徐明明等
摘要:目的: 探討抑制性神經(jīng)元的表達改變在FMR1基因敲除鼠癲癇發(fā)病機制的作用。方法:應(yīng)用免疫組織化學(xué)染色檢測2 周齡FMR1基因敲除型(KO)和同齡野生型(WT)小鼠海馬抑制性神經(jīng)元數(shù)目(Glutamic acid decarboxylase (GAD) and parvalbumin(PV)陽性神經(jīng)元);應(yīng)用Western blot法檢測上述小鼠海馬組織GAD和PV的含量。結(jié)果:FMR1 KO鼠和WT鼠海馬GAD神經(jīng)元數(shù)目的比較:FMR1 KO鼠海馬GAD神經(jīng)元81±20.134,WT鼠96.11±15.186,差異具有統(tǒng)計學(xué)意義(P=0.006);CA1區(qū)FMR1 KO鼠33.24±7.710,WT鼠40.59±5.738,差異具有統(tǒng)計學(xué)意義(P=0.001);CA3區(qū)FMR1 KO鼠30.94±11.028,WT鼠36.06±6.432差異具有統(tǒng)計學(xué)意義(P=0.041);DG區(qū)FMR1 KO鼠17.82±5.703, WT鼠21.44±3.245,差異不具有統(tǒng)計學(xué)意義(P=0.0669); FMR1 KO鼠和WT鼠的GAD65.67的蛋白水平的比較,GAD65: t=5.152,P=0.000;GAD67: t=4.723,P=0.000, FMR1 KO鼠的GAD65.67的蛋白水平均高于WT鼠。FMR1 KO鼠和WT鼠海馬PV陽性神經(jīng)元數(shù)目的比較:FMR1 KO鼠和WT鼠海馬GAD神經(jīng)元數(shù)目的比較:FMR1 KO鼠海馬PV陽性神經(jīng)元77±16.321,WT鼠90.13±13.126,差異具有統(tǒng)計學(xué)意義(P=0.005);CA1區(qū)FMR1 KO鼠24.13±6.635,WT鼠35.63±6.635,差異具有統(tǒng)計學(xué)意義(P=0.002);CA3區(qū)FMR1 KO鼠25.94±9.123,WT鼠30.12±5.357差異具有統(tǒng)計學(xué)意義(P=0.038);DG區(qū)FMR1 KO鼠14.73±4.473, WT鼠16.12±3.149,差異不具有統(tǒng)計學(xué)意義(P=0.068); KO小鼠海馬中PV表達量分別較WT小鼠減少,KO鼠0.132±0.011,WT鼠0.155±0.013,差異有統(tǒng)計學(xué)意義(P=0.0386)。結(jié)論:FMR-1KO小鼠海馬抑制性神經(jīng)元數(shù)目的減少以及PV蛋白表達減低,可能是誘發(fā)該種小鼠癲癇發(fā)病率高重要原因。KO鼠GAD表達增高,考慮和KO鼠代償性增高有關(guān)。
關(guān)鍵詞 :脆性X綜合征;抑制性神經(jīng)元;癲癇;谷氨酸脫羧酶; 小白蛋白
中圖分類號:R54 文獻標(biāo)識碼: A 文章編號:
深圳市科技計劃項目 項目編號 201303187 FMR1基因敲除鼠PV陽性神經(jīng)元變化及其癲癇發(fā)病機制的研究
Abstract : Objective.This study focused on the expression differences of Glutamic acid decarboxylase (GAD) and parvalbumin(PV) expression between FMR1KO and wild-type (WT) mice in hippocampus. Methods. 2 weeks mice were randomly obtained for immunohistochemistry and Western blotting to detect the expression.Results Number of GAD interneurons in hippocampus : FMR1 KO mice81±20.134,WT mice 96.11±15.186 (P=0.006); CA1: FMR1 KO mice33.24±7.710, WT mice 40.59±5.738(P=0.001); CA3: FMR1 KO mice 30.94±11.028,WT mice 36.06±6.432 (P=0.041); DG: FMR1 KO mice 17.82±5.703, WT mice 21.44±3.245 (P=0.0669); The expressions of GAD65 and GAD67 were significantly increased in the hippocampus tissues in FMR-1KO when compaired to that in WT mice (P=0.000).Number of parvalbumin(PV)-expressing interneuron in hippocampus : FMR1 KO mice 77±16.321,WT mice 90.13±13.126 (P=0.005); CA1: FMR1 KO mice 24.13±6.635, WT mice 35.63±6.635(P=0.002); CA3: FMR1 KO mice 25.94±9.123,WT mice 30.12±5.357 (P=0.038); DG: FMR1 KO mice14.73±4.473, WT mice16.12±3.149 (P=0.068); The expressions of PV were reduced in the hippocampus tissues in FMR-1KO when compaired to that in WT mice (P=0.0386).Conclusion The decreased number of inhibitory interneurons in the hippocampus of FMR1 KO mice demonstrates that interneurons is possible to play an critical role in increased susceptibility to epileptic seizures of FXS.Increased expression of GAD of KO mice is compensatory.
