本文選題：海馬神經(jīng)元 + 星形膠質(zhì)細(xì)胞�。� 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：第一部分體外共培養(yǎng)癲癇放電細(xì)胞模型的建立 目的：獲得一種操作簡(jiǎn)單、高效穩(wěn)定的神經(jīng)元和星形膠質(zhì)細(xì)胞共培養(yǎng)方法，，從而建立穩(wěn)定的癲癇細(xì)胞模型。 方法：選用清潔級(jí)孕16-17d的SD孕鼠，清潔級(jí)出生24h以內(nèi)的SD新生鼠，分別采用海馬神經(jīng)元與星形膠質(zhì)細(xì)胞共同培養(yǎng)的混合培養(yǎng)方法（混合培養(yǎng)組）、改良Banker共培養(yǎng)方法（改良Banker共培養(yǎng)組）和插入式培養(yǎng)皿的方法（插入式培養(yǎng)皿組）制作海馬神經(jīng)元與星形膠質(zhì)細(xì)胞共培養(yǎng)模型，進(jìn)而對(duì)三組培養(yǎng)的細(xì)胞分別應(yīng)用無(wú)鎂細(xì)胞外液及正常細(xì)胞外液誘導(dǎo)3h，制作癲癇模型及正常對(duì)照，每種方法重復(fù)3次。采用免疫熒光檢測(cè)共培養(yǎng)中神經(jīng)元特異性烯醇化酶(neuron specificenolase,NSE)及星形膠質(zhì)細(xì)胞膠質(zhì)纖維酸性蛋白(glial fibrillary acidicprotein，GFAP）表達(dá)情況，鑒定比較培養(yǎng)體系中神經(jīng)元及星形膠質(zhì)細(xì)胞純度；應(yīng)用膜片鉗全細(xì)胞模式的電流鉗記錄不同培養(yǎng)組中神經(jīng)元細(xì)胞驚厥放電情況并比較放電成功率。 結(jié)果：（1）插入式培養(yǎng)皿組神經(jīng)元純度為[94.3%-100%]星形膠質(zhì)細(xì)胞純度為[93%-100%],較改良Banker共培養(yǎng)組得到的神經(jīng)元[87.6%-95%]及星膠[88.2%-93%]純度更純。（2）插入式培養(yǎng)皿組95％左右的海馬神經(jīng)元（ｎ＝10）能夠記錄到陣發(fā)性持續(xù)棘波樣爆發(fā)和陣發(fā)性去極化樣偏移（Paroxysmal depolarizing shifts,PDSs）樣發(fā)作；改良Banker共培養(yǎng)組有93%左右的海馬神經(jīng)元（ｎ＝10）能夠記錄到陣發(fā)性持續(xù)棘波樣爆發(fā)和PDSs；混合培養(yǎng)組這一比例為90%左右。（3）經(jīng)無(wú)鎂細(xì)胞外液處理后72h，插入式培養(yǎng)皿組仍有85％的神經(jīng)元（ｎ＝10）能夠記錄到陣發(fā)性持續(xù)棘波樣爆發(fā)和PDSs。而改良Banker共培養(yǎng)組有82%左右的海馬神經(jīng)元（ｎ＝10）能夠記錄到陣發(fā)性持續(xù)棘波樣爆發(fā)和PDSs；混合培養(yǎng)組這一比例有80%左右。 結(jié)論：三種方法均可建立成功的體外共培養(yǎng)癲癇細(xì)胞模型，但插入式培養(yǎng)皿共培養(yǎng)方法建立的模型更簡(jiǎn)便，細(xì)胞培養(yǎng)純度更高。 第二部分：體外共培養(yǎng)癲癇細(xì)胞模型中沉默突觸的轉(zhuǎn)化及AMPA受體亞基的變化 目的：采用體外共培養(yǎng)癲癇細(xì)胞模型證實(shí)癲癇發(fā)作可否導(dǎo)致沉默突觸的激活轉(zhuǎn)化及AMPA受體各亞基的組成變化。 方法：在前期證實(shí)插入式培養(yǎng)皿方法建立穩(wěn)定的體外共培養(yǎng)細(xì)胞模型的基礎(chǔ)上，對(duì)插入式培養(yǎng)皿共培養(yǎng)的神經(jīng)元及星形膠質(zhì)細(xì)胞分為兩組，無(wú)鎂實(shí)驗(yàn)組采用無(wú)鎂細(xì)胞外液培養(yǎng)3h誘導(dǎo)反復(fù)自發(fā)性驚厥樣癲癇放電模型。正常對(duì)照組以含鎂正常細(xì)胞外液代替無(wú)鎂細(xì)胞外液培養(yǎng)3h，實(shí)驗(yàn)重復(fù)3次。應(yīng)用Synapto GreenTM C4(FM1-43）及突觸素(Synaptophysin,SYN）雙標(biāo)檢測(cè)有無(wú)突觸前沉默突觸的變化；膜片鉗記錄-氨基-3-羥基-5-甲基-4-異惡唑丙酸（a-mino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor，AMPA)受體介導(dǎo)的微興奮性突觸后電流（micro excitatory postsynapticcurrents,mEPSCs）及NR1與GLuR1亞基免疫熒光雙標(biāo)反映有無(wú)突觸后沉默突觸的改變；SYN分別與AMPA受體的GLuR1、GLuR2、GLuR3、GLuR4亞基進(jìn)行免疫熒光雙標(biāo)，檢測(cè)細(xì)胞膜上AMPA受體各亞基表達(dá)情況。 結(jié)果：FM1-43及SYN雙標(biāo)檢測(cè)顯示SYN陽(yáng)性而FM1-43陰性熒光顆粒占SYN陽(yáng)性熒光顆粒的比例，無(wú)鎂實(shí)驗(yàn)組較正常對(duì)照組有所減少（P0.05）；無(wú)鎂實(shí)驗(yàn)組AMPA受體mEPSCs的頻率及幅度均較正常對(duì)照組出現(xiàn)明顯增加（P0.05）；無(wú)鎂實(shí)驗(yàn)組GLuR1陰性NR1陽(yáng)性的熒光顆粒占NR1陽(yáng)性熒光顆粒比例較正常對(duì)照組發(fā)生減少（P0.05）；無(wú)鎂實(shí)驗(yàn)組可見(jiàn)GLuR2、GLuR4亞基與SYN重合的熒光顆粒個(gè)數(shù)/細(xì)胞核個(gè)數(shù)較正常對(duì)照組減少（P0.05）,GLuR1、GLuR3亞基與SYN重合的熒光顆粒個(gè)數(shù)/細(xì)胞核個(gè)數(shù)較正常對(duì)照組增加（P0.05）。 結(jié)論：無(wú)鎂誘導(dǎo)的癲癇發(fā)作可導(dǎo)致突觸前沉默突觸及突觸后沉默突觸的減少，減少的沉默突觸極有可能激活轉(zhuǎn)化為功能突觸，并成為癲癇發(fā)生發(fā)展的病理基礎(chǔ)；無(wú)鎂誘導(dǎo)共培養(yǎng)神經(jīng)元癲癇放電后可使膜GLuR2、GLuR4亞基減少,膜GLuR1、GLuR3亞基增加。
[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 .

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.1

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本文編號(hào)：1753187