【作者】 罗景华；

【导师】 任榕娜； 杨朋范；

【作者基本信息】 福建医科大学 ， 儿科学， 2010， 硕士

【摘要】 【目的】研究胶质纤维酸性蛋白(GFAP)、谷氨酸-胱氨酸转运体(GLAST)及神经胶质谷氨酸转运体(GLT-1)在颞叶内侧癫痫患者海马区胶质细胞中的表达情况,探讨难治性癫痫的发病机制;探讨影响颞叶癫痫患者海马组织中神经元丢失的相关因素。【方法】第一、二部分:取40例难治性颞叶内侧癫痫患者在手术中切除的海马组织,根据在光镜下观察到的神经元丢失情况,分为海马硬化组(A组)23例和海马非硬化组(B组)17例。通过免疫组化法检测两组中GFAP、GLAST、GLT-1蛋白阳性表达的情况;通过逆转录-聚合酶链反应(RT-PCR)方法检测两组中GLASTmRNA及GLT-1mRNA的表达情况。利用quantity one凝胶成像管理系统进行图像分析,检测GLAST基因,GLT-1基因,β-actin条带的灰度值,结果以GLAST/β-actin,GLT-1/β-actin条带的灰度值比进行半定量分析。第三部分:对55例颞叶癫痫患儿临床资料进行回顾性病例对照研究,运用单因素及多因素方法分析其海马组织中神经元丢失的发生与临床病理因素间的关系。【结果】1、硬化组(A组)与非硬化组(B组)相比,A组中胶质细胞明显增生、肥大,突起增多,有些胶质细胞突起交错成网状。2、A组中海马总区域、CA1区、CA2区及齿状回中GFAP蛋白的表达量O.D.值为别为:(55.70±26.92)、(102.52±23.55)、(34.46±8.69)、(48.01±6.24 );B组中相应区域中GFAP的表达量为别为: (26.54±7.85)、(36.05±3.71)、(17.03±1.87)、(24.44±2.98)。两组间比较,各区之间差别有统计学意义(P<0.01)。3、A组中海马总区域、齿状回中GLAST蛋白的表达量O.D.值分别为:(40.50±6.02)、(36.73±6.50);B组中相应区域中GLAST蛋白的表达量分别为:(41.09±5.92)、(43.29±10.61)。两组间比较差别无统计学意义(P>0.05)。A组中CA1区、CA2区中GLAST蛋白的表达量O.D.值分别为:(45.84±7.23)、(52.67±11.51);B组中相应区域中GLAST蛋白的表达量分别为:(48.56±10.62)、(39.39±9.48)。两组间比较差别有统计学意义(P<0.05)。4、A组中海马总区域、CA1区、CA2区中GLT-1蛋白的表达量O.D.值分别为:(102.54±3.78 )、(50.57±28.60)、(114.53±3.84);B组中海马总区域、CA1区、CA2区中GLT-1蛋白的表达量O.D.值分别为:(178.17±61.23)、(123.40±3.58)、(109.27±4.14)。两组间比较差异有统计学意义(P<0.05)。A组B组与中齿状回中GLT-1蛋白的表达量分别为:( 107.41±3.97)、(107.44±4.21)。两组间比较差异无统计学意义(P>0.05)。5、A组中GLASTmRNA的相对表达量为(0.665±0.22),与B组(0.703±0.16)比较,两组间差别无显著性(P>0.05)。6、A组中GLT-1mRNA的相对表达量为(1.025±0.18),与B组(1.032±0.21)比较,两组间差别无显著性(P>0.05)。7、55例颞叶癫痫患儿中,海马神经元丢失47例,总发生率85.45%(47/55) ,单因素分析发现性别、癫痫家族史、有无先兆、有无损伤因子、用药的种类(单药或联合用药)与癫痫患者海马神经元的丢失程度无关,而发病年龄(≤3岁)、发病病程(>2年)、发作频率(≥1次/月)、发作持续时间(≥60s)、发作的类型(全身性发作)等为其影响因素。8、多因素Logistic回归结果显示发作频率、发作持续时间、发作的类型为海马神经元丢失发生的独立影响因素。【结论】1、难治性颞叶内侧癫痫患者海马各区中GFAP表达增加,提示癫痫反复发作后胶质细胞反应性增生。2、GLAST、GLT-1在海马CA1区表达减少、CA2区表达量反而增加,提示癫痫反复发作后海马区谷氨酸转运体的重新分布可能是难治性颞叶癫痫发病机制之一。3、谷氨酸转运体GLASTmRNA及GLT-1mRNA没有变化,而其蛋白表达减少,提示谷氨酸转运体转录后机制异常可能是难治性颞叶癫痫的发病机制之一。4、多因素分析结果证实癫痫患者海马神经元丢失的发生与发病年龄、发病病程、发作频率、发作持续时间、发作的类型等有关,对癫痫患者早期干预治疗十分重要。更多还原