Keywords:Fragile X syndrome; interneurons; Epilepsy; Glutamic acid decarboxylase
脆性X綜合征FXS(Fragile X syndrome,F(xiàn)XS)是最常見的遺傳性智力低下疾病之一,它是一種外顯率不完全的X-連鎖遺傳病(1),最初由細胞遺傳學(xué)檢查發(fā)現(xiàn)X染色體上存在脆性位點而得名,男性發(fā)病率明顯高于女性,臨床突出表現(xiàn)為中至重度的智力發(fā)育障礙,臉狹長及下頜突出等典型面部特征,以及青春期后的巨大睪丸,同時伴有適應(yīng)能力下降、孤獨樣癥狀等異常行為,患者可出現(xiàn)癲癇發(fā)作,探索其發(fā)病機制及藥物治療途徑是目前國際上研究的熱點問題。[1]FMR1 KO鼠的許多行為學(xué)表現(xiàn)與脆性X綜合征患者非常相似,最明顯的包括自發(fā)活動增加、曠場習(xí)慣能力下降、聽源性驚厥易感性增強 [2]。神經(jīng)環(huán)路中興奮性與抑制性功能的協(xié)調(diào)平衡對維持正常大腦的整體功能是必要的,GAD神經(jīng)元抑制系統(tǒng)的減弱與癲癇相關(guān)[3]。Leah Selby等在一周歲KO鼠中研究發(fā)現(xiàn)GABA能中間神經(jīng)元的變化[4]。本研究擬通過研究抑制性神經(jīng)元的改變,為脆性X綜合征的癲癇發(fā)病機制提供進一步的依據(jù),為人們認識脆性X綜合征的發(fā)病機制打下基礎(chǔ)。
1 材料與方法
1.材料
1.1 實驗動物及分組
FMR1基因敲除型(KO)純合子(-/-)及其野生型(WT)純合子(+/+)FVB近交系小鼠由荷蘭伊拉斯塔斯大學(xué)細胞生物學(xué)及遺傳學(xué)研究中心Oostra BA 教授惠贈。實驗前取鼠尾巴進行PCR和Western Blot進行基因型鑒定。
2.方法
2.1免疫組化漂浮法:將FVB小鼠350mg/kg水合氯醛麻醉后,4%多聚甲醛灌注固定腦組織,常規(guī)脫水、冰凍切片,漂洗盒孔內(nèi)注入PBS,各取保存于PBS中的切片6-9片放入孔內(nèi)。用PBS換洗3次,每次5分鐘。每片加1滴(50ul)過氧化物酶阻斷劑(S-P試劑盒中A液),室溫下置振蕩器振蕩孵育30分鐘后,用PBS換洗3次。每片加1滴(50ul)正常非特異性血清(S-P試劑盒中B液),室溫下置振蕩器振蕩孵育60分鐘后,每片加100ul 一抗( GAD 1∶400 PV1:8000以0.2%Triton液稀釋) ,室溫下振蕩器振蕩60分鐘后放入4℃冰箱中,孵育過夜。PBS沖洗7次,吸干孔內(nèi)殘余液體,每片加1滴(50ul)生物素標(biāo)記的二抗(C液),室溫下振蕩孵育60分鐘。PBS沖洗3次,吸干孔內(nèi)殘余液體,每片加1滴(50ul)鏈霉菌抗生物素-過氧化物酶溶液(D液),室溫下振蕩器振蕩孵育30分鐘。PBS沖洗3次,吸干孔內(nèi)殘余液體,DAB液顯色,顯微鏡下觀察顯色結(jié)果,3-5分鐘用PBS液終止反應(yīng),PBS換洗3次,將切片貼于明膠處理過的載玻片上,自然晾干,脫水透明,中性樹膠封片??瞻讓φ諏嶒灒河肞BS代替一抗,其余步驟不變。
