposes were to investigate the changes and probable mechanisms of growth of astrocytes administrated with lipopolysaccharide (LPS), the expression of toll-like receptors.Methods: LPS was administrated to rat astrocytes for 60min, 6h, 24h and 48h, and the changes of growth of astrocytes were detected by MTT method. Meanwhile, the long-term changes of growth of astrocytes after administration with LPS for 24h were also studied. Moreover, the effects of inhibitor of NF-κB (SN50) and MAPK pathway (PD98059) on proliferation of astrocytes were observed. The expression of toll-like receptors in astrocytes was detected by immunocytochemistry, western blotting and RT-PCR. NF-κB p65 and phospho-p38 proteins expressed in astrocytes were assayed with western blotting.Results: Changes of astrocytes growth were observed only when LPS had been administrated for 24h. LPS of low concentration could promote proliferation of astrocytes and increase the cell viability(P<0.05 vs control), while LPS of high concentration inhibited the proliferation of astrocytes. Being administrated for 24h, LPS could promote the proliferation of astrocytes in short-term. As for the long-term effects, LPS inhibited the proliferation of astrocytes in a concentration-dependent manner. Pretreatment with SN50 or PD98059 could block the effects of LPS on astrocytes. Astrocytes expressed both cell surface and intracellular TLR3, low-level TLR4 in normal circumstance. LPS increased the expression of TLR4 of astrocytes in a concentration-dependent manner, while the expression of TLR3 kept constant. P-p38 and p65 protein expressed most intensely in 24h group, and decreased subsequently. Pretreatment with PD98059 (inhibitor of MAPK) or SN50 (inhibitor of NF-κB) could block the effect.Conclusion: LPS of low concentration could promote the proliferation of astrocytes in short-term and LPS of high concentration could inhibit it. The expression of toll-like receptors in astrocytes was not homogeneous but rather tailored environmental signal. The molecular mechanism of the changes may be related to the activation of pathway of MAPK and NF-κB.Part II Astrocytes exerted a dual effect on the damage of dopaminergic neurons induced by LipopolysaccharideObjectives: The objectives were to investigate the role of astrocytes in the lipopolysaccharide- induced damage of dopaminergic neurons.Methods: After lipopolysaccharide was applied to the third generation of rat astrocytes for 24h, supernatants of astrocytes were collected. We obtained primary mesencephalic dopaminergic neuron-enriched culture systems by neurobasal and ara-c and established coculture system of both astrocytes and neurons by transwell inserts. We administrated lipopolysaccharide into neuron-enriched systems and coculture systems and detected the change of dopaminergic neurons. At the same time, the supernatants of astrocytes were administrated into the neuron-enriched systems, and the survival of dopaminergic neurons and the expression of tyrosine hydroxylase mRNA were observed.Results: Lipopolysaccharide had a negative effect on the survival of dopaminergic neurons concentration-dependently. Both astrocytes and supernatants of astrocytes promoted the survival of dopaminergic neurons, and the former was better than the latter. In the preoccupation of existence of astrocytes, low-concentration lipopolysaccharide promoted the survival of dopaminergic neurons, while high-concentration decreased. The change of the expression of tyrosine hydroxylase mRNA was similar to the survival of dopaminergic neurons. Conclusions: Astrocytes played a protective role in the damage of dopaminergic neurons induced by lipopolysaccharide, and suitable activation of astrocytes could increase the protective effect while excessive activation of astrocytes could attenuate the effect.Part III IL-6 could medicated the dual effect of astrocytes administrated with lipopolysaccharide on the growth of PC12 by IL-6Objectives: After PC12 cells were treated with astrocyte conditioned medium (ACM) which was dealt with LPS, the changes of growth and the probable mechanisms were investigated.Methods: Firstly, the astrocyte conditioned medium (ACM) was obtained after astrocytes were administrated with lipopolysaccharides (LPS) over 24h. Then, the ACM was stratified by ultrafree MC filter units. Subsequently, the changes of the growth of PC12 cells cultivated with the ACM were detected by MTT method. The cytokines secreted by astrocytes after administration with LPS for 24h were also studied. Finally, the effect of the ACM pretreated with recombinant rat IL-6 or rabbit anti-rat IL-6 monoclone antibody on the growth of PC12 cells was observed. In addition, ACM added IL-6 neutralizing antibody was administrated into the neuron-enriched systems, and the survival of dopaminergic neurons and the expression of TH mRNA were analyzed.Results: ACM treated with LPS of low concentration could promote proliferation of PC12 and increase the cell viability(P<0.05 vs control), while ACM treated with LPS of high concentration attenuated the proliferation of PC12. The ACM of molecular weight from 5 to 30KDa had a biggest effect on the proliferation of PC12. Being administrated for 24h, LPS could promote the astrocytes to secrete IL-6 in a concentration-dependent manner, while the secretions of GDNF, TNF-α, NO had no significant changes. Low concentration in LPS could up-regulated the production of GSH and the activity of GPx, but high concentration down-regulated. Recombinant rat IL-6 could simulate the change, while rabbit anti-rat IL-6 monoclone antibody could block the effect. Anti-rat IL-6 neutralizing antibody itself had no effect on the survival of mesencephalic dopaminergic neurons, but significantly changed the effect of ACM. ACM administrated with IL-6-neutralizing antibody promoted the survival of dopaminergic neurons dose-independently, but significantly lower than that of ACM. Moreover, after ACM was administrated with IL-6-neutralizing antibody, dual effect of ACM disappeared.Conclusion: Astrocytes played a protective role on PC12 cells, and suitable activation of astrocytes increased the proliferation while excessive activation decreased. It is production of IL-6 mainly, not secretion of GDNF, that medicated astrocytic dualism.更多还原