体外血脑屏障及模拟代谢系统对丙烯酰胺诱导的SH-SY5Y凋亡的影响  

Effects of Blood-brain Barrier and Simulated metabolic system on Apoptosis of SH-SYSY Induced by Acrylamide in Vitro

作　　者：陈宵[1] 朱丹[1] 杨义光[1] 李忠生[1] 张意[1] 肖经纬[1] 李斌[1] Chen Xiao;Zhu Dan;Yang Yiguang;Li Zhongsheng;Zhang Yi;Xiao Jingwei;Li Bin(National Institute for Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,北京100050

出　　处：《中华劳动卫生职业病杂志》2018年第6期401-407,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金面上项目（81773474）;国家重点研发计划（2017YFF0211201）

摘　　要：目的通过体外细胞整体模型模拟体内神经系统对外源性化学物质的屏障效应和代谢微环境，评价丙烯酰胺对神经细胞凋亡发生的影响。方法用人脐静脉内皮细胞（HUVEC）和大鼠胶质瘤细胞（c6）建立非接触式共培养体外血脑屏障（blood brain bamer，BBB）模型，通过测定跨内皮阻抗（ Trans-endotheilal electrical resistance, TEER）以及辣根过氧化物酶（Horseradish Peroxidase，HRP）的示踪效应分别验证建立的BBB模型的紧密连接性及通透性。将人肾皮质近曲小管上皮细胞（HK一2）、人正常肝细胞（L．02）和人神经母细胞瘤细胞（SH—SY5Y）接种于自制的多器官细胞共培养板中，观察细胞生长情况，建立多器官细胞共培养（integrate discrete multiple organ cell co-cuhure, idMOC）模型。通过观察共培养条件下各组织细胞与单独培养条件下生长曲线情况，对idMOC模型进行验证。用不同浓度的丙烯酰胺（Acrylamide，ACR）对SH．SY5Y细胞直接染毒、经BBB模型及idMOC模型后对SH—SY5Y细胞间接染毒，用流式细胞仪分析2种情况下SH—SY5Y细胞凋亡率的变化，探索模型对典型神经毒物ACR损伤效应的影响。结果HUVEC细胞在c6细胞的作用下，间隙可以形成广泛的紧密连接的复合体而抑制外加电场下电流的跨内皮运动，TEER值均数在4、5、6d时均高于内皮细胞单独培养组，差异有统计学意义（R0．05）；20min后各时间点HRP的通透率在共培养组均小于单独培养组，差异有统计学意义（P〈0．05），表明共培养组的屏障功能高于单独培养组。各细胞间可通过培养板的交换孔进行物质交换，细胞生长状态良好，无明显死亡。且各细胞单独培养与在共培养组培养时生长曲线基本一致，差异无统计学意义（P〉0．05）。不同浓度ACR（140、270μg／m1）染毒情况下，与直接染毒组比较，经BBB模型和idMOC模型的间接染毒组�Objective To evaluate the effect of acrylamide on the apoptosis of nerve cells by integral cell modelling in vitro which simulates the barrier effect and metabolic micro-environment. Methods A non-contact and co-cultured in vitro blood brain barrier（BBB） model was established by using human umbilical vein endothelial cells （HUVEC） and rat glioma cells （C6）. The trans-endotheilal electrical resistance （TEER） and horseradish peroxidase （HRP） tracer effects were measured to verify the tight connectivity and permeability of the established BBB model. An integrate discrete multiple organ cell co-culture （idMOC） model was established by inoculating the human renal cortical proximal tubular epithelial cells （HK-2）, human normal hepatocytes （L-02） and human neuroblastoma ceils （SH-SYSY） into the self-made multi-organ plate for co-culturing. Then the model was verified by observing the growth curve of various tissue cells under co-culturing or culturing individually. SH-SY5Y cells were exposed to different concentrations of acrylamide directly and indirectly （through BBB model and idMOC model）. The changes of cell apoptosis rate were analyzed by flow cytometry to explore the impact of model on Acrylamide （ACR） injury of typical neurotoxic agents. Results HUVEC cells can form a wide range of close-connected complex and then inhibit the external electric field under the crossendothelial movement, and the mean was lower than that of endothelial cell culture group at 4, 5 and 6 days （P〈O.05）; After 20 rain, the penetration rate of HRP in the co-culture group was less than that in the individual culture group, and the difference was statistically significant （P〈0.05）, indicating that the barrier function of the co-culture group was higher than that of the individual culture group. All cells can exchange substances through the exchange hole of the culture plate, the cells grow well and there was no obvious death. The growth curve in individual culture group and co-culture

关 键 词：血脑屏障 多器官细胞共培养 SH—SY5Y 丙烯酰胺 神经毒性 

分 类 号：R13[医药卫生—劳动卫生]