發(fā)布時(shí)間：2018-06-19 08:23

  本文選題：彌漫性軸索損傷 + 促炎因子　； 參考：《河北醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:彌漫性軸索損傷(diffuse axonal injury,DAI)是指頭部遭受特殊鈍性外力產(chǎn)生加速運(yùn)動(dòng)時(shí),在剪應(yīng)力的作用下腦內(nèi)發(fā)生的廣泛分布的以神經(jīng)軸索腫脹、斷裂為特征的一系列病理生理變化,可單獨(dú)出現(xiàn)也可同其他病理改變相伴發(fā)生。其典型表現(xiàn)為意識(shí)障礙,甚至可長期處于去大腦強(qiáng)直或植物生存狀態(tài),預(yù)后差。80%的交通工具所致的顱腦損傷和50%的嚴(yán)重顱腦損傷病人存在彌漫性軸索損傷,有學(xué)者提出1/3的顱腦損傷死亡案例與彌漫性軸索損傷有關(guān)[1,2]。彌漫性軸索損傷的致死率和致殘率均高,發(fā)病機(jī)制復(fù)雜,臨床卻缺乏有效的檢測(cè)手段。此外,彌漫性軸索損傷還可導(dǎo)致智能缺損、雙側(cè)感覺運(yùn)動(dòng)障礙、交感神經(jīng)增強(qiáng)病癥及癲癇等多種并發(fā)癥[3～6],成為法醫(yī)學(xué)損傷評(píng)定的難題。 已有研究表明,彌漫性軸索損傷造成外傷后的腦功能障礙不僅是由于最初的機(jī)械外力對(duì)組織的剪切擠壓等作用所致的原發(fā)性損傷,很大程度上與損傷后數(shù)小時(shí)甚至數(shù)天中發(fā)生的復(fù)雜的神經(jīng)元“二次打擊”(神經(jīng)元繼發(fā)性損傷)有關(guān)[7,8]。本實(shí)驗(yàn)室前期實(shí)驗(yàn)已經(jīng)觀察到彌漫性軸索損傷后大量的小膠質(zhì)細(xì)胞被激活,離體實(shí)驗(yàn)也已經(jīng)證實(shí),促炎性細(xì)胞因子能激活小膠質(zhì)細(xì)胞。但是,在彌漫性軸索損傷整體模型中,促炎因子的表達(dá)特點(diǎn)如何?其與小膠質(zhì)細(xì)胞的活化關(guān)系如何,小膠質(zhì)細(xì)胞的活化與軸索的繼發(fā)性損傷的關(guān)系如何?其將值得深入研究。 本實(shí)驗(yàn)旨在參照1994年[9]Marmarou A等的方法建立大鼠彌漫性軸索損傷模型,通過免疫組織化學(xué)方法,觀察彌漫性軸索損傷后激活的小膠質(zhì)細(xì)胞分布特點(diǎn)及小膠質(zhì)細(xì)胞和神經(jīng)元表達(dá)炎癥介質(zhì)IL-1β、TNF-α和IFN-γ在損傷后不同時(shí)間點(diǎn)表達(dá)的動(dòng)態(tài)變化,探討DAI中腦干區(qū)域促炎細(xì)胞因子IL-1β、TNF-α和IFN-γ的動(dòng)態(tài)變化,及其與小膠質(zhì)細(xì)胞激活的關(guān)系。 方法:Sprague-Dawley大鼠(體重: 260±21g)隨機(jī)分為11組:正常對(duì)照組、手術(shù)對(duì)照組、損傷組;包括打擊后不同時(shí)相組:30min、1h、3h、6h、12h、24h、48h、72h、7d。參照Marmarous A等的方法制備大鼠腦彌漫性軸索損傷模型。 正常對(duì)照組大鼠不作任何處理;手術(shù)對(duì)照組大鼠給予手術(shù)及打擊前處理,但不予打擊;損傷組按上述方法給予打擊,根據(jù)打擊后按不同時(shí)間給予10%水合氯醛麻醉后全身灌注固定取全腦,制作石蠟切片,采用HE染色、GLESS氏嗜銀染色方法觀察大鼠腦組織的病理改變。經(jīng)以上方法驗(yàn)證符合標(biāo)準(zhǔn)的大鼠列入打擊組,用免疫組織化學(xué)的方法測(cè)定腦組織中腦干損傷區(qū)IL-1β、IFN-γ和TNF-α的表達(dá)狀況。 統(tǒng)計(jì)數(shù)據(jù)采用均數(shù)士標(biāo)準(zhǔn)差(Mean士SD)表示,用SPSS統(tǒng)計(jì)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,各組均數(shù)的比較行單因素方差分析(ANOVA),用最小顯著差法(LSD)作兩兩比較,以p0.05為有顯著性差異。 結(jié)果: 1成功建立彌漫性軸索損傷模型:常規(guī)HE染色觀察,腦干的損傷最嚴(yán)重,因而采用連續(xù)切片對(duì)比觀察腦干HE染色及GLESS氏嗜銀染色的形態(tài)學(xué)改變,結(jié)果發(fā)現(xiàn)HE染色組織結(jié)構(gòu)疏松水腫的部位,GLESS氏嗜銀染色可見軸索的腫脹、扭曲等改變;而且水腫越嚴(yán)重,軸索腫脹、扭曲、斷裂就越明顯。 