【摘要】：目的通過(guò)觀察戊四氮點(diǎn)燃大鼠模型海馬FAKpY397蛋白的動(dòng)態(tài)表達(dá)變化,結(jié)合苔蘚纖維出芽的程度,探討其在苔蘚纖維出芽及癲癇致癇過(guò)程中的作用。 方法將135只健康的成年雄性Sprague-Dawley (SD)大鼠,隨機(jī)分為實(shí)驗(yàn)組90只和對(duì)照組45只。實(shí)驗(yàn)組每天腹腔注射亞驚厥劑量的PTZ(即戊四氮—35mg/kg,對(duì)照組每天腹腔注射同等量生理鹽水,成功點(diǎn)燃后停止注射(癇性發(fā)作等級(jí)評(píng)定標(biāo)準(zhǔn)參照Racine標(biāo)準(zhǔn))。根據(jù)首次腹腔注射后3天,1周,2周,4周和6周將實(shí)驗(yàn)組及對(duì)照組分別隨機(jī)分為五個(gè)亞組。根據(jù)不同的實(shí)驗(yàn)方法及用途再將每個(gè)不同的再亞組隨機(jī)細(xì)分為三個(gè)小組：一組用于免疫熒光及免疫組化染色,檢測(cè)各不同時(shí)間點(diǎn)實(shí)驗(yàn)組與對(duì)照組FAKpY397蛋白在海馬的表達(dá)分布及表達(dá)量的變化；一組用于Western Blot,檢測(cè)不同時(shí)間點(diǎn)不同組別大鼠海馬區(qū)FAKpY397蛋白總表達(dá)量及其變化趨勢(shì)；另一組用于Timm染色,檢測(cè)海馬苔蘚纖維出芽情況并進(jìn)行評(píng)分。其中實(shí)驗(yàn)組每小組各6只,對(duì)照組每小組各3只。 結(jié)果 1.動(dòng)物行為學(xué) 隨著每天相同亞驚厥劑量PTZ經(jīng)腹腔注射給藥,實(shí)驗(yàn)組大鼠所表現(xiàn)的癇性發(fā)作反應(yīng)逐漸加重,多數(shù)給藥后第21-28天(23.1±-2.3天)被成功點(diǎn)燃。PTZ致癇模型點(diǎn)燃率為93.75%,而死亡率為4.44%。被成功點(diǎn)燃的大鼠可見Racine I至RacineⅢ級(jí)的自發(fā)癇性發(fā)作。對(duì)照組大鼠未出現(xiàn)癇性發(fā)作,行為正常。 2.Western Blot結(jié)果顯示：在PTZ點(diǎn)燃致癇過(guò)程中,海馬FAKpY397蛋白總表達(dá)量于再PTZ腹腔注射后3d稍下降,1周時(shí)開始增加,4周達(dá)高峰,6周下降,仍高于對(duì)照組。除3天組外(0.05),實(shí)驗(yàn)組各時(shí)間點(diǎn)與對(duì)照組間比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。 3.免疫熒光及組化結(jié)果顯示：FAKpY397在海馬各區(qū)有表達(dá),主要分布于胞漿；免疫組化半定量分析示CA3區(qū)FAKpY397蛋白表達(dá)于PTZ腹腔注射后3d開始增加,4周達(dá)高峰,6周下降,仍高于對(duì)照組。實(shí)驗(yàn)組中除1周組與2周組進(jìn)行比較無(wú)統(tǒng)計(jì)學(xué)差異(P0.05),其余各時(shí)間點(diǎn)兩兩比較及實(shí)驗(yàn)組與對(duì)照組對(duì)應(yīng)時(shí)間點(diǎn)比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。 4.苔蘚纖維出芽評(píng)分 對(duì)照組CA3區(qū)未見苔蘚纖維出芽或僅見輕微出芽,苔蘚纖維出芽評(píng)分在各時(shí)間點(diǎn)間兩兩比較均無(wú)統(tǒng)計(jì)學(xué)差異。實(shí)驗(yàn)組CA3區(qū)苔蘚纖維出芽評(píng)分從PTZ腹腔注射后3天開始增加,4周時(shí)達(dá)高峰,6周下降。實(shí)驗(yàn)組中除4周與6周間比較無(wú)統(tǒng)計(jì)學(xué)差異(0.05),其余各時(shí)間點(diǎn)兩兩比較及實(shí)驗(yàn)組與對(duì)照組對(duì)應(yīng)時(shí)間點(diǎn)比較均有統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論 FAKpY397蛋白在海馬CA3區(qū)表達(dá)增多可能對(duì)于苔蘚纖維出芽具有促進(jìn)作用進(jìn)而誘發(fā)癲癇發(fā)作。
[Abstract]:Objective to investigate the role of pentylenetetrazol (PTZ) in the process of mossy fiber sprouting and epilepsy by observing the dynamic expression of FAKpY397 protein in hippocampus of rats with pentylenetetrazol (PTZ) kindling and combining with the degree of mossy fiber sprouting. Methods 135 healthy male Sprague-Dawley (SD) rats were randomly divided into experimental group (n = 90) and control group (n = 45). Subconvulsant dose of PTZ (pentylenetetrazo-35 mg / kg) was injected intraperitoneally every day in the experimental group, and the control group was injected with the same amount of normal saline daily. The injection was stopped after successful kindling (Racine criterion was used to evaluate the grade of epileptic seizures). According to 3 days, 1 week, 2 weeks, 4 weeks and 6 weeks after the first intraperitoneal injection, the experimental group and the control group were randomly divided into five subgroups. Each subgroup was subdivided randomly into three groups according to different experimental methods and uses: one group was used for immunofluorescence and immunohistochemical staining. The expression distribution and quantity of FAKpY397 protein in hippocampus of experimental group and control group were detected at different time points. One group was used to detect the total expression of FAKpY397 protein and its changing trend at different time points in different groups of rats, the other group was used for Timm staining to detect and score the budding of mossy fibers in hippocampus. There were 6 rats in each group in the experimental group and 3 rats in the control group. Result 1. With the same daily subconvulsive dose of PTZ administered intraperitoneally, the epileptic seizure response in the experimental group increased gradually. Most of the drugs were ignited 21-28 days (23.1 鹵-2.3 days) after administration. The kindling rate of epileptic model induced by PTZ was 93.755.The death rate was 4.44g. Spontaneous epileptic seizures of Racine I to Racine 鈪，

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