【Abstract】 【Objective】To determine the expression level of glial fibrillary acidic protein (GFAP), Glutamate - cystine transporter (GLAST) and Glial glutamate transporter 1 (GLT-1) in human mesial temporal lobe epilepsy. And to find the independent factors for the neuron loss in hippocampus in human mesial temporal epilepsy.【Methods】1. Part 1 and 2: The medial temporal cortex and hippocampus from 40 patients with mesial temporal lobe epilepsy were harvested in surgery. The surgically resected hippocampus was classified into two groups. The expression and alteration of GFAP, GLAST, and GLT-1 in the specimens was assessed using immunohistochemical staining. The expression and alteration of GLASTmRNA and GLT-1mRNA in the specimen was assessed using reverse transcription polymerase chain reaction (RT-PCR). The results were observed and compared withβ-acti, quantified and compared by the Software Quantity-One.2. Part 3: A case-control study was carried out in 55 cases with temporal lobe epilepsy diagnosed by EEG ,clinic and pathological .Unvaried analysis and multivariate logistic regression analysis were performed to find the correlation between the loss of neuron in hippocampus and clinic pathological factors.【Results】1. The expression of GFAP increased in various districts of the hippocampus. A change in astrocyte morphology was observed, with astrocyte cell bodies becoming larger and processes becoming more stellate and often longer in length.2.The levels of GFAP were(55.70±26.92)(,102.52±23.55)(,34.46±8.69)(,48.01±6.24 )in total hippocampal area,CA1,CA2 and dentate gyrus in the specimens from A, respectively. Compared with group A, levels of GFAP were (26.54±7.85),(36.05±3.71),(17.03±1.87),(24.44±2.98)in the specimens from B, There were significantly difference in the two groups (P both < 0.01 ).3.The expressing of GLAST in CA1 and CA2 indicated decreases(P<0.05) from A, whereas, non-significantly change in total hippocampal area and DG compare to group B (P>0.05). the levels of GLAST were(40.50±6.02)(,45.84±7.23)(,52.67±11.51),(36.73±6.50)in total hippocampal area,CA1,CA2 and DG in the specimens from A while(41.09±5.92),(48.56±10.62),(39.39±9.48),(43.29±10.61)in group B.4.The expression of GLT-1 in the sclerotic hippocampus (CA1) decreases (P<0.05), however, the there were up regulation of them in the CA2 (P<0.05) in the specimens from A. the total expression of GLT-1 in the sclerotic hippocampus showed (150.57±28.60), with (50.57±28.60), (114.53±3.84) in CA1 and CA2 respectively; In group B, the total expression showed (178.17±61.23), with (123.40±3.58), (109.27±4.14) in CA1 and CA2 respectively.5. The expression of GLASTmRNA were (0.665±0.22),(0.703±0.16)in group A and group B respectively. There was non-significant change in the two groups.6. the expression of GLT-1mRNA were(1.025±0.18), (1.032±0.21) in group A and group B respectively. There was also non-significant change in the two groups.7. The incidence of the loss of neuron in hippocampus in temporal lobe epilepsy was 47 (85%). Single variable analysis showed that sex, familial history of epilepsy , aura, damage factor and Medication are not concerned with the loss of neuron in hippocampus, however ,age of onset (≤3 years) , course of disease(>2 years) , seizure frequency(≥1moth) , persistence time(≥60s), generalized seizure all associated with the incidence of the loss of neuron in hippocampus.8. Multivariate logistic regression analysis showed that the independent influencing factors for the loss of neuron in hippocampus in children with temporal epilepsy Included seizure frequency, persistence time and the tape of seizure.【Conclusions】1. The expression of GFAP increased in various districts of the hippocampus.2. While GLAST and GLT-1 reduction in the hippocampus CA1 area but increased in CA2 area in mesial temporal lobe epilepsy. It suggesting that the redistribution of gial glutamate transporter in hippocampus after seizures may be associated with the pathogenesis of intractable temporal lobe epilepsy.3. These data suggest that the post-transcription Modification of GLT-1mRNA and GLASTmRNA in hippocampus may be associated with the pathogenesis of intractable temporal lobe epilepsy.4. The development of the loss of neuron in hippocampus may be associated with many factors including age of onset, course of disease, seizure frequency, persistence time and generalized seizure. In order to lower the incidence of the loss of neuron, early intervening treatment is very important.更多还原