2β-APP的免疫組織化學(xué)染色:正常對(duì)照組和假手術(shù)對(duì)照組軸索無陽性表達(dá),神經(jīng)元胞體中可見弱表達(dá)。損傷組30min-6h組大鼠腦干神經(jīng)元胞體、軸突可見多量β-APP的陽性表達(dá);48h-72h時(shí)腫脹、斷裂的軸索β-APP表達(dá)量達(dá)到高峰,周圍多量小膠質(zhì)細(xì)胞聚集。β-APP陽性表達(dá)是軸漿運(yùn)輸障礙的結(jié)果,也是神經(jīng)元功能受損的表現(xiàn)。 3正常對(duì)照組與假手術(shù)對(duì)照組大鼠腦組織IL-1β弱表達(dá),陽性神經(jīng)元胞漿淡染,呈棕黃色;損傷后30min腦干腹側(cè)及其深部陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)明顯增高,與對(duì)照組比有顯著性差異(P0.05)。陽性表達(dá)在6h達(dá)到高峰,其后神經(jīng)元和小膠質(zhì)細(xì)胞表達(dá)呈逐漸下降趨勢(shì),24h形成低谷,但較對(duì)照組仍然有顯著性差異(P0.05),48h后神經(jīng)元陽性細(xì)胞數(shù)表現(xiàn)呈上升趨勢(shì),與24h比有顯著性差異(P0.05),在72h形成小高峰,7d陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)逐漸回落至低水平,較對(duì)照組仍有顯著性差異(P0.05)。 4正常對(duì)照組與假手術(shù)對(duì)照組大鼠腦組織IFN-γ弱表達(dá),陽性神經(jīng)元淡染,呈棕黃色;損傷后30min腦干腹側(cè)及其深部陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)明顯增高,與對(duì)照組比有顯著性差異(P0.05)。陽性表達(dá)在6h達(dá)到高峰,其后神經(jīng)元和小膠質(zhì)細(xì)胞表達(dá)呈逐漸下降趨勢(shì),在24h形成低谷,但較對(duì)照組仍然有顯著性差異(P0.05),48h以后神經(jīng)元陽性細(xì)胞數(shù)表現(xiàn)略呈上升趨勢(shì),與24h比有顯著性差異(P0.05),在72h形成小高峰,7d陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)逐漸回落至低水平,較對(duì)照組仍有顯著性差異(P0.05)。 5正常對(duì)照組與假手術(shù)對(duì)照組大鼠腦組織TNF-α弱表達(dá),陽性神經(jīng)元淡染,呈棕黃色;損傷后30min腦干腹側(cè)及其深部陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)明顯增高,與對(duì)照組比有統(tǒng)計(jì)學(xué)意義(P0.05)。陽性表達(dá)在6h達(dá)到高峰,其后神經(jīng)元和小膠質(zhì)細(xì)胞表達(dá)呈逐漸下降趨勢(shì),在24h形成低谷,但較對(duì)照組仍然有明顯的差異(P0.05),48h以后神經(jīng)元陽性細(xì)胞數(shù)表現(xiàn)略呈上升趨勢(shì),與24h比有顯著性差異(P0.05),在72h形成小高峰,7d陽性神經(jīng)元和小膠質(zhì)細(xì)胞數(shù)逐漸回落至低水平,較對(duì)照組仍有顯著性差異(P0.05)。 結(jié)論:采用Marmarou A的自由落體撞擊模型復(fù)制了大鼠彌漫性軸索損傷模型,觀察到DAI后大鼠腦干軸索損傷區(qū)域IL-1β、TNF-α、IFN-γ的表達(dá)。結(jié)果顯示,促炎性細(xì)胞因子IL-1β、TNF-α、IFN-γ在軸索損傷后表達(dá)增加,且呈一定的時(shí)間規(guī)律性,說明小膠質(zhì)細(xì)胞活化和促炎性細(xì)胞因子的表達(dá)增加在DAI后繼發(fā)性腦組織損傷中發(fā)揮一定得作用。
[Abstract]:Objective : diffuse axonal injury ( DAI ) refers to a series of pathological changes of axonal swelling and fracture in the brain under the action of shear stress .



