【作者】 张莹；

【导师】 孙黎光；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2008， 博士

【摘要】 目的重金属铅是一种普遍存在的环境神经毒物,低水平的慢性铅暴露对婴幼儿中枢神经系统不可逆的损害以及对学习记忆能力的影响是一个备受关注的重大问题。婴幼儿的消化道对铅的吸收率较成人约高5倍,而排泄率较成人低,加之儿童血脑屏障发育不全,因此铅极易在婴幼儿神经系统发生蓄积。0～6岁儿童的大脑智力发育对铅的敏感性高于成人30倍,我国很多城市儿童铅中毒已达到30～40%,是美国儿童的10余倍。研究表明当儿童的血铅由0.48μM升高到0.97μM时,智商平均下降2～6分。神经胶质细胞占人类脑细胞的90%,是神经系统的支持细胞,在构成髓鞘、促进神经细胞发育、神经组织修复和再生等方面有重要作用,其中的一大类细胞星形胶质细胞对神经元的发育、迁移、突触的形成、递质传递的调节、能量供应以及维持中枢神经系统内环境稳定,都起着十分重要的作用。目前研究认为星形胶质细胞（astrocyte）较内皮细胞对铅更为敏感,认为铅可能是通过损害星形胶质细胞,进而损害内皮细胞,从而增加血-脑屏障通透性而进入脑,最终损害脑功能的。研究发现,缺氧、低糖、重金属等应激信号可以使内质网（endoplasmicreticulum,ER）内错误折叠蛋白增多,可诱发未折叠蛋白反应（unfolded proteinreaction,UPR）,通过上调分子伴侣如葡萄糖调节蛋白78（glucose regulated proteins,GRP78）等帮助蛋白正确折叠以减轻细胞损伤。当应激强度过大,ER功能严重受损时细胞将启动生长抑制DNA损伤诱导因子（growth arrest and DNA damageinducible gene,GADD153）等信号触发细胞凋亡。有实验证实,暴露于较高浓度铅环境的幼鼠脑中的铅大部分被隔离在星形胶质细胞中,并可以检测出一种分子量为23KD的新蛋白可以结合铅,另外,也有学者提出,星形胶质细胞可能通过内质网应激蓄积大量的铅,从而保护更为敏感的神经元。这些证据表明,在非生理性金属的刺激下,机体很可能启动了一种耐受机制,即染铅后星形胶质细胞通过某些蛋白的高表达与铅螯合,作为铅蓄积库以保护中枢神经系统的正常功能。本研究用醋酸铅作用于原代培养的脑星形胶质细胞,观察其对细胞增殖、细胞周期和CyclinD1、GADD153及GRP78表达量的影响。为了探讨GRP78是细胞内质网上铅作用的靶蛋白,本研究还观察了细胞内铅离子与GRP78的螯合作用,以及铅诱导GRP78表达的一些信号转导通路,以证明GRP78是细胞内质网上的一种铅结合蛋白。方法1、Wistar大鼠脑星形胶质细胞的原代培养,分别加入醋酸铅、PD98059（ERK上游激酶MEK1的特异性抑制剂）及Wortmannin（PKB上游激酶PI3K的特异性抑制剂）培养不同时间,加入醋酸钠作为对照。2、免疫组化法观察细胞GFAP表达以鉴定原代培养星形胶质细胞的纯度。3、MTT比色法观察醋酸铅对星形胶质细胞增殖的影响。4、流式细胞技术分析醋酸铅对星形胶质细胞的细胞周期进程的影响。5、Western blot法检测醋酸铅诱导CyclinD1和GADD153蛋白表达变化。6、免疫沉淀方法纯化出醋酸铅诱导的GRP78蛋白,Western blot方法检测纯化的GRP78蛋白量变化,石墨炉原子吸收法检测纯化产物中的铅含量。7、胶体金免疫电镜法观察醋酸铅诱导的GRP78定位的改变。8、Western blot和RT-PCR方法检测铅诱导的ERK和PKB活性的变化,以及其诱导GRP78表达的信号转导通路。结果1、免疫组化法结果显示:原代培养脑星形胶质细胞经过差速粘附处理后,传代2～4代后,细胞状态稳定。经显微镜下细胞计数观察,95%以上的细胞胞浆着黄褐色,胞核着蓝色,证明绝大多数培养的原代细胞为星形胶质细胞。2、MTT比色法结果显示:醋酸铅可抑制星形胶质细胞增殖,且与铅的水平呈剂量依赖关系,1.0μM醋酸铅浓度作用细胞8天起,细胞增殖比下降具有统计学意义（P＜0.05）。3、流式细胞技术结果显示:与醋酸钠对照相比,经1.0μM醋酸铅处理8、12、15、30 d后,星形胶质细胞的细胞周期停滞在G1期（58.64±1.75%→69.81±1.56%,70.80±1.27%,90.59±1.25%,80.9±1.11%）。4、Western blot结果显示:（1）醋酸铅使星形胶质细胞CyclinD1蛋白表达呈时效依赖性下降趋势（P＜0.05）。（2）1.0μM醋酸铅作用于细胞30天以后,星形胶质细胞GADD153蛋白开始表达。（3）醋酸铅能诱导星形胶质细胞内质网GRP78蛋白表达增加,此诱导作用具有量效依赖性和时效依赖性。0.2、1.0、10μM的醋酸铅作用于细胞24小时,都可诱导GRP78表达的增加,1.0和10μM醋酸铅效果显著（P＜0.01）。1.0μM的醋酸铅作用于细胞24小时后,GRP78表达量显著增高,4～8天左右达到峰值（P＜0.01）,约12天左右表达量开始下降,至染铅30天时仍高于对照组（P＜0.05）。PI3K抑制剂wortmannin和MEK1抑制剂PD98059均能抑制醋酸铅诱导的GRP78蛋白表达的增加（P＜0.05）,wortmannin的抑制效果更明显。（4）醋酸铅能诱导星形胶质细胞ERK蛋白活性增高,具有时效依赖性和量效依赖性。0.2、1.0、10μM的醋酸铅作用于细胞30分钟,都可诱导ERK活性增高,以1.0μM的醋酸铅效果最明显（P＜0.05）。1.0μM的醋酸铅作用于细胞5分钟时ERK活性开始增高,30分钟时达到峰值,随后又逐渐降至正常水平。MEK1抑制剂PD98059能抑制醋酸铅诱导的ERK活性增高（P＜0.05）。（5）醋酸铅能诱导星形胶质细胞PKB蛋白活性增高,具有时效依赖性和量效依赖性。0.2、1.0、10μM的醋酸铅作用于细胞30分钟,都可诱导PKB活性增高,以1.0μM的醋酸铅效果最明显（P＜0.05）。1.0μM的醋酸铅作用于细胞5分钟时PKB活性开始增高,30分钟时达到峰值,随后又逐渐降至正常水平。PI3K抑制剂wortmannin能抑制醋酸铅诱导的PKB活性增高（P＜0.05）。5、免疫沉淀及石墨炉原子吸收法结果显示:GRP78能紧密螯合进入细胞内的铅离子。1.0μM醋酸铅处理细胞后,用亲和层析法将细胞裂解液中的GRP78蛋白纯化,western blot方法检测纯化产物为分子量78KD的单一蛋白质。纯化后的产物用石墨炉原子吸收仪检测铅含量,1.0μM醋酸铅作用于细胞24小时后纯化产物中的铅含量开始显著增高,8天左右达到峰值（P＜0.01）,随后铅含量下降,但仍高于对照组（P＜0.05）。6、胶体金免疫电镜结果显示:1.0μM醋酸铅作用于细胞24小时后,星形胶质细胞内GRP78的表达量开始显著增加,并且其定位由内质网转移至胞核周围胞浆。10μM醋酸铅作用于细胞10小时后,细胞也有类似变化。