2.2 Western Blot法:用RIPA常規(guī)提取皮層、海馬組織的蛋白。15%變性聚丙烯酰胺分離凝膠分離(120V,90分鐘),采用PVDF膜恒壓100V轉(zhuǎn)膜60分鐘, 5%脫脂奶粉溶液封閉膜1h,加入TTBS稀釋的一抗(FMRP 1:1000;PV 1:16000;GAD 1:32000;抗β-actin抗體1:2000),4℃孵育輕搖過夜。加入二抗(抗鼠抗體1:4000,抗兔抗體1:4000),室溫下與膜孵育1h(輕搖),ECL發(fā)光液孵育,x光膠片曝光顯影并進行掃描。
3.數(shù)據(jù)采集和統(tǒng)計
使用SPSSl7.0 for Windows軟件包(SPSS lnc.,Chicago,IL,USA)進行統(tǒng)計分析,兩樣本均數(shù)比較采用t檢驗。以P<0.05具有統(tǒng)計學(xué)意義。
2 結(jié)果
2.1 FMR1 KO鼠海馬GAD神經(jīng)元數(shù)目的變化
FMR1 KO鼠和WT鼠海馬GAD神經(jīng)元數(shù)目的比較:FMR1 KO鼠海馬GAD神經(jīng)元81±20.134,WT鼠96.11±15.186,差異具有統(tǒng)計學(xué)意義(P=0.006);CA1區(qū)FMR1 KO鼠33.24±7.710,WT鼠40.59±5.738,差異具有統(tǒng)計學(xué)意義(P=0.001);CA3區(qū)FMR1 KO鼠30.94±11.028,WT鼠36.06±6.432差異具有統(tǒng)計學(xué)意義(P=0.041);DG區(qū)FMR1 KO鼠17.82±5.703, WT鼠21.44±3.245,差異不具有統(tǒng)計學(xué)意義(P=0.0669)(見表1)
表1 FMR1 KO和WT鼠海馬GAD神經(jīng)元數(shù)目比較
2.2 海馬組織GAD蛋白表達的變化
WT鼠和FMR1 KO鼠海馬組織GAD蛋白的表達量
FMR1 KO鼠和WT鼠的GAD65.67的蛋白水平的比較,GAD65: t=5.152,P=0.000;GAD67: t=4.723,P=0.000, FMR1 KO鼠的GAD65.67的蛋白水平均高于WT鼠。(見表2 圖1)
表2 WT鼠和FMR1 KO鼠海馬組織GAD蛋白的表達量
FMR1 KO鼠的GAD65和GAD67的蛋白水平均高于WT鼠,*P<0.05
圖1 WT鼠和FMR1 KO鼠GAD65 67蛋白印跡
2.3 KO與WT小鼠腦組織PV陽性中間神經(jīng)元數(shù)的表達
FMR1 KO鼠和WT鼠海馬PV陽性神經(jīng)元數(shù)目的比較:FMR1 KO鼠和WT鼠海馬GAD神經(jīng)元數(shù)目的比較:FMR1 KO鼠海馬PV陽性神經(jīng)元77±16.321,WT鼠90.13±13.126,差異具有統(tǒng)計學(xué)意義(P=0.005);CA1區(qū)FMR1 KO鼠24.13±6.635,WT鼠35.63±6.635,差異具有統(tǒng)計學(xué)意義(P=0.002);CA3區(qū)FMR1 KO鼠25.94±9.123,WT鼠30.12±5.357差異具有統(tǒng)計學(xué)意義(P=0.038);DG區(qū)FMR1 KO鼠14.73±4.473, WT鼠16.12±3.149,差異不具有統(tǒng)計學(xué)意義(P=0.068)(見表3)
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