几种激素和IL—1β对人脑星形胶质细胞IL—6、TNF—α

目的：观察T3等因素对体培养人胎大脑星形胶质细胞分泌IL－6、TNF－α的调节作用。方法：纯化培养人胎大脑星形胶质细胞,应用酶联免疫分析ELISA）方法检测培养上清液中IL－6、TNF－α的水平。结果：（1）星形胶质细胞（AC）在体外培养条件下可自发分泌IL－6、而TNF－α则几乎检测不到。（2）LPS（0．1μg/mL)即可诱导AC产生IL－6,TNF－α。（3）IL－1β是IL－6分泌的主要诱导剂,但不诱导TNF－α分泌。（4）氢化可的松可明显抑制AC分泌IL－6、TNF－α。（5）T3在72h可刺激IL－6的分泌。（6）胰岛素对IL-6的分泌没有明显调节作用,结论：AC可通过分泌细胞因子与炎症反应等病理过程并维持中枢神经系统的正常发育、内环境的稳定,且受多种因素的调节,在中枢神经系统中T3、胰岛素主要参与调节发育和代谢,可能不直接参与炎症和免疫机制调节。     

几种激素和IL-1β对人脑星形胶质细胞IL-6 、TNF-α分泌的影响

目的 观察T3等因素对体外培养人胎大脑星形胶质细胞分泌IL-6、TNF-α的调节作用。方法 纯化培养人胎大脑星形胶质细胞,应用酶联免疫分析(ELISA)方法检测培养上清液中IL-6、TNF-α的水平。结果 (1)星形胶质细胞(AC)在体外培养条件下可自发分泌IL-6,而TNF-α则几乎检测不到。(2)LPS(0.1μg/mL)即可诱导AC产生IL-6和TNF-α。(3)IL-1β是IL-6分泌的主要诱导剂,但不诱导TNF-α分泌。(4)氢化可的松可明显抑制AC分泌IL-6、TNF-α。(5)T3在72h可刺激IL-6的分泌。(6)胰岛素对IL-6的分泌没有明显的调节作用。结论 AC可通过分泌细胞因子参与炎症反应等病理过程并维持中枢神经系统的正常发育、内环境的稳定,且受多种因素的调节。在中枢神经系统中T3、胰岛素主要参与调节发育和代谢,可能不直接参与炎症和免疫机制调节。     

TGF—β1基因转染对未成熟树突状细胞表型和功能的影响

目的探讨转化生长因子（TGF）-β1基因转染对未成熟树突状细胞（imDC）细胞表型和功能的影响。方法分别从Wistar大鼠骨髓培养imDC及成熟树突状细胞（mDC），用TGF-β1-pcDNA3质粒转染imDC，透射电镜观察细胞超微结构，流式细胞术检测其细胞表型，ELISA法检测IL-10、IL-12、TGF-β1水平，观察其对脂多糖（LPS）刺激的反应，混合淋巴细胞反应比较其在体外刺激抗原特异性T淋巴细胞增殖的能力。结果TGF-β1-imDC在LPS刺激后仍能保持未成熟的细胞形态，表面抗原CD86，CD80，CD40及MHC Ⅱ表达都有明显下降（P〈0．01），分泌TGF-β-1水平为（793．82±206．38）ng／L，明显高于imDC和mDC（P〈0．01），分泌IL-10、IL-12明显低于mDC（P〈0．01），而与imDC无显著差异（P〉0．05），LPS刺激TGF-β1-imDC 24h后分泌IL-10、IL-12的能力显著低于imDC（P〈0．01），在体外刺激抗原特异性T淋巴细胞增殖的能力明显低于imDC和mDC（P〈0．01）。结论TGF-β1-imDC的表面抗原表达明显下降，抗原递呈能力降低，分泌免疫抑制因子增加，能够维持细胞于未成熟状态。     