7、RT-PCR分析结果显示:（1）醋酸铅能诱导星形胶质细胞内质网的GRP78 mRNA表达增加,具有时效依赖性和量效依赖性;PI3K抑制剂wortmannin和MEK1抑制剂PD98059都可抑制醋酸铅的作用（P＜0.05）,wortmannin的抑制效果更为明显。（2）醋酸铅能诱导星形胶质细胞的ERK₂ mRNA表达增加（P＜0.05）。MEK1抑制剂PD98059可抑制此作用。结论1、在原代培养的鼠脑星形胶质细胞中,醋酸铅可诱导未折叠蛋白反应,使细胞CyclinD1蛋白水平下降,细胞周期停滞在G1期;长时间铅暴露甚至可诱导GADD153蛋白表达,但并未观察到细胞的凋亡。2、醋酸铅诱导星形胶质细胞内质网GRP78 mRNA水平和蛋白水平表达增加,PI3K抑制剂wortmannin和MEK1抑制剂PD98059都可阻断此诱导作用。醋酸铅可依赖MEK1/ERK、PI3K/PKB通路促进GRP78表达。3、细胞内的GRP78可以与铅离子紧密螯合,此作用具有量效依赖性。4、醋酸铅经MEK途径迅速激活星形胶质细胞ERK活性,经PI3K途径迅速激活PKB活性。醋酸铅对ERK和PKB活性的改变是通过快速磷酸化激酶蛋白实现的,并不是促进蛋白合成的增加。更多还原

【Abstract】 ObjectiveHeavy metal lead is a widespread environmental toxicant;never reversible neurotoxic and learning memory effects from chronic exposure to low levels of lead are a problem of significant magnitude in children and infants.The absorptivity to lead of alimentary canal in children is higher than that in adults,and excretion rate of children is lower than that of adults.In addition,the agenesis of blood and brain barrier in children,so lead concentrates easily in children’s neural system.The sensitivity to lead that development of brains for 0 to 6 years old of children is 30 times higher than that of adults.Nearly 30～40%of children in many cities of china have the trouble of lead poisoning which is almost 10 times of children in America.Studies indicate that when the concentration of Pb in the blood increases from 0.48μM to 0.97μM,the IQ of these children averagely decreases 2～6 points.90%of brain cells are glia cells,which are neural sustaining cells.The important functions of glia include that myelination,promoting development of neuron,rehab and rebirth of neuron,etc.The primary cells of glia are astrocyte,whose functions includes that helping the development and movement of neuron,forming of synapse, accommodation of neurotransmitter transfer and energy,maintaining the stabilization of central nerve system.Some studies considered that maybe lead damaged astrocyte firstly and then damaged endodermis so that to pass the blood brain barrier easily,and damaged the brain function ultimately.It is well known that stress can bring about alterations of the endoplasmic recticulum（ER）homeostasis which cause ER stress.Unfolded protein reaction（UPR） is an important genomic response to ER stress by synthesis ER chaperones proteins such as glucose regulated proteins 78（GRP78）,which plays critical roles in cell survival as part of UPR.When the ER function is severely impaired,the organelle elicits apoptosis signal activated by growth arrest and DNA damage inducible gene （GADD153）and other signals.Some studies have shown that most of Pb could be isolated to the astroglial cells when rat children were exposed to a higher level of Pb in the environment,while at the same time,a kind of new protein which could bind to Pb was found.In addition,some scholar have considered that astroglia could cumulate a great deal of Pb by ER stress so that to protect the more sensitive neuron.All these evidences indicate that cells are capable of activating