[1]李　敏,楚人杰,张　姣,等.γ-氨基丁酸Rho2受体在神经母细胞瘤细胞中的稳定表达及其在谷氨酸盐致细胞死亡中的作用[J].新乡医学院学报,2019,36(1):013-18.[doi:10.7683/xxyxyxb.2019.01.003]
　LI Min,CHU Ren-jie,ZHANG Jiao,et al.Expression of gamma-aminobutyric acid Rho2 receptor in neuroblastoma cells and its role in the cell death induced by glutamate[J].Journal of Xinxiang Medical University,2019,36(1):013-18.[doi:10.7683/xxyxyxb.2019.01.003]
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γ-氨基丁酸Rho2受体在神经母细胞瘤细胞中的稳定表达及其在谷氨酸盐致细胞死亡中的作用
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
36
期数:
2019年1
页码:
013-18
栏目:
基础研究
出版日期:
2019-01-05

文章信息/Info

Title:
Expression of gamma-aminobutyric acid Rho2 receptor in neuroblastoma cells and its role in the cell death induced by glutamate
作者:
李　敏; 楚人杰; 张　姣; 韩思乐; 李超堃
（新乡医学院基础医学院生理学与神经生物学教研室，河南　新乡　453003）
Author(s):
LI Min; CHU Ren-jie; ZHANG Jiao; HAN Si-le; LI Chao-kun
（Department of Physiology and Neurobiology，Xinxiang Medical University，Xinxiang 453003，Henan Province，China）
关键词:
真核表达载体; γ-氨基丁酸; 神经母细胞瘤; SH-SY5Y细胞; 细胞克隆; 基因表达
Keywords:
eukaryotic expression vector; γ-aminobutyric acid; neuroblastoma; SH-SY5Y cell; cell clone; gene expression
分类号:
R739.4
DOI:
10.7683/xxyxyxb.2019.01.003
文献标志码:
A
摘要:
目的　构建人γ-氨基丁酸（GABA）Rho2受体（GABRR2）真核表达载体，并转染神经母细胞瘤 SH-SY5Y 细胞，建立稳定表达细胞株，探讨GABRR2在谷氨酸盐致细胞死亡中的作用。方法　采用快速克隆方法将人脑组织GABRR2基因克隆至pIRES2-eGFP质粒，构建重组质粒pIRES2-eGFP-Rho2，利用X-fect试剂盒将其转染至SH-SY5Y细胞中，使用遗传霉素进行筛选，建立稳定表达pIRES2-eGFP-Rho2的SH-SY5Y细胞株。使用流式细胞术检测稳定转染过表达pIRES2-eGFP-Rho2的细胞，利用免疫荧光法鉴定稳定转染的SH-SY5Y细胞中pIRES2-eGFP-Rho2的表达。将野生型SH-SY5Y细胞分为A1、B1、C1组，稳定表达pIRES2-eGFP-Rho2的SH-SY5Y细胞分为A2、B2、C2组，每组设3个平行孔，其中A1、A2组细胞使用达尔伯克改良伊格尔培养基（DMEM）孵育，B1、B2组细胞使用含有谷氨酸盐的DMEM孵育，C1、C2组细胞使用含有谷氨酸盐和GABA的DMEM孵育，将各组细胞置于细胞培养箱中培养24 h，应用乳酸脱氢酶试剂盒检测各组细胞的存活率。结果　重组真核表达载体构建正确，获得了稳定表达pIRES2-eGFP-Rho2的细胞株。A1、B1、C1组细胞存活率比较差异有统计学意义（F=5.542，P<0.05）；其中B1、C1组细胞存活率显著低于A1组（P<0.05），C1组细胞存活率显著高于B1组（P<0.05）。A2、B2、C2组细胞存活率比较差异有统计学意义（F=6.588，P<0.05）；其中B2、C2组细胞存活率显著低于A2组（P<0.05），C2组细胞存活率显著高于B2组（P<0.01）。A2组细胞存活率与A1组比较差异无统计学意义（P>0.05），B2组细胞存活率显著低于B1组（P<0.05），C2组细胞存活率显著低于C1组（P<0.05）。结论　成功构建pIRES2-eGFP-Rho2真核表达载体，建立了稳定表达pIRES2-eGFP-Rho2的SH-SY5Y细胞株，为体外研究GABRR2的功能提供了实验基础，并证明使用GABA可缓解谷氨酸盐对细胞的损伤，为缺血性脑损伤的分子生物学治疗提供了靶点。
Abstract:
Objective　To construct human gamma aminobutyric acid （GABA） Rho2 receptor （GABRR2） eukaryotic expression vector and transfect the neuroblastoma SH-SY5Y cell to establish stable expression cell line，and to investigate the role of GABRR2 in the cell death induced by glutamate.Methods　The recombinant plasmid pIRES2-eGFP-Rho2 was constructed by cloning the human brain tissue GABRR2 gene into pIRES2-eGFP plasmid by rapid cloning method.The recombinant