激光扫描共聚焦显微镜测定培养神经细胞脂氢过氧化物含量

目的探讨一种既可定量又可动态测定培养神经细胞内脂氢过氧化物(LPO)含量变化的新方法。方法应用激光扫描共聚焦显微镜测定β-淀粉样蛋白(AB1-42)对PC12细胞内LPO含量，及星形胶质细胞条件培养液(ACM)对叔丁基脂氢过氧化物(tbOOH)引发的PC12细胞内LPO动态变化的影响。结果观察到Aβ1-42诱导了PC12细胞LPO含量的增加，ACM可抑制tbOOH引发的LPO上升。结论利用激光扫描共聚焦显微镜既可用定量测定又可动态观察培养活神经细胞内LPO含量变化。     

NOGO—A在PC12细胞分化过程中的表达及意义

目的探讨NOGO—A在PC12细胞生长发育过程中的表达及意义。方法培养大鼠嗜铬细胞瘤细胞系PC12细胞，用神经生长因子诱导其分化，并于倒置显微镜下随机取20视野计数观察细胞增值和轴突生长情况。采用免疫荧光染色、逆转录酶聚合酶链反应（RT—PCR）及免疫印迹法等方法检测诱导后第1d、第3d、第5d、第7d PC12细胞中NOGO—AmRNA及蛋白的表达及变化，并留取细胞培养液检测多巴胺水平。结果未分化的PC12细胞中未检测到NOGO—A mRNA及蛋白表达。经神经生长因子诱导的PC12细胞，细胞轴突不断生长，NOGO—AmRNA及蛋白的表达逐渐增高（P〈0．05）。PC12细胞在分化过程中多巴胺（DA）分泌水平无明显差别。结论PC12细胞向交感神经元分化的过程中NOGO—A的表达逐渐增强，推测NOGO—A在神经元发育早期可能促进轴突生长。但对多巴胺激素释放的调节不明显。     

白细胞介素-2基因佐剂对单纯疱疹病毒糖蛋白D DNA疫苗的免疫增强作用

目的探讨自细胞介素-2（IL-2）基因佐剂对单纯疱疹病毒Ⅰ型（HSV—Ⅰ）糖蛋白D（gD）DNA疫苗的免疫调节作用。方法将表达HSV—Ⅰ gD基因的DNA疫苗质粒IRES-gD以及HSV-Ⅰ gD与IL-2基因共表达的重组质粒IRES-gD-IL-2分别注入BALB／c鼠股四头肌，检测特异性抗体和中和抗体的产生情况，并于第3次注射后14d用滴度100组织培养半数感染量（TCID50）的HSV—Ⅰ病毒感染小鼠，观察小鼠的生存情况。结果IRES-gD组和IRES-gD-IL-2组均可刺激小鼠产生抗HSV—Ⅰ gD的特异性抗体和中和抗体，抗体滴度随免疫次数增加而升高IRES-g D-IL-2组小鼠产生的抗体及中和抗体滴度明显高于IRES-gD组，两组小鼠的生存率差异无统计学意义。绪论IL-2基因佐剂可促进HSV—Ⅰ gD DNA疫苗诱导抗体的产生，并具有免疫增强作用。     

神昌注射液对谷氨酸诱导损伤PC12细胞的保护作用

目的探讨神昌注射液对谷氨酸（Glu）引起PC12细胞损伤的保护作用。方法细胞培养完成后随机分为5组;每组12份,除正常组外其余四组均用10mmol/L Glu作用于PC12细胞制成细胞损伤模型,留12份作对照,另三组给予不同剂量神昌注射液观察神昌注射液对其细胞形态、存活力、细胞凋亡率和IL-6、IL-8水平的影响。结果神昌注射液可有效改善谷氨酸诱导的PC12细胞的形态损伤,提高细胞存活力、降低细胞凋亡率及细胞因子IL-6、IL-8的水平,其中以中、高剂量更明显。结论神昌注射液对谷氨酸所致PC12细胞损伤具有保护作用,以中剂量组（19.4ul/ml）效果最优。机制可能与其能提高细胞存活力及抑制凋亡、降低IL-6、IL-8水平有关。     

