【作者】 朱涛；

【导师】 舒强； 方向明；

【作者基本信息】 浙江大学 ， 儿科学， 2013， 博士

【摘要】 脓毒性脑病(sepsis-associated encephalopathy,SAE)是感染后全身炎症反应所致的弥漫性大脑功能障碍和意识改变,是多脏器功能不全综合征(multiple organ dysfunction syndrome,MODS)的重要组成部分及患者预后不良的独立危险因素。SAE的病因和病理机制非常复杂,以白细胞浸润、神经元细胞变性坏死、星型胶质细胞和小胶质细胞激活为标志的炎症反应起到了重要的作用。其中星形胶质细胞在中枢神经系统损伤后呈现保护或毒性作用的“双相”性及活化的时空性特点和SAE的临床表现有很好的契合性。星形细胞激活后出现反应性胶质化(reactive gliosis)、增殖迁移等状态,胞内游离ca2+浓度的升高起了重要作用。星形细胞过度活化可诱发凋亡,其中定位于线粒体参与非Caspase依赖凋亡途径的BNIP3/AIF/EndoG通路介导神经凋亡亦被证实和钙超载有关。瞬时感受器电位M2型(Transient Receptor Potential2Channels,TRPM2)通道是位于细胞膜上的一种多功能钙离子通透性的非选择性阳离子通道,通过调节胞内钙离子浓度可以调节氧化应激或再灌注等病理损伤中的细胞死亡。TRPM2通道很可能参与调节了LPS诱导的星形胶质细胞的活化和凋亡。本研究通过建立LPS腹腔注射致小鼠脑内炎症的模型及引入[RPM2基因敲除小鼠,研究脓毒症在小鼠脑内的炎症改变及星形胶质细胞增殖和胶质化的时间规律,TRPM2在LPS致炎小鼠脑内星形胶质细胞增殖和胶质化的调节作用及参与BNIP3/AIF/EndoG介导LPS致炎小鼠海马区神经细胞的凋亡；以及通过星形胶质细胞培养及TRPM2-siRNA转染,进一步研究LPS是否可以诱导星形胶质细胞的适应性表达,以及TRPM2-siRNA转染是否可以有效抑制LPS诱导的体外星形胶质细胞的活化和致炎性细胞因子TNF-α、IL-1β、IL-6的分泌。从而明确星形胶质细胞TRPM2通道与脓毒性脑病的关系和分子机制,期待为防治脓毒症及脓毒性脑病提供理论依据。目的：研究LPS致炎后野生型和基因敲除(TRPM2)小鼠的一般行为学、脑内炎症改变、星形胶质细胞活化、促炎因子分泌及凋亡的差异；同时研究LPS诱导后星形胶质细胞TRPM2的表达特点及基因沉默后的差异,探讨LPS致炎对小鼠星形胶质细胞的活化影响、诱导TRPM2的适应性表达及TRPM2通道在星形胶质细胞活化中的作用及对神经细胞凋亡的影响。方法：1.动物模型：成年雄性C57BL/6小鼠野生型小鼠分别腹腔注射LPS5mg/kg、10mg/kg、25mg/kg、50mg/kg、75mg/kg、100mg/kg;对照组注入等量生理盐水。监测小鼠体温、神经行为学改变、惊厥发作、摄食改变及死亡率；24h后取脑HE染色观察神经元及胶质细胞病理变化特点和观察脑膜、脑室及脑实质炎症反应；免疫荧光检测小鼠海马区GFAP阳性细胞数量；TUNEL法检测小鼠海马区神经细胞凋亡。2.在体实验：成年雄性C57BL/6小鼠野生型及同种系TRPM2小鼠分别腹腔注射LPS50mg/kg,对照组注入等量生理盐水,监测小鼠一般神经行为学改变、死亡率；并于注射后12h,24h,48h时间点取小鼠脑组织,通过免疫组化、Western blot方法检测LPS致炎后12h,24h,48h时间点野生型及TRPM2小鼠大脑海马区GFAP和TRPM2的表达变化和差异；荧光定量RT-PCR技术检测不同实验组小鼠脑组织TNF-a, IL-1β,IL-6mRNA的表达变化；Western blot方法检测海马BNIP3/AIF/EndoG的蛋白质表达变化。3.离体实验：培养纯化鉴定小鼠星形胶质细胞；MTT法检测不同浓度(LPS: Oμg/ml,1.0μg/ml,3.0μg/ml,5.0μg/ml,7.0μg/ml,10.0μg/ml,12.0μg/ml,15.0μg/ml)及不同时间(2h,6h,12h,24h,2d及3d)LPS刺激后星形胶质细胞存活率；免疫荧光法检测星形胶质细胞GFAP的表达,荧光定量RT-PCR法检测TRPM2-mRNA的表达,Western blot方法检测TRPM2蛋白的表达,ELISA法检测星形细胞培养液细胞因子(TNF-α、IL-1β、IL-6)浓度变化。以75ng TRPM2-siRNA和3μl HiPerFect Transfection Reagent转染星形胶质细胞进行基因沉默,以10.0μg/ml LPS分别诱导细胞0h、24h、48h后,荧光定量RT-PCR技术检测小鼠星形胶质细胞上TRPM2mRNA表达水平的变化,Western blot方法检测TRPM2蛋白表达,ELISA法检测星形胶质细胞培养液细胞因子(TNF-a, IL-1β, IL-6)浓度。结果：1.小鼠LPS腹腔注射后有相应体温改变,出现相应神经行为学改变及惊厥发作、拒食现象以及死亡,均较对照组明显,其中LPS (50mg/kg)腹腔注射组死亡率达到33.3%；LPS致炎后小鼠脑内出现典型炎症性病理改变,TUNEL结果还发现小鼠海马区神经元出现凋亡现象较对照组显著(P<0.05)；随着LPS刺激剂量增加小鼠海马区GFAP阳性细胞数显著增加,细胞胞浆明显增加、突起增多(P<0.05)。2.野生型小鼠海马区GFAP阳性细胞数与TRPM2表达在脓毒症脑损伤后显著增加,LPS致炎野生型(L组)GFAP与TRPM2表达在12h即有增多,24h达到高峰,48h略有减少,均较生理盐水对照组(S组)显著增多(P<0.05)。LPS致炎后TRPM2基因敲除小鼠较野生型病死率增加,一般神经行为学指标改善。TRPM2基因敲除小鼠GFAP表达在脓毒症脑损伤后显著降低,TRPM2基因敲除LPS致炎组(KO/L组)小鼠海马区GFAP阳性细胞较野生型LPS致炎组(L组)显著减少(P<0.05)。小鼠海马区可见GFAP细胞与TRPM2细胞有共聚现象。LPS致炎后24h TRPM2基因敲除较野生型小鼠脑组织TNF-α,IL-1β, IL-6mRNA的表达下降。脓毒症脑损伤后4个实验组小鼠海马区BNIP3(F(3.16)=176.53, P<0.01、AIF (F(3.16)=98.61, P<0.01)\EndoG (F(3,16)=60.55, p<0.01)表达均有显著性差异。BNIP3蛋白质表达L组较S组显著增加(P<0.05),KO/L组较KO/S组(P<0.05)和L组(P<0.05)均显著降低,S组与KO/S组比较无统计学差异(P>0.05)。AIF蛋白质表达L组较S组显著增加(P<0.05),KO/L组较KO/S组(P<0.05)和L组(P<0.05)均显著降低。Endo G蛋白质表达L组较S组显著增加(P<0.05),KO/L组较L组显著降低(P<0.05),S组与KO/S组比较无统计学差异(P>0.05)。3.不同浓度LPS刺激24h后星形胶质细胞存活率差异具有统计学意义(F(7,72)=9.39,P<0.01)；持续刺激24h后10.0μg/ml浓度的LPS是星形胶质细胞增殖活性的临界点。