plasmid pIRES2-eGFP-Rho2 was transfected into SH-SY5Y cells by X-fect kit，and the SH-SY5Y cell lines with stable expression of pIRES2-eGFP-Rho2 was established by geneticin screening.The number of SH-SY5Y cells over-expressing pIRES2-eGFP-Rho2 was detected by flow cytometry.The expression of pIRES2-eGFP-Rho2 in stable transfected SH-SY5Y cells were was identified by immunofluorescence.The wild-type SH-SY5Y cells were divided into group A1,group B1 and group C1,and the SH-SY5Y cells stably expressing pIRES2-eGFP-Rho2 were divided into group A2,group B2 and group C2,with three parallel holes for each group.The cells in the group A1 and group A2 were incubated with Dulbecco′s modified Eagle′s medium(DMEM).The cells in the group B1 and group B2 were incubated with DMEM containing glutamate.The cells in the group C1 and group C2 were incubated with DMEM containing glutamate and GABA.The cells in all groups were cultured for 24 hours in cell incubator.The survival rate of wild type SH-SY5Y cells and the SH-SY5Y cells with stable expression of GABRR2 were detected by lactate dehydrogenase kit.Results　The recombinant eukaryotic expression vector was constructed correctly，and the SH-SY5Y cell line with stable expression of pIRES2-eGFP-Rho2 was obtained.The survival rate of the wild-type SH-SY5Y cells in group A1,B1 and C1 was significantly different(F=5.542,P<0.05).The survival rate of cells in group B1 and C1 was significantly lower than that in group A1(P<0.05).The cell survival rate of group C1 was significantly higher than that of group B1(P<0.05).There was a significant difference in cell viability among the group A2,B2,and C2 of SH-SY5Y cells stably expressing pIRES2-eGFP-Rho2 (F=6.588,P<0.05).The survival rate of cells in group B2 and C2 was significantly lower than that in group A2(P<0.05).The cells survival rate in group C2 was significantly higher than that in group B2 (P<0.01).There was no significant difference in cell survival rate between group A2 and group A1(P>0.05).The cell survival rate in group B2 was significantly lower than that in group B1(P<0.05).The cell survival rate in the group C2 was significantly lower than that in the group C1(P<0.05).Conclusion　The pIRES2-eGFP-Rho2 eukaryotic expression vector was successfully constructed,and the SH-SY5Y cell line with stable expression of pIRES2-eGFP-Rho2 was established.It provide the experimental basis for the study of the function of GABRR2 in vitro.This study prove that GABA could alleviate the cell damage caused by glutamate，which provide the target for clinical treatment of molecular biological treatment.