tolerance mechanisms to the nonphysiologic metal,i.e.astroglia cells could increase expression of certain protein and chelate with Pb when cells were exposed to Pb,which were as the Pb cumulated pool to protect the normal function of CNS.To evaluate the impact of acetate Pb induced stress in ER;we investigate the effects of acetate Pb on morphological,cell proliferation,cell-cycle and the expressions of cyclinD1,GADD153 and GRP78.In order to discuss if GRP78 is the target protein on the ER for Pb,we observe the chelate of GRP78 with Pb and some certain signal pathways through which Pb induces the express of GRP78.Methods1.Wistar rat brain astrocyte cells were primary cultured in medium with or without acetate Pb which blocked by PD98059（MEK1 inhibitor）or Wortmannin （PI3K inhibitor）,acetate sodium substituted acetate Pb as the comparison.2.The hallmark of the primary culture astrocyte was estimate by observing the expression of glia fibrillary acidic protein（GFAP）in the astrocyte through the method of immunochistochemistry（IC）.3.The effect of acetate Pb on the cell proliferation in astrocyte was estimated by MTT analysis.4.The effect of acetate Pb on the cell-cycle in astrocyte was estimated by FCM analysis.5.The protein expressions of cyclinD1 and GADD153 induced by acetate Pb were accessed by western blotting.6.The chelate of GRP78 with Pb was examined by immunoprecipitation（IP）, western blotting and atomic absorption spectrophotometer（AAS）. 7.The localizations transformation of GRP78 was observed with colloid gold immunoelectron microscopy.8.The changes of the activity of ERK and PKB,and the signal pathway which GRP78 was induced by Pb were determined by reverse transcription PCR（RT-PCR） and western blotting.Results1.By immunohistochemical staining and observing under the microscope,more than 95%primary culture cells were positively stained with GFAP as witnessed by the brown DAB reaction product in the cytoplasm,indicating that they were astroglia.2.MTT assay showed that:Treated astrocyte cells with acetate Pb,the cell proliferate rate decreased time dependently.When 1.0μM acetate Pb treated cells for 8 day,the cell proliferate rate decreased statistics（P＜0.05）.3.FCM assay showed that:Treated astrocyte cells with acetate Pb for 8,12,15 or 30 day,the cell-cycle progression was inhibited at G1 phase（P＜0.01）.4.Western blot showed that:（1）Treated astrocyte cells with acetate Pb,CyclinD1 protein expression decreased time dependently（P＜0.05）.（2）1.0μM acetate Pb treated astrocyte for 30 day,GADD153 protein began to be expressed.（3）Acetate Pb could induce the increasing expression of GRP78 dose and time dependently.0.2,1.0 and 10μM acetate Pb treated astrocyte cells for 24h,the GRP78 protein expression would be increased,1.0 and 10μM acetate Pb group have statistics significance（P＜0.01）.1.0μM acetate Pb treated cells for 24 h,the expression of GRP78 increased significance,and the peak was at treated for 4～8 day（P＜0.01）, nearly at 12 day,the expression of GRP78 began to decrease which was still higher than the normal level（P＜0.05）.PD98059 and Wortmannin could inhibit the effects of acetate Pb treatment,and the effect of Wortmannin was better.