体外共培养癫痫细胞模型中沉默突触的转化及AMPA受体亚基的变化

发布时间：2018-04-15 07:43

本文选题：海马神经元 + 星形胶质细胞　；参考：《重庆医科大学》2014年硕士论文

【摘要】：第一部分体外共培养癫痫放电细胞模型的建立目的：获得一种操作简单、高效稳定的神经元和星形胶质细胞共培养方法，，从而建立稳定的癫痫细胞模型。方法：选用清洁级孕16-17d的SD孕鼠，清洁级出生24h以内的SD新生鼠，分别采用海马神经元与星形胶质细胞共同培养的混合培养方法（混合培养组）、改良Banker共培养方法（改良Banker共培养组）和插入式培养皿的方法（插入式培养皿组）制作海马神经元与星形胶质细胞共培养模型，进而对三组培养的细胞分别应用无镁细胞外液及正常细胞外液诱导3h，制作癫痫模型及正常对照，每种方法重复3次。采用免疫荧光检测共培养中神经元特异性烯醇化酶(neuron specificenolase,NSE)及星形胶质细胞胶质纤维酸性蛋白(glial fibrillary acidicprotein，GFAP）表达情况，鉴定比较培养体系中神经元及星形胶质细胞纯度；应用膜片钳全细胞模式的电流钳记录不同培养组中神经元细胞惊厥放电情况并比较放电成功率。结果：（1）插入式培养皿组神经元纯度为[94.3%-100%]星形胶质细胞纯度为[93%-100%],较改良Banker共培养组得到的神经元[87.6%-95%]及星胶[88.2%-93%]纯度更纯。（2）插入式培养皿组95％左右的海马神经元（ｎ＝10）能够记录到阵发性持续棘波样爆发和阵发性去极化样偏移（Paroxysmal depolarizing shifts,PDSs）样发作；改良Banker共培养组有93%左右的海马神经元（ｎ＝10）能够记录到阵发性持续棘波样爆发和PDSs；混合培养组这一比例为90%左右。（3）经无镁细胞外液处理后72h，插入式培养皿组仍有85％的神经元（ｎ＝10）能够记录到阵发性持续棘波样爆发和PDSs。而改良Banker共培养组有82%左右的海马神经元（ｎ＝10）能够记录到阵发性持续棘波样爆发和PDSs；混合培养组这一比例有80%左右。结论：三种方法均可建立成功的体外共培养癫痫细胞模型，但插入式培养皿共培养方法建立的模型更简便，细胞培养纯度更高。第二部分：体外共培养癫痫细胞模型中沉默突触的转化及AMPA受体亚基的变化目的：采用体外共培养癫痫细胞模型证实癫痫发作可否导致沉默突触的激活转化及AMPA受体各亚基的组成变化。方法：在前期证实插入式培养皿方法建立稳定的体外共培养细胞模型的基础上，对插入式培养皿共培养的神经元及星形胶质细胞分为两组，无镁实验组采用无镁细胞外液培养3h诱导反复自发性惊厥样癫痫放电模型。正常对照组以含镁正常细胞外液代替无镁细胞外液培养3h，实验重复3次。应用Synapto GreenTM C4(FM1-43）及突触素(Synaptophysin,SYN）双标检测有无突触前沉默突触的变化；膜片钳记录-氨基-3-羟基-5-甲基-4-异恶唑丙酸（a-mino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor，AMPA)受体介导的微兴奋性突触后电流（micro excitatory postsynapticcurrents,mEPSCs）及NR1与GLuR1亚基免疫荧光双标反映有无突触后沉默突触的改变；SYN分别与AMPA受体的GLuR1、GLuR2、GLuR3、GLuR4亚基进行免疫荧光双标，检测细胞膜上AMPA受体各亚基表达情况。结果：FM1-43及SYN双标检测显示SYN阳性而FM1-43阴性荧光颗粒占SYN阳性荧光颗粒的比例，无镁实验组较正常对照组有所减少（P0.05）；无镁实验组AMPA受体mEPSCs的频率及幅度均较正常对照组出现明显增加（P0.05）；无镁实验组GLuR1阴性NR1阳性的荧光颗粒占NR1阳性荧光颗粒比例较正常对照组发生减少（P0.05）；无镁实验组可见GLuR2、GLuR4亚基与SYN重合的荧光颗粒个数/细胞核个数较正常对照组减少（P0.05）,GLuR1、GLuR3亚基与SYN重合的荧光颗粒个数/细胞核个数较正常对照组增加（P0.05）。结论：无镁诱导的癫痫发作可导致突触前沉默突触及突触后沉默突触的减少，减少的沉默突触极有可能激活转化为功能突触，并成为癫痫发生发展的病理基础；无镁诱导共培养神经元癫痫放电后可使膜GLuR2、GLuR4亚基减少,膜GLuR1、GLuR3亚基增加。
[Abstract]:Establishment of the first part in vitro co - cultured epileptic discharge cell model

Objective : To obtain a simple and highly stable method for co - culture of neurons with astrocytes and to establish a stable model of epileptic cells .