参考文献/References:

［1］　王琪，李超堃，李敏，等.γ-氨基丁酸受体ρ2亚基重组蛋白构建及表达［J］.新乡医学院学报，2018，35（1）：1-5.
［2］　BHATTACHARYA P，PANDEY A K，PAUL S，et al.Alleviation of glutamate mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia：a possible mechanism of GABA agonism［J］.J Physiol Biochem，2014，70（4）：901-913.
［3］　LIU B，LI L，ZHANG Q，et al.Preservation of GABAA receptor funtion by PTEN inhibition protects against neuronal death in ischemic stroke［J］.Stroke，2010，41（5）：1018-1026.
［4］　MELE M，RIBEIRO L，INCIO A R，et al.GABAA receptor dephosphorylation followed by internalization is coupled to neuronal death in in vitro isischemia［J］.Neurobiol Dis，2014，65：220-232.
［5］　XU Q，YANG J，CAO Y.Acupuncture improves locomotor function by enhancing GABA receptor expression in transient focal cerebral ischemia rats［J］.Neurosci Lett，2015，588：88-94.
［6］　CLARKSON A N.Perisynaptic GABA receptor the overzealous protector［J］.Adv Pharmacol Sci，2012，708：42-48.
［7］　CHI O Z，HUNTER C，LIU X，et al.Effects of GABAA receptor blockade on regional cerebral blood flow and blood-brain barrier disruption in focal cerebral ischemia［J］.J Neuro Sic，2011，301 （1/2）；66-70.
［8］　CARVER C M，WU X，GANGISETTY O，et al.Perimenstrual-like hormonal regulation of extrasynaptic δ-containing GABAA receptors mediating tonic inhibition and neurosteroid sensitivity［J］.J Neurosci，2014，34（43）：14181-14197.
［9］　XU X，SEPICH C，LUKAS R J.et al.Emamectin is a non-selective allosteric activator of nicotinic acetylcholine receptors and GABAA/C receptors［J］.Biochem Biophys Res Commun，2016，473（4）：795-800.
［10］　TSUJI M，TAGUCHI A，OHSHIMA M，et al.Progesterone and allopregnanolone exacerbate hypoxic-ischemic brain injury in immature rats［J］.Exp Neurol，2012，233（1）：214-220.
［11］　ZIEMBA A M，FORMAN S A.Correction for inhibition leads to an allosteric co-agonist model for pentobarbital modulation and activation of α1β3γ2L GABAA receptors［J］.PLoS One，2016，11（4）：e0154031.
［12］　PAN B，LIAN J M，HUANG X F，et al.Aripiprazole increases the PKA signalling and expression of the GABAA receptor and CREB1 in the nucleus accumbens of rats［J］.J Mol Neurosci，2016，59（1）：36-47.
［13］　ROSAS-ARELLANO A，PARODI J，MACHUCA-PARRA A I，et al.The GABAA receptors in hippocampal spontaneous activity and their distribution in hippocampus，amygdala and visual cortex［J］.Neurosci Lett，2011，500（1）：20-25.
［14］　LI C，WEN A，SHEN B，et al.Fast cloning：a highly simplified，purification-free，sequence- and ligation-independent PCR cloning method［J］.BMC Biotechnol，2011，11：92.
［15］　MARTNEZ-DELGADO G，REYES-HARO D，ESPINO-HARO D，et al.Dynamics of GABAρ2 receptors in retinal bipolar neurons and cerebellar astrocytes［J］.Neuroreport，2011，22（1）：4-9.
［16］　姚扬，杨洋，张涛，等.γ氨基丁酸治疗脑缺血的研究进展［J］.继续医学教育，2005，19（12）：59-63.
［17］　DOMINGOS-SOUZA G，MESCHIARI C A，BUZELLE S L，et al.Sodium and water intake are not affected by GABAC receptor activation in the lateral parabrachial nucleus of sodium-depleted rats［J］.J Chem Neuroan，2016，74：47-54.
［18］　DENTER D G，HECK N，RIEDEMANN T，et al.GABAC receptors are functionnally expressed in the intermediate zone and regulate radial migration in the embryonic mouse neocortex［J］.Neuroscience，2010，167（1）：24-34.

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更新日期/Last Update: 2019-01-05