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  • Title: [Effects of Blood-brain Barrier and Simulated metabolic system on Apoptosis of SH-SY5Y Induced by Acrylamide in Vitro].
    Author: Chen X, Zhu D, Yang YG, Li ZS, Zhang Y, Xiao JW, Li B.
    Journal: Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi; 2018 Jun 20; 36(6):401-407. PubMed ID: 30248731.
    Abstract:
    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 cells (SH-SY5Y) 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 cross-endothelial movement, and the mean was lower than that of endothelial cell culture group at 4, 5 and 6 days (P<0.05) ; After 20 min, 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 group were basically the same, the difference was not statistically significant (P>0.05) . Under the condition of different concentrations of ACR (140, 270 g/ml) , compared with the direct exposure group, the apoptosis rate of the BBB model and the idMOC model were significantly decreased, and the difference was statistically significant (P<0.05) . Conclusion: Based on HUVEC cells and C6 cells co-culture system, a blood-brain barrier model in vitro was established and based on co-culture of HK-2, L-02 and SH-SY5Y, the idMOC model was established. The toxicity and toxic action characteristics of ACR on SH-SY5Y cells were evaluated by validation tests. 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 barrier，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-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-culture，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、6 d时均高于内皮细胞单独培养组，差异有统计学意义（P<0.05）；20 min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、270 μg/ml）染毒情况下，与直接染毒组比较，经BBB模型和idMOC模型的间接染毒组凋亡率均明显降低，差异有统计学意义（P<0.05）。 结论： 建立了基于HUVEC细胞和C6细胞共培养体系的体外BBB模型以及基于HK-2细胞、人L-02细胞和SH-SY5Y细胞共培养体系的体外idMOC模型，并通过验证实验评价了ACR对SH-SY5Y细胞的毒性及毒作用特点。.
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