不同刺激时间点的星形胶质细胞存活率差异具有统计学意义(F(1,18)=6.41,P<0.05),早期随着LPS刺激时间的延长,星形胶质细胞增殖活性逐渐增加,且刺激24h时达高峰,在LPS刺激48h以后星形胶质细胞增殖活性明显降低,至72h达到最低。故10.0μg/ml浓度LPS刺激48h是星形胶质细胞从增殖转向凋亡的临界点。LPS不同刺激时间点星形胶质细胞培养液中细胞因子TNF-a (F(1.18)=66.21, P<0.01)、IL-1β(F(1.18)=430.76, P<0.01)、IL-6(F(1.18)=12725.06, P<0.01的浓度变化具有统计学意义。LPS刺激12h后培养液中TNF-a浓度显著上升,6h后IL-1β浓度显著上升,2h后IL-6浓度显著上升。LPS能诱导星形胶质细胞TRPM2mRNA和TRPM2蛋白表达显著增加(P<0.05)。TRPM2-siRNA转染能显著降低星形胶质细胞TRPM2mRNA和TRPM2蛋白表达(P<0.05)。TRPM2-siRNA转染后细胞GFAP蛋白表达显著低于非转染组细胞GFAP蛋白表达(P<0.05)。TRPM2-siRNA转染能抑制LPS诱导星形胶质细胞分泌TNF-a, IL-1β和IL-6。LPS+TRPM2-siRNA组TNF-a, IL-1β和IL-6浓度较LPS组均显著降低(P<0.05)。结论：1.小鼠LPS (50mg/kg)腹腔注射建模可诱发明显的脑内炎症和神经行为学改变,并最接近临床脓毒症标准。2.LPS可诱导星形胶质细胞增殖、胶质化及凋亡,并在时间上有一定的规律性；LPS可诱导星形胶质细胞TRPM2-mRNA和TRPM2蛋白的表达,这种表达在星形胶质细胞属于适应性表达；TRPM2通道参与LPS致炎小鼠脑内星形胶质细胞增殖和胶质化调节、星形胶质细胞致炎性细胞因子TNF-α、IL-1β、IL-6分泌调节及可能参与了BNIP3/AIF/EndoG介导的LPS致炎小鼠海马区神经细胞凋亡。3.PRPM2-siRNA转染基因沉默后可以有效抑制LPS诱导的体外星形胶质细胞TRPM2-mRNA和TRPM2蛋白的表达；抑制LPS诱导的体外星形胶质细胞的活化和致炎性细胞因子TNF-α、IL-1β、IL-6的分泌。更多还原

【Abstract】 Sepsis associated encephalopathy (SAE) is a diffuse cerebral dysfunction resulted by systemic inflammatory response.SAE is the most common form of encephalopathy in critical ill patients with poor prognosis. The pathogenesis of SAE is very complex and the local inflammation play an important role with leukocyte infiltration, degenerated and necrotic neurons, activation of astrocyte and microglia. Clinical and experimental studies have shown significant associations between biphasic activation of astrocyte and clinical feature. Reactive gliosis, proliferation and migration are a characteristic response of astrocytes to inflammation and trauma of the central nervous system resulted by increase of intracellular calcium concentration [Ca2+];. Caspase-independent cell death in CNS cells and activation of BNIP3/AIF/Endo G pathway have been proved associations with calcium overload. Biphasic effect of lipopolysaccharide can lead to the astrocytes activation or apoptosis, and activation of astrocytes on neuronal protection and toxicity. Calcium homeostasis and abnormal calcium signaling may be a key factor in the activation of astrocytes. Transient receptor potential melastatin2(TRPM2) is a Ca2+-permeable ion channel of the melastatin-related TRP channels. TRPM2may participate into the regulation of activation and apoptosis of astrocytes. Thus, in order to investigate the inflamation in CNS and time law of astrocyte activation and apoptosis, we developed a model of lipopolysaccharide (LPS) induced sepsis in mouse to observe hippocampus dentate gyrus neuron apoptosis and activation of astrocytes in the LPS induced brain damage. Further, the mechanism of TRPM2pathway in LPS induced brain damage will be demonstrated. Both in vitro and in vivo data point to the role of astrocytes as both major source and target of LPS induced brain damage signaling. In this study, understanding the astroglial inflammatory response occurring in brain tissue may provide new strategies for targeting astrocyte-mediated LPS induced brain damage. Inhibition of cell TRPM2channel activation may improve the survival rate of