【摘要】：目的探討特異性si RNA沉默Nogo-66受體(Ng R)對宮內(nèi)感染所致早產(chǎn)大鼠腦損傷修復(fù)的影響及作用機制。方法孕15 d Sprague-Dawley大鼠分別應(yīng)用RU486和LPS誘導(dǎo)早產(chǎn),隨機選取RU486誘導(dǎo)的早產(chǎn)大鼠為對照組,將LPS誘導(dǎo)的宮內(nèi)感染致腦損傷早產(chǎn)大鼠隨機分為模型組、空載體組和Ng R-si RNA組,每組36只。對照組和模型組僅給予常規(guī)飼養(yǎng),空載體組和Ng R-si RNA組均于出生后第1天(P1)經(jīng)側(cè)腦室1次性注入慢病毒空載體和Ng R-si RNA慢病毒載體后常規(guī)飼養(yǎng)。各組分別于P3、P7、P14時隨機選取8只早產(chǎn)大鼠斷頭取腦。RT-PCR檢測Ng R m RNA表達(dá),Western blot測定活性Rho A蛋白表達(dá),免疫熒光組化檢測小膠質(zhì)細(xì)胞活化程度和少突膠質(zhì)前體細(xì)胞(OPCs)形態(tài),蘇木精-伊紅染色觀察腦組織病理學(xué)改變,P30時行動物行為學(xué)評分。結(jié)果 P3時,Ng R-si RNA組腦組織Ng R m RNA表達(dá)量、活性Rho A蛋白水平顯著低于模型組和空載體組(P0.05);各組Ng R m RNA表達(dá)量與活性Rho A蛋白水平均呈正相關(guān)性(分別r=0.792、0.747、0.827、0.825,P0.05)。免疫熒光組化結(jié)果顯示,Ng R-si RNA組P3時小膠質(zhì)細(xì)胞CD11b熒光強度值較模型組和空載體組明顯減弱(P0.05);各組O4抗體標(biāo)記的OPCs細(xì)胞形態(tài)主要呈現(xiàn)三極突起形態(tài)。病理學(xué)結(jié)果顯示,對照組腦室周圍白質(zhì)結(jié)構(gòu)正常,染色清晰;模型組和空載體組白質(zhì)結(jié)構(gòu)疏松,纖維紊亂,可見軟化灶;Ng R-si RNA組白質(zhì)結(jié)構(gòu)疏松,纖維紊亂相對較輕,膠質(zhì)細(xì)胞增生不明顯,無明顯軟化灶。行為學(xué)評分顯示,Ng R-si RNA組的懸吊實驗評分、活動總路程、平均速度和跨格次數(shù)大于模型組和空載體組,而斜坡實驗時間及中心區(qū)活動時間和路程明顯少于模型組和空載體組(P0.05),但同對照組比較差異均無統(tǒng)計學(xué)意義(P0.05)。結(jié)論 Ng R特異性si RNA可有效沉默宮內(nèi)感染所致腦損傷早產(chǎn)大鼠Ng R基因表達(dá),在腦損傷后的修復(fù)中具有顯著神經(jīng)保護作用。
[Abstract]:Objective to investigate the effect and mechanism of specific si RNA silencing Nogo-66 receptor (Ng R) on brain injury and repair induced by intrauterine infection in premature rats. Methods Sprague-Dawley rats on the 15th day of gestation were induced preterm delivery by RU486 and LPS, respectively. The premature rats induced by RU486 were randomly divided into model group, no-carrier group and Ng R-si RNA group. There are 36 rats in each group. The control group and the model group were only given routine feeding, while the empty carrier group and Ng R-si RNA group were fed with lentivirus empty vector and Ng R-si RNA lentivirus vector through the lateral ventricle on the first day after birth (P1). The brains of 8 premature rats were randomly selected at P3, P7 and P14. The expression of Ng R m RNA, Western blot was detected by RT-PCR. The expression of active Rho A protein was detected. The activation of microglia and the morphology of oligodendrocytes (OPCs) were detected by immunofluorescence histochemical method. The pathological changes of brain tissue were observed by hematoxylin-Ehong staining. The animal behavioral score was evaluated at P30. Results at P3, the expression of Ng R m RNA and the level of active Rho A protein in Ng R-si RNA group were significantly lower than those in model group and empty carrier group (P 0.05). The expression of Ng R m RNA was positively correlated with the level of active Rho A protein (r 鈮，

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