白细胞介素-6与脑胶质瘤关系的研究

目的:通过观察人脑胶质瘤细胞是否可以自发分泌白细胞介素-6(interleukin-6,IL-6),为进一步探讨IL-6在脑胶质瘤发生、发展中的可能作用提供线索。方法:对4例脑胶质瘤病人的手术标本进行原代细胞培养,分不同时相收获上清,并对其上清进行IL-6活性检测。结果:各组上清中均有IL-6活性,IL-6生物活性以D_(0～6)上清活性最高(1.131U/L),D_(0～6)上清活性最低(0.18U/L)。人脑胶质瘤细胞受细菌脂多糖刺激后,分泌IL-6的能力略有增强。IL-6单克隆抗体可中和脑胶质瘤上清对B9.9细胞的增殖作用。结论:原代培养的人脑胶质瘤细胞可自发分泌IL-6。     

栀子甙在炎症诱导的多巴胺能神经元损伤中的作用及机制

目的 研究栀子甙在脂多糖(LPS)介导的星形胶质细胞(AC)过度激活时对多巴胺能神经元保护作用的影响及可能机制.方法 建立高纯度多巴胺能神经元培养体系、多巴胺能神经元和AC混合培养体系并分别经栀子甙预处理后再予以LPS作用24 h,同时设立对照组,观察多巴胺能神经元的生存率,TH mRNA的表达及培养基中TNF-α、NO、IL-6、基质金属蛋白酶-9(MMP-9)和胶质源性神经生长因子(GDNF)含量的变化.结果 AC促进多巴胺能神经元的存活.栀子甙不能增加高纯度多巴胺能神经元培养体系中多巴胺能神经元的存活率,却可剂量依赖地增加混合培养体系中多巴胺能神经元存活率,同对照组相比,40 mg/L组增加细胞存活率从203.0%±17.4%升高到256.7%±15.2%(F=17.22,P=0.001).LPS作用24 h后混合培养体系中TNF-α、NO、GDNF含量并无显著变化,IL-6、MMP-9含量显著上升,栀子甙可明显下调这种反应.同对照组相比,40 mg/L组IL-6含量下降到原来的67.2%±6.4%(F=12.89,P=0.001),MMP-9下降到原来的77.3%±9.8%(F=8.27,P=0.001).结论 LPS通过过度激活AC而增加炎性因子的分泌,从而削弱其对多巴胺能神经元的保护作用,栀子甙通过抑制AC分泌炎性介质,不增加GDNF的分泌,上调AC对多巴胺能神经元的保护作用.     

布美他尼对LPS诱导的小胶质细胞活化及炎性分泌的影响

目的观察钠-钾-氯共转运体(Na+-K+-2Cl-cotransporter 1,NKCC1)的特异性抑制剂布美他尼(Bumetanide)对LPS(脂多糖)诱导的小胶质细胞活化及炎性因子分泌的影响。方法差速贴壁法培养大鼠原代小胶质细胞,纯化的小胶质细胞分为对照组,LPS组(1.0μg/ml)和布美他尼干预组(NKCC1抑制剂,10μM),在干预1、3、6和12 h固定细胞爬片,免疫荧光双标显示小胶质细胞活化形态;各时间点收取上层培养基,用ELISA法测定各组培养基中TNF-α和IL-1β的水平。结果 LPS干预后小胶质细胞活化,体积增大,布美他尼干预后小胶质细胞活化受到抑制。小胶质细胞分泌的TNF-α和IL-1β在LPS干预1 h时开始明显增加,TNF-α的分泌在6 h达到最高峰;IL-1β的分泌在3 h时达到最高峰(P0.05)。布美他尼干预后与LPS组比较,TNF-α和IL-1β分泌明显减少,其中在3、6、12 h时TNF-α分泌显著降低(P0.05),而IL-1β的分泌在1、3、6、12 h时明显降低(P0.05)。结论布美他尼可减少LPS诱导的小胶质细胞活化及炎性因子分泌增加,提示NKCC1通路在小胶质细胞活化及炎性因子分泌中发挥了重要作用。     