astrocytes. Taken together, the present research will provide new targets for the future treatment and prevention of SAE.Objective:To study the changes characteristics of neurological behavior, cerebral inflammatory, activation of astrocytes and production of proinflammatory cytokines between C57BL/6wild-type and TRPM2-/-mice, and to investigate the characteristics of astrocyte TRPM2after LPS stimulated, and the expression of TRPM2mRNA and protein, cytokine (TNF-α, IL-1β, IL-6), BNIP3, AIF, Endo G after infection/inflammation, and to study the effect of TRPM2-siRNA to inhibit astrocyte proliferation and astrogliosis.Methods:1. Animal model:All C57BL/6male adult mice were intraperitoneal injected with5mg/kg、10mg/kg、25mg/kg、50mg/kg、75mg/kg、100mg/kg LPS and control group were injected with equal amount saline solution instead. Investigate the change of body temperature, neurological behavior, convulsive seizures, feeding behavior and mortality before and after LPS intraperitoneal injection. After24h, the mice brains were immediately collected and were studied by pathological examination. Histological characteristics of the cerebral inflammation of the mice were studied by hematoxylin-eosin (HE) staining and immunohistochemistry was used for evaluation of GFAP expression and nerve cells apoptosis were detected by TUNEL method.2. In vivo experiments:C57BL/6wild-type and TRPM2mice were intraperitoneal injected with50mg/kg LPS. After12h,24h,48h, the mice brains were immediately collected and were studied by immunohistochemistry, Western blot method to study the difference in levels of GFAP,TRPM2and BNIP3/AIF/EndoG proteins expression in any brain regions were found at12h,24h,48h between the two groups.3. In vitro experiment:MTT method were used for evaluation of survival rate of astrocytes after incubated for different times(2h,6h,12h,24h,2d and3d) with different concentrations (0μg/ml,1.0μg/ml,3.0μg/ml,5.0μg/ml,7.0μg/ml,10.0μg/ml,12.0μg/ml,15.0μg/ml) of LPS, and immunofluorescence methods were used for evaluation of GFAP expression after incubated with LPS, and the concentrations of cytokines(TNF-α, IL-1β, IL-6) in the samples of culture medium were analyzed by Enzyme-linked immunosorbent assays (ELISA). Further, real-time quantitative RT-PCR was used to analyze TRPM2-mRNA, and Western blot method was used to analyze TRPM2proteins of astrocytes after incubated with LPS.4. TRPM2-siRNA transfection:After PTGS (post-transcriptionalgene silencing) with TRPM2-siRNA and exposed to LPS, immunofluorescence methods were used for evaluation of GFAP expression, and the concentrations of cytokines (TNF-α, IL-1β, IL-6) in the samples of culture medium were analyzed by Enzyme-linked immunosorbent assays (ELISA).Results:1. Mice with LPS intraperitoneal injection were showed body temperature increase, abnormal neurological and feeding behavior, convulsive seizines, and33.3%mortality (50mg/kg LPS). Inflammation of the brain was induced after LPS intraperitoneal injection, but all cases of the control group had no histologic evidence of