本文關(guān)鍵詞： 免疫組織化學(xué) E-選擇素 皮膚切傷 死后穩(wěn)定性　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 研究背景及目的:皮膚作為人體最大的器官,與外界直接接觸,對(duì)機(jī)體具有重要的屏障和保護(hù)作用,其最易遭受機(jī)械性損傷.在法醫(yī)學(xué)實(shí)踐中,皮膚損傷時(shí)間的推斷常是法醫(yī)工作者需要解決的關(guān)鍵問(wèn)題。正因?yàn)槿绱?關(guān)于皮膚損傷時(shí)間推斷方面的研究,一直以來(lái)是國(guó)內(nèi)外法醫(yī)病理學(xué)者的研究熱點(diǎn)問(wèn)題之一。E-選擇素是細(xì)胞粘附分子(adhesion molecule,AM)中選擇素家族(selectin family,SF)成員之一。在體內(nèi)分布非常局限,主要由毛細(xì)血管、后微靜脈的內(nèi)皮細(xì)胞分泌,其胞內(nèi)無(wú)儲(chǔ)存。它只在外傷、炎癥等刺激后在血管內(nèi)皮細(xì)胞表面表達(dá),且具有特異性。E-選擇素產(chǎn)生后,參與記憶T淋巴細(xì)胞、單核細(xì)胞、嗜酸性粒細(xì)胞和嗜堿性粒細(xì)胞的聚集,以及內(nèi)皮細(xì)胞增殖、遷移、管狀形成和毛細(xì)血管的生成,但最主要是介導(dǎo)血液中中性粒細(xì)胞粘附。E-選擇素參與損傷早期修復(fù)作用,目前國(guó)內(nèi)外對(duì)E-選擇素的研究多側(cè)重于臨床方面,而對(duì)于它在法醫(yī)學(xué)領(lǐng)域方面對(duì)皮膚損傷后不同時(shí)間的表達(dá)變化及死后穩(wěn)定性的研究甚少。尤其是國(guó)內(nèi)對(duì)E-選擇素在皮膚機(jī)械性損傷修復(fù)時(shí)間的推斷方面幾乎未見(jiàn)報(bào)道。本研究通過(guò)建立大鼠皮膚切創(chuàng)損傷模型,運(yùn)用免疫組化技術(shù)及其陽(yáng)性細(xì)胞計(jì)數(shù)法對(duì)損傷組織進(jìn)行E-選擇素的檢測(cè)與分析,探討在皮膚創(chuàng)傷愈合過(guò)程中其表達(dá)的時(shí)序性變化規(guī)律,以及死后穩(wěn)定性狀況,有可能為法醫(yī)病理學(xué)皮膚損傷時(shí)間的推斷,鑒別生前傷和死后傷提供一個(gè)新的理論依據(jù)。 方法:采用完全隨機(jī)分組方法,分為實(shí)驗(yàn)組和對(duì)照組。實(shí)驗(yàn)組又分為生前造創(chuàng)組、死后造創(chuàng)組、死后穩(wěn)定性-20℃和25℃組。根據(jù)造創(chuàng)后處死時(shí)間不同,將實(shí)驗(yàn)大鼠隨機(jī)分為傷后30min、1h、2h、3h、5h、7h、9h、12h、1d、3d、5d、7d共12亞組;死后造創(chuàng)組分為0.5h,1h,3h共3亞組;另設(shè)一組為正常對(duì)照組,即未造創(chuàng)組。生前造創(chuàng)組在大鼠頭部制做全層切創(chuàng),在預(yù)定時(shí)間分別頸椎脫臼法處死后即刻取材;死后造創(chuàng)組于處死大鼠后在預(yù)定時(shí)間造創(chuàng)取材。死后穩(wěn)定性組中將大鼠造創(chuàng)后于1d將它們頸椎脫臼法處死。隨機(jī)分為14亞組,其中9亞組置于-20℃環(huán)境中,于6h、12h和1d、2d、3d、4d、5d、6d、7d取材;另5亞置于25℃環(huán)境中,于6h、12h和1d、2d、3d取材。取材皮膚組織用多聚甲醛-PBS液中固定后,石蠟包埋切片,進(jìn)行常規(guī)HE染色光鏡觀察;采用免疫組織化學(xué)染色法進(jìn)行E-選擇素染色,采用SPSS11.0統(tǒng)計(jì)軟件包對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果:1、HE染色觀察:傷后1h,創(chuàng)口周?chē)?xì)血管擴(kuò)張、充血,組織間水腫;傷后12h內(nèi),可見(jiàn)大量炎性細(xì)胞游出,主要以中性粒細(xì)胞浸潤(rùn)為主;傷后1～3d,肉芽組織逐漸形成并大量增生,炎性細(xì)胞以單核-巨噬細(xì)胞為主;傷后5～7d,肉芽組織成熟,成纖維細(xì)胞生成,炎性細(xì)胞消失,創(chuàng)口基本愈合。死后造創(chuàng)組及正常對(duì)照組鏡下無(wú)上述改變。死后在-20℃下6小時(shí)至7d,皮膚損傷部位組織結(jié)構(gòu)與斷頸處死即刻基本一樣,沒(méi)有發(fā)生明顯變化,在第6、7d可見(jiàn)其內(nèi)極少數(shù)炎癥細(xì)胞結(jié)構(gòu)欠清晰,核固縮、粹裂,但邊緣、組織結(jié)構(gòu)較清楚;在25℃下,皮膚損傷部位組織中炎癥細(xì)胞、部分組織細(xì)胞逐漸自溶;以至損傷邊緣結(jié)構(gòu)模糊不清。2、免疫組織化學(xué)SABC法染色觀察:傷后1h,創(chuàng)緣周?chē)?xì)血管內(nèi)皮細(xì)胞可見(jiàn)棕黃色陽(yáng)性表達(dá),以后持續(xù)升高,至傷后1d,陽(yáng)性表達(dá)達(dá)到高峰;以后表達(dá)逐漸減弱,至傷后7d,仍可見(jiàn)有少量陽(yáng)性表達(dá)。-20℃死穩(wěn)定性組中,不同時(shí)間陽(yáng)性表達(dá)與傷后1d處死即刻沒(méi)有明顯變化;25℃環(huán)境中,隨著時(shí)間延長(zhǎng),組織及細(xì)胞的自溶,各亞組的表達(dá)情況與傷后1d處死即刻有明顯變化,陽(yáng)性表達(dá)逐漸減少,在2d時(shí)未見(jiàn)表達(dá)。