咪唑克生对脂多糖诱导的皮质星形胶质细胞相关炎症细胞因子的影响

目的：了解咪唑克生（Ida）对炎症状态下的星形胶质细胞相关的炎症细胞因子的调节作用。方法：将纯度〉95％星形胶质细胞随机分为空白对照组、脂多糖刺激组和Ida炎症干预组，每组7瓶细胞。Ida 100μmol·L^-1，脂多糖10μg·mL^-1。Ida预处理6h、药物共处理12h、脂多糖处理12和24h时留上清液检测细胞因子，细胞提取RNA检测胶质酸性纤维蛋白和核转录因子κB（NF-κB）水平。结果：Ida能抑制促炎细胞因子（P〈0．01）和NF-κB的表达（P〈0．05）。结论：Ida具有抗炎作用。     

星形胶质细胞与Aβ1-40诱导凋亡的PC12细胞共育条件培养液对神经干细胞分化的影响机制

目的 将星形胶质细胞与β-淀粉样蛋白(Aβ1-40)诱导凋亡的PCI2细胞共育,观察星形胶质细胞条件培养液(ACM)对胚胎大鼠皮层神经干细胞(NSCs)体外定向分化为神经元的比例影响及机制,探讨神经营养素家族蛋白[包括脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养素-3(NT-3)]是否参与此过程. 方法 PC12细胞分别经10 μg/mLAβ1-40诱导不同时间(0、4、6、12、24h)后分为两部分,第一部分应用流式细胞技术检测不同时间点PC12细胞凋亡率;第二部分分别与星形胶质细胞共育2 d,将收集的ACM分为两部分.一部分应用ELISA法检测ACM中BDNF、NGF、NT-3蛋白含量,另一部分以1;3比例同DMEM/F12堵养基混合,对NSCs进行体外诱导分化,应用激光共聚焦显微镜、NSE免疫荧光技术鉴定和计数神经元分化比例. 结果 在Aβ1-40作用6 h时间点,PC12细胞凋亡率达高峰,与其共育的ACM中BDNF蛋白总量明显增高,诱导的NSCs神经元分化比例明显升高,与其他组比较,差异均有统计学意义(P<0.05). 结论 星形胶质细胞与Aβ1-40诱导凋亡的PCI2细胞共育后.ACM提高了NSCs向神经元的分化比例,ACM中BDNF可能参与了这一过程.     

Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia

Oligodendrocytes are the primary cells injured in periventricular leukomalacia (PVL), a predominant form of brain white matter lesion in preterm infants. To explore the possible linkage between white matter injury and maternal infection, purified rat O-2A progenitor (Oligodendrocyte-type 2 astrocyte progenitor) cell cultures were used as a model in studying the effects of lipopolysaccharide (LPS), an endotoxin, on survival and differentiation of oligodendrocytes and the involvement of other glial cells in the effects of LPS. O-2A progenitor cells were cultured from optic nerves of 7-day-old rat pups in a chemically defined medium (CDM). Astrocyte and microglia cell cultures were prepared from the cortex of 1-day-old rat brains in the CDM. Direct treatment of LPS (1 microg/ml) to O-2A cells had no effect on viability or differentiation of these cells. When O-2A progenitor cells were cultured in the conditioned medium obtained from either astrocyte or microglial cell cultures for 48 hr, survival rate and differentiation of O-2A cells into mature oligodendrocytes were greatly enhanced as measured by the MTT assay and immunocytochemistry. The conditioned medium obtained from astrocytes or microglia treated with LPS for 48 hr, however, failed to show such a promotional effect on viability and differentiation of O-2A cells. When 5 microg/ml LPS was used to stimulate astrocytes or microglia, the conditioned medium from these glial cell cultures caused O-2A cell injury. The overall results indicate that astrocytes and microglia may promote viability and differentiation of O-2A progenitor cells under physiological conditions, but they may also mediate cytotoxic effects of LPS on oligodendrocytes under an infectious disease biochemical environment.     