sepsis. The nerve cells apoptosis were detected significantly increase (P<0.05) compared with the control group after LPS injection by TUNEL methods.2. Compared with the control group, the numbers of GFAP+cells were significantly increased after exposed for12h with LPS (10μg/ml)(P<0.05), and close to its peak at24h, and the numbers of GFAP+cells were slightly decreased after exposed for48h with LPS (P<0.05).Compared with the control group, the expression of TRPM2mRNA and TRPM2protein in astrocytes were significantly increased after exposed for12h,24h and72h with LPS (10μg/ml)(P<0.05). Compared with the wild-type mice, TRPM2mice were detected significantly decreased in CNS inflammation and the numbers of GFAP+cells.Compared with the control group, the expression of BNIP3(F(3,16)=176.53, P<0.01), AIF (F(3,16)=98.61, P<0.01), EndoG (F(3,16)=60.55, P<0.01) proteins in hippocampus regions were significantly increased of LPS-induced group (L group). However, the expression of BNIP3, AIF and Endo G of KO/L group were significantly decreased compared with KO/S group and L group (P<0.05).3. Compared with the control group, the survival rate of astrocytes after incubated for different times(2h,6h,12h,24h,2d and3d)(F(7)72)=9.39, P<0.01)with different concentrations (Oμg/ml,1.0μg/ml,3.0μg/ml,5.0μg/ml,7.0μg/ml,10.0μg/ml,12.0μg/ml,15.0μg/ml)(F(1,18)=6.41, P<0.05) of LPS were significantly difference. As the concentration of LPS increased, the numbers of GFAP+cells were significantly increased after exposed for12h with LPS (10μg/ml)(P<0.05), and close to its peak at24h, and the numbers of GFAP+cells were slightly decreased after exposed for48h with LPS, closed to the trough for72h (P<0.05).Compared with the control group, the concentrations of cytokines (TNF-α, IL-1β, IL-6) in the samples of culture medium exposed for LPS were significantly difference(TNF-a:F(1.18)=66.21, P<0.01),(IL-Iβ:F(1.18)=430.76, P<0.01),(IL-6: F(1,18)=12725.06, P<0.01). Compared with the control group, the expression of TRPM2mRNA and TRPM2protein in astrocytes were significantly increased after exposed for LPS (P<0.05).After PTGS with TRPM2-siRNA, the expression of TRPM2mRNA and TRPM2protein in astrocytes were significantly decreased after exposed for LPS (P<0.05), the expression of GFAP in astrocytes decreased (P<0.05),and he concentrations of cytokines (TNF-a, IL-1β, IL-6) in the samples of culture medium exposed for LPS were significantly decreased (P<0.05).Conclusion:1. Mice with LPS intraperitoneal injection (50mg/kg) might sepsis mouse model with CNS inflamation.2. LPS could increase astrocyte activation, the expression of TRPM2and the concentrations of cytokines (TNF-a, IL-1p, IL-6). TRPM2protein expression in astrocyte is adaptive expression. TRPM2might play the important role on BNIP3/AIF/Endo G signalling pathway in LPS-induced nerve cells apoptosis of astrocvtes.3. TRPM2-siRNA transfection could inhibited the effect LPS on astrocytes. These results suggested that TRPM2channel might play important roles in the process of astrocyte activation.更多还原