在正常對(duì)照組及死后傷組中均未見(jiàn)E-選擇素表達(dá)。3、陽(yáng)性細(xì)胞計(jì)數(shù)法結(jié)果:陽(yáng)性細(xì)胞率(PR值)在傷后1h到傷后1d逐漸上升,傷后1d達(dá)到高峰,傷后3d～7d,PR值逐漸下降。4、統(tǒng)計(jì)學(xué)處理結(jié)果:方差分析結(jié)果表明生前創(chuàng)傷各相鄰兩組之間E-選擇素PR值相比均具有顯著差異(P0.01)。其中生前創(chuàng)傷1d組與其他各時(shí)間組相比均有顯著差異(P0.01)。 結(jié)論:大鼠皮膚切創(chuàng)后E-選擇素可迅速增量表達(dá),且與時(shí)間相關(guān),呈一定規(guī)律性,呈上升-高峰-下降的時(shí)序變化;在傷后1d達(dá)到高峰。E選擇素在生前及死后均不表達(dá),選擇素在皮膚創(chuàng)傷后表達(dá)的時(shí)序性變化規(guī)律并具有特異性,有望作為皮膚損傷發(fā)生后個(gè)體存活時(shí)間推斷的一個(gè)客觀有效的指標(biāo)。且在死后E選擇素表達(dá)穩(wěn)定性與否受溫度影響很大,在-20℃下,其穩(wěn)定性好,但在25℃環(huán)境中,其穩(wěn)定性差。
[Abstract]:Background and purpose: the skin is the largest organ in the body, direct contact with the outside world, with a barrier and an important protective effect on the body, the most vulnerable to mechanical injury. In forensic practice, skin injury time estimation is often the key issues of forensic practitioners need to solve. Because of this, the research on the skin the time of injury, has been a research focus at home and abroad scholars of forensic pathology.E- selectin is a cell adhesion molecule (adhesion, molecule, AM) in the selectin family (selectin family, SF). A member of the in vivo distribution is very limited, mainly composed of capillaries, venules of endothelial cell secretion. No storage in the cells. It is only after trauma, inflammation stimulated expression in vascular endothelial cells, and the specificity of.E- selectin was produced, in memory T cells, monocytes, eosinophils Neutrophils and basophils aggregation, and endothelial cell proliferation, migration, tube formation and capillary formation, but the most important is mediated by blood neutrophil adhesion.E- selectin is involved in the repair of early damage, both at home and abroad on the study of E- focused on the clinical aspects, and to learn it in the field of forensic Research on expression and death in different time after skin injury after stability is little. Especially for the domestic E- selectin in time to repair the skin mechanical injury that was rarely reported. This study established a rat skin wound injury model, using immunohistochemical technique and positive cell counting method E- choice of detection and Analysis on tissue injury, to investigate the healing time regularity of its expression in the process of skin wound, and after the death of stability condition, are possible for the method A new theoretical basis is provided to identify the time of pathological skin injury and to differentiate between pre - and postmortem injuries.
鏂規(guī)硶:閲囩敤瀹屽叏闅忔満鍒嗙粍鏂規(guī)硶,鍒嗕負(fù)瀹為獙緇勫拰瀵圭収緇，

本文編號(hào)：1471509