NPPB对星形胶质细胞增殖的抑制作用

目的观察体积调节性阴离子通道阻滞剂NPPB对体外培养星形胶质细胞增殖的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与NPPB干预组;在不同时间点(0、6、12、24、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,NPPB干预组在12、24h时星形胶质细胞增殖率较正常对照组降低(P<0.05),同时处于S期细胞的百分率亦相对正常对照组明显下降(P<0.01)。结论体积调节性氯离子通道阻滞剂NPPB可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC通道参与了体外培养星形胶质细胞的增殖。     

胶质细胞对共培养的脑微血管内皮细胞增殖及功能的影响

目的 研究体外培养条件下，胶质细胞对脑微血管内皮细胞(BMEC)增殖及功能的影响。方法 模拟血-脑脊液屏障结构及内皮细胞、胶质细胞间相互影响的途径，建立内皮细胞与胶质细胞共培养模型，采用细胞计数、细胞活性检测、酶含量与细胞吞饮量测定对内皮细胞增殖和功能进行研究。结果 共培养和条件培养时，内皮细胞增殖能力减弱，细胞活性以及酶含量增高，细胞吞饮量则无明显变化。结论 胶质细胞可通过两种途径影响内皮细胞的生长。胶质细胞可诱导和维持微血管内皮细胞的脑表型，但并不能促进内皮细胞生长。     

Astroglia-released factor shows similar effects as benzodiazepine inverse agonists

Media conditioned by cultured neonatal cerebral cortex microexplants (CCM) or astrocytes (ACM) contain low molecular weight (<1,000 Da) substance(s) which inhibits the gamma aminobutyric acid (GABA)-induced inward current recorded in cerebellar granule cells and hippocampal neurons in culture using the whole-cell patch-clamp technique. This effect is specific for CCm and ACM, as medium conditioned by PC12 cells (PC12CM) does not affect the GABA response of these cells. It is also specific for GABA-induced currents because glutamate-induced currents do not change either in amplitude or in shape in the presence of CCM or ACM. The inhibitory effect on the GABA response in cerebellar granule cells of both ACM and CCM could be suppressed by flumazenil, a specific benzodiazepine (BZD) antagnoist and could be mimicked by two BZD inverse agonists. These data thus demonstrate the presence of a BZD inverse agonist-like activity in CCM and ACM. This effect of ACM on different neuronal cell types was heterogenous since no detectable effect could be observed on the GABA-induced current in GABA-responsive dorsal root ganglion (DRG) neurons, presumably reflecting a functional heterogeneity of the GABA_A receptors present in these different neuronal subsets. By the release of such an endogenous BZD inverse agonist like activity, glia cells could possibly modulate GABA_A receptor-mediated responses. © 1994 Wiley-Liss, Inc.     

β淀粉样蛋白（1-42）促进α-突触核蛋白在Ser129位点磷酸化和聚集

目的：通过转染人α-突触核蛋白（α-syn）A53T突变基因的PC12细胞（A53T-PC12细胞）模型,探讨β-淀粉样蛋白1-42（Aβ1-42）对A53T-PC12细胞在Ser29位点α-syn磷酸化（Pα-syn）水平、聚集程度的影响。方法：分别予H2O2、PBS、Aβ1-42干预A53T-PC12细胞（将A53T-PC12细胞分为：PBS干预组;5μmol.L-1Aβ1-42干预组;5μmol.L-1Aβ1-42＋300μmol.L-1维生素C干预组;200μmol.L-1H2O2干预组）,观察细胞内活性氧自由基（ROS）水平变化。并通过双重免疫荧光染色观察A53T-PC12细胞内α-syn聚集情况,并通过Western blot半定量分析A53T-PC12细胞内Ser129位点磷酸化Pα-syn水平。结果：Aβ1-42增加细胞内ROS。免疫荧光染色提示Aβ1-42干预后促进A53T-PC12细胞内α-syn聚集;Western blot半定量分析显示Pα-syn较PBS干预组升高（P〈0.001,P〈0.05）。结论：Aβ1-42促进A53T-PC12细胞内α-syn在Ser129位点磷酸化和聚集。     

