1、電針治療對(duì)大鼠脊髓損傷后Nogo_A蛋白表達(dá)的影響         08-11-29 15:32:00     編輯：studa20             作者：李振鵬，孔抗美，陳育春，郭天明，陳澤鋒，劉加貝   【摘要】  目的：探討電針治療對(duì)大鼠脊髓損傷(SCI后Nogo_A蛋白表達(dá)的影響。方法：以96只2月齡Wistar

2、大鼠為受試對(duì)象，SCI組于T9處行脊髓全橫斷，治療組予電針治療，采用Western blot測(cè)定各時(shí)間點(diǎn)及各組脊髓Nogo_A表達(dá)及變化,并以蘇木精_伊紅和免疫組化染色對(duì)受損脊髓進(jìn)行形態(tài)學(xué)觀察。結(jié)果：SCI后Nogo_A蛋白表達(dá)呈遞增趨勢(shì)，于14d后最強(qiáng)；經(jīng)電針治療，Nogo_A表達(dá)減少。結(jié)論：Nogo_A是SCI后脊髓神經(jīng)纖維再生的主要抑制因子，電針治療對(duì)Nogo_A表達(dá)具有明顯的抑制作用。 【關(guān)鍵詞】  電針；脊髓損傷；Nogo_A蛋白    AbstractObjective:  To study the effect of electr

3、oacupuncture on the expression of Nogo_A protein in rats with spinal cord injury(SCI. Methods: Ninety_six adult Wistar rats were randomly assigned into control group(A, SCI group(B, sham_treatment group(Cand electroacupuncture treatment group(D. Group B, C and D were received transect SCI at the T9

4、vertebrae level while group A was just opened the vertebra and their spinal cords were not wounded. Group D was treated with electroacupuncture. All the spinal cords of the trauma section were taken out at different time points after o_peration. Western blot and immunohistochemical methods were used

5、 to examine the change of the expression of Nogo_A in these tissue. Results： Just minim Nogo_A was found in the control group, while the manifold expression of Nogo_A was found after operation，especially at the end of the second week, the expression was inhibited by electroacupuncture. Conclusion: N

6、ogo_A is a major suppressor factor of spinal cord nerve fiber regeneration after SCI, and the electroacupuncture can obviously inhibit the expression of Nogo_A protein.    Key Wordselectroacupuncture，spinal cord injury，Nogo_A protein    脊髓損傷(SCI臨床常見(jiàn)，致癱率較高。電針是治療SCI的重要方法,

7、其療效已獲得臨床實(shí)踐和動(dòng)物實(shí)驗(yàn)1_2的證實(shí)，但其作用機(jī)制尚未完全明確。本實(shí)驗(yàn)通過(guò)電針治療SCI大鼠，于不同時(shí)間點(diǎn)采集大鼠受傷脊髓段，以Western blot測(cè)定其Nogo_A蛋白表達(dá)，探討電針治療SCI的機(jī)制。    1  材料與方法    1.1  動(dòng)物模型制作    Wistar大鼠(廣東省實(shí)驗(yàn)動(dòng)物中心提供96只(2月齡，體質(zhì)量220270，雌雄各半，隨機(jī)分為對(duì)照組(A組。24只和SCI組(72只。SCI組于T9處行脊髓全橫斷，然后再隨機(jī)分為損傷組(B組，硬膜外腔不留置電極、治療

8、對(duì)照組(C組，留置電極但不通電和電流刺激治療組(D組，損傷部位上下端的椎間隙硬膜外腔留置電極，予G6805_2型多用治療儀治療，強(qiáng)度以大鼠有輕微肌肉抽動(dòng)和無(wú)明顯不安嘶叫為適，每天治療30min，療程至處死動(dòng)物取標(biāo)本前為止，各組24只。各組分別于術(shù)后1、7、14和28d取各損傷部位的脊髓組織(每組3只置_70冰箱保存?zhèn)溆谩?#160;   1.2  Western blot測(cè)定Nogo_A蛋白    取各組脊髓樣本40mg，加入細(xì)胞裂解液30L，冰浴下研磨勻漿，煮沸后7500r/min離心取上清液，再加入等體積的2×SDS上樣

9、緩沖液，提取總蛋白，電泳，結(jié)束后按常規(guī)轉(zhuǎn)膜。在膜封閉液內(nèi)封閉，4保存6h，加一抗，與封閉液混勻后4冰箱過(guò)夜。TBS液清洗3次，加入封閉液后滴加二抗(1400稀釋，室溫振蕩混勻1h，TBS清洗膜3次，加化學(xué)發(fā)光劑，在暗室條件下顯影和定影制成X光片。將各組X光片掃描后采用C_IMAGING SYSTEMS軟件進(jìn)行灰度分析，以Nogo_A蛋白和內(nèi)標(biāo)蛋白(actin灰度的比值作為Nogo_A蛋白的相對(duì)表達(dá)量。    1.3  病理標(biāo)本制備    術(shù)后1、7、14和28d時(shí),隨機(jī)取大鼠每組3只，質(zhì)量濃度20g/L戊巴比妥鈉(40mg/

10、kg腹腔注射麻醉后開(kāi)胸，自左心室插管至主動(dòng)脈，剪開(kāi)右心耳，先灌注PBS 150mL，再灌注質(zhì)量濃度40g/L多聚甲醛250mL，固定。2后取出以傷段為中心的長(zhǎng)約20mm(810脊髓組織，放入相同固定液繼續(xù)固定，24后常規(guī)石蠟包埋，連續(xù)切片(厚約4m，分別進(jìn)行蘇木精_伊紅及Nogo_A抗體免疫組化染色，觀察受損節(jié)段組織學(xué)改變。    1.4  免疫組化染色    免疫組化染色采用ABC法，切片脫蠟至水，微波修復(fù)抗原，滴加封閉液，及抗Nogo_A工作液，室溫37 1d，PBS沖洗，DAB顯色，蘇木精輕度對(duì)比染色，脫水、封固。實(shí)驗(yàn)中設(shè)立嚴(yán)格的正常血清對(duì)照和陰性對(duì)照。    1.5  統(tǒng)計(jì)學(xué)處理    結(jié)果以±s表示，分別比較A、B、C組各時(shí)間點(diǎn)Nogo_A表達(dá)，采用t檢驗(yàn)，0.05，SPSS10.0統(tǒng)計(jì)軟件分析。    第3期  李振鵬等.電針治療對(duì)大鼠脊髓損傷后Nogo_A蛋白表達(dá)的影響    2  結(jié)果&#