（4）Acetate Pb could increase activation of ERK dose and time dependently.0.2, 1.0 and 10μM acetate Pb treated astrocyte cells for 30 min,the activation of ERK would be increased,1.0μM acetate Pb group has the most statistics significance（P＜ 0.05）.When 1.0μM acetate Pb treated cells for 5min,the activation of ERK began to increase and the peak was at 30min,and then the level of ERK activation gradually decreased to the normal level.PD98059 could prevented the increasing of ERK activation induced by acetate Pb（P＜0.05）.（5）Acetate Pb could increase activation of PKB dose and time dependently.0.2, 1.0 and 10μM acetate Pb treated astrocyte cells for 30min,the activation of PKB would be increased,1.0μM acetate Pb group has the most statistics significance（P＜0.05）.When 1.0μM acetate Pb treated cells for 5min,the activation of PKB began to increase and the peak was at 30 min,and then the level of PKB activation gradually decreased to the normal level.Wortmannin could prevented the increasing of PKB activation induced by acetate Pb（P＜0.05）.5.Immunoprecipitation and method of AAS showed that:GRP78 could chelate with Pb ion which came into cells tightly.The immunoprecipitated GRP78 protein was analyzed by Western blotting which could detect the protein was a single protein of 78KD.The immunoprecipitated content was detected by AAS,Pb content began to increase when cells were treated by acetate Pb for 24 h,and the peak was at 8 d（P＜0.01）,and then Pb content decreased gradually,but still higher than the normal（P＜0.05）.6.Colloid gold immunoelectron microscopy assay showed that:1.0μM acetate Pb treated cells for 24 h,GRP78 protein expression began to remarkably increase whose location transferred from ER to the cytosol around the nuclei.10μM acetate Pb treated cells for 10 h,cells would have the similar changes.7.RT-PCR analysis showed that:（1）Acetate Pb could induce the increasing of mRNA expression for GRP78 dose and time dependently.PD98059 and Wortmannin could inhibit the effects of acetate Pb treatment,and the effect of Wortmannin was better.（2）Acetate Pb could upregulate mRNA expression of ERK₂（P＜0.05）,which was prevented by PD98059 effectively. Conclusion1.Acetate Pb could induce the UPR in the primary culture astrocyte ER,and could down-regulate the expression of protein cyclinD1 and the stagnation of cell-cycle.A long time of exposing to Pb could induce the expression of GADD153, but we still couldn’t observe apoptosis.2.Acetate Pb up-regulated GRP78 mRNA and protein expression through PI3K/PKB and MEK1/ERK signal transduction pathway.3.GRP78 in astrocyte could strongly chelate with Pb ion dose dependently.4.Acetate Pb activated MEK/ERK and PI3K/PKB by phosphorylating the kinases immediately rather than increasing their protein synthesis.更多还原