Cultured neurons release an inhibitor of astroglia proliferation (astrostatine)

Using in vitro techniques, we looked for a possible downregulation of rat astroglia proliferation by neuronal cells. We demonstrate that medium conditioned by 7-day-old rat cerebellar granule neurons or by 16-day-old rat embryo hippocampal neurons strongly inhibits the proliferation of cultured astroglial cells. Two neuronal cell lines, the PC12 rat pheocromocytoma and the neuro 2A (N2A) murine neuroblastoma also release such an activity. This release in N2A-conditioned medium (CM) occurs when the cells are at high density and show a low proliferation rate. This activity is present in media conditioned by neuronal cells, but not in media conditioned by normal astrocytes, by two glioma cell lines, or by one fibroblastic cell line. This proliferation inhibitor addresses normal astrocytes: the proliferation of two glioma cell lines, of a fibroblastic cell line, and of the two neuronal cell lines (PC12, N2A) is not inhibited by N2A CM. Moreover, this activity is directed against type 1 astrocytes, but not against type 2. Using three different assays, we demonstrate that DNA synthesis by astroglial cells is inhibited. N2A CM has no cytotoxic effect on astrocytes and does not modify their overall protein synthesis. Using affinity and gel filtration chromatography, we show that this activity is associated with a protein whose molecular weight ranges between 15 and 20 kDa. The possible relationship between this N2A cell-derived astroglia proliferation inhibitor and other types of potential glial proliferation inhibitors has been investigated. A brain glycoprotein immunologically related to epidermal growth factor receptor (EGFR) was reported to inhibit astroglial cell proliferation in vitro. Using polyclonal and monoclonal antibodies against EGFR, we were unable to immunoprecipitate the astrocyte proliferation inhibitor in N2A CM or to demonstrate by immunoblotting the presence of an EGFR-like immunoreactivity in the N2A CM or in the active chromatographic fractions of N2A CM. Transforming growth factor beta (TGF beta) is a well-known modulator of the proliferation of various cell types and was shown to be present in N2A CM. Using a polyclonal anti-TGF beta antibody that recognizes TGF beta on Western blots of N2A CM, we were unable to immunoprecipitate the astrocyte proliferation inhibitor of N2A CM. It seems thus far that the neuronal astroglia proliferation inhibitor is a new protein for which we propose the name astrostatine.     

Effects of astrocytes on neuronal attachment and survival shown in a serum-free co-culture system.

In order to study the neurosupportive effects of glial cells, we optimized a glial-neuron non-contact co-culture method. Astrocyte conditioned medium (ACM) and an astrocyte feeder layer were used to promote neuronal attachment and neuronal survival respectively. Neuron-enriched cultures were prepared from cortices of E-18 day rat embryos. Instead of plating cells in serum-supplemented medium, as an indispensable first-step procedure for many serum-free culture protocols, we found that coating the coverslips briefly with ACM was sufficient for the healthy attachment and neurite outgrowth of the dissociated neurons in serum-free medium. A high survival rate of the low density (4x10(4) cells/cm(2)) neuronal cultures was achieved by co-culturing primary neurons with an astrocyte feeder layer. This non-contact co-culture method could be easily implemented with ordinary culture dishes. Our serum-free chemically defined medium was MEM supplemented with insulin, transferrin, selenium and pyruvate. In this serum-free medium, glial cells did not proliferate and a neuron-enriched population was obtained without the need for mitotic inhibitors. Our experimental results reveal a critical role for astrocytes in neuronal attachment and growth. This method can be used to study glial-neuron interactions as well as culturing low-density population of pure neurons.