It has been shown that the post - traumatic brain dysfunction caused by diffuse axonal injury is not only the primary injury due to the initial mechanical external force on the shear extrusion of the tissue , but also the complex neuron " secondary attack " ( secondary injury in neurons ) which occurred in several hours or even days after the injury . The experimental study of the present lab has shown that the pro - inflammatory cytokines can activate the microglial cells . However , the relationship between the activation of the microglial cells and the secondary injury of the axonal injury in the diffuse axonal injury is worth further study .



In this experiment , the dynamic changes of IL - 1尾 , TNF - 偽 and IFN - 緯 at different time points after injury were observed by immunohistochemical method , and the dynamic changes of IL - 1尾 , TNF - 偽 and IFN - 緯 were observed in DAI , and their relationship with microglial activation was discussed .



Methods : Sprague - Dawley rats ( body weight : 260 鹵 21g ) were randomly divided into 11 groups : normal control group , operation control group and injury group .



In normal control group , no treatment was done in the normal control group ; the operation control group was given operation and pre - attack treatment , but it was not blow - down ; the injured group was treated by the above - mentioned method . According to the method , the pathological changes of brain tissue were observed with HE staining and GLESS ' s silver staining method . The expression of IL - 1尾 , IFN - 緯 and TNF - 偽 in brain stem cells was measured by immunohistochemical method .



Statistical analysis was carried out by SPSS statistical analysis software . The statistical analysis was carried out by SPSS statistical analysis software , and the mean difference of the mean number of each group was compared with that of the single factor analysis ( ANOVA ) , and the difference was significant between the two comparisons with the least significant difference method ( LSD ) .



Results : 1 A diffuse axonal injury model was established successfully : HE staining was observed and the damage of brain stem was the most serious .



There was no positive expression of 尾 - APP in normal control group and sham - operated control group .



The expression of IL - 1尾 in brain tissue of normal control group and sham - operated control group was significantly higher than that in control group ( P0.05 ) .



Compared with the control group , there was a significant difference in the number of neuron - positive neurons and microglial cells ( P0.05 ) .



The expression of TNF - 偽 in brain tissue of normal control group and sham - operated control group was significantly higher than that in control group ( P0.05 ) .



Conclusion : The expression of IL - 1尾 , TNF - 偽 and IFN - 緯 in the injured brain stem of rats after DAI was observed by the free fall impact model of Marmarou A . The results showed that the expression of IL - 1尾 , TNF - 偽 , IFN - 緯 increased after axonal injury , and the expression of pro - inflammatory cytokines IL - 1尾 , TNF - 偽 and IFN - 緯 increased after axonal injury .
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：D919

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉學(xué)彬,馮璞,謝蜀生;髓鞘堿性蛋白的提取鑒定及其誘導(dǎo)口服耐受[J];北京醫(yī)科大學(xué)學(xué)報(bào);1998年04期

2 �；萑A,楊思達(dá),何馨,何旦莎,夏學(xué)峰,鄧維意;中樞神經(jīng)系統(tǒng)疾病患兒血清和腦脊液髓鞘堿性蛋白的定量分析[J];中國當(dāng)代兒科雜志;1999年04期

3 賀曉生,易聲禹,章翔,費(fèi)舟,張劍寧,梁景文,楊利孫;大鼠頭顱瞬間側(cè)向旋轉(zhuǎn)后腦干水腫的形成及其超微形態(tài)學(xué)基礎(chǔ)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);1998年02期

4 李衛(wèi);陳善成;汪志剛;宋秀豹;王玉蘋;張梅;;Anti-MBP與大鼠腦組織損傷后腦干繼發(fā)性脫髓鞘病變的關(guān)系[J];南方醫(yī)科大學(xué)學(xué)報(bào);2008年06期

5 于曉軍,陳國弟,茍清,吳梅筠;大鼠閉合性腦損傷后血清髓鞘堿性蛋白研究[J];法醫(yī)學(xué)雜志;1999年02期

6 陳俊杰，王若菡，李昌隆，能赤一，王德恭，，林佳，章全偉，徐靜媛，姚佳，凌立輝;簡易的髓鞘堿性蛋白及其抗體酶聯(lián)免疫吸附同步定量測(cè)定法[J];華西醫(yī)科大學(xué)學(xué)報(bào);1995年02期

7 張莉,杜冠華;細(xì)胞因子在腦缺血損傷機(jī)制中的作用[J];國外醫(yī)學(xué)(腦血管疾病分冊(cè));2004年05期

8 顧琴;湯繼宏;王浙東;胡斯明;李巖;;實(shí)驗(yàn)性癲癇發(fā)作后早期血清NSE、S-100β、MBP的變化[J];蘇州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2008年02期

9 李英姬;崔松彪;樸虎男;吳光;;慢性酒精中毒患者血清S-100β、MBP含量測(cè)定的臨床研究[J];中風(fēng)與神經(jīng)疾病雜志;2008年04期

10 賀曉生,易聲禹,章翔,費(fèi)舟,張劍寧,梁景文,楊利孫;腦彌漫性軸索損傷軸索內(nèi)鈣超載及鈣拮抗劑的治療作用[J];中華神經(jīng)外科雜志;2000年01期

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