Methods : SD rats with gestational age 16 - 17d were selected and SD neonatal rats within 24 hours were cultured in clean grade . The models of co - culture of hippocampal neurons and astrocytes were prepared by modified Banker co - culture method ( modified Banker co - culture group ) and plug - in culture dish ( insert culture dish group ) .
Using the current forceps of the whole - cell mode of patch clamp , the discharge of neurons in different culture groups was recorded and the success rate of discharge was compared .

Results : ( 1 ) The purity of neurons in the inserted culture dish group was 94 . 3 % -100 % , the purity of astrocytes was 93 % -100 % , the purity of the neurons obtained by the modified Banker co - culture group was 87.6 % -95 % , and the purity was more pure .
In the modified Banker co - culture group , 93 % of the hippocampal neurons ( n = 10 ) could be recorded in the burst and PDSs .
( 3 ) After treated with magnesium - free cell , 85 % of neurons ( n = 10 ) in the inserted culture dish group were able to record the burst and PDSs , while the improved Banker co - culture group had about 82 % of hippocampal neurons ( n = 10 ) .
The proportion of the mixed culture group was about 80 % .

Conclusion : Three methods can establish a successful model of co - culture of epileptic cells in vitro , but the model established by the co - culture method of the inserted culture dish is simpler and the cell culture purity is higher .

Part Two : The Transformation of Silent Synapses and the Changes of AMPA Receptor Subunits in the Model of In Vitro Co - culture of Epilepsy Cells

Objective : To establish a model of epileptic cell in vitro to confirm whether the seizure could lead to the activation transformation of silent synaptic and the composition of AMPA receptor subunit .

Methods : The cultured neurons and astrocytes were divided into two groups on the basis of establishing stable in vitro co - culture cell model by inserting culture dish method in the previous stage . The rats were cultured for 3 hours with magnesium - free cell culture solution for 3 hours . The normal control group was cultured for 3 hours with magnesium - containing normal cell , and the experiment was repeated 3 times . SynaptoGreen TM C4 ( FM1 - 43 ) and Synaptophysin ( SYN ) were used to detect the changes of presynaptic silent presynaptic presynaptic presynaptic synaptic ;
Patch clamp recording - amino - 3 - hydroxy - 5 - methyl - 4 - isoxazolepropionic acid receptor ( AMPA ) receptor - mediated micro - excitatory postsynaptic currents ( mEPSCs ) and NR1 and GLuR1 subunit immunofluorescence double standards reflect the change of synaptic silent synaptic ;
SYN and GLuR1 , GLuR2 , GLuR3 and GLuR4 subunits of AMPA receptors were used to detect the expression of AMPA receptors on the cell membrane .

Results : SYN - positive and FM1 - 43 negative fluorescent particles accounted for SYN positive and FM1 - 43 negative fluorescent particles accounted for the proportion of SYN - positive fluorescent particles , and magnesium - free experimental group decreased significantly compared with control group ( P0.05 ) .
The frequency and amplitude of AMPA receptor mEPSCs in the experimental group were significantly higher than those in the normal control group ( P0.05 ) .
Compared with the control group , the proportion of NR1 negative NR1 negative NR1 negative NR1 positive fluorescent particles decreased ( P0.05 ) .
Compared with the control group , the number of fluorescent particles and the number of nuclei of GLuR2 , GLuR4 subunit and SYN coincide with those of the normal control group ( P0.05 ) , and the number of fluorescent particles and the number of nuclei of GLuR1 and GLuR3 subunit and SYN increased significantly ( P0.05 ) .

Conclusion : No magnesium - induced seizures can lead to the decrease of presynaptic silent presynaptic and postsynaptic silencing , and the decreased silencing synaptic poles may be activated to function synaptic connections and become a pathological basis for the development of epilepsy .
The GLUR2 , GLUR 4 subunit decreased , GLuR1 and GLUR3 subunit increased after induced by magnesium - free co - cultured neurons .

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】