本文選題：法醫(yī)病理學(xué) + 腦挫傷��； 參考：《中國(guó)醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 前言 腦挫傷是法醫(yī)學(xué)工作中常見的損傷類型,推斷腦挫傷時(shí)間對(duì)刑事案件的偵查及審判具有重要意義,然而準(zhǔn)確推斷腦挫傷時(shí)間一直是法醫(yī)學(xué)中的難題,至今尚未圓滿解決。基質(zhì)金屬蛋白酶3(matrix metaUoproteinase-3,MMP-3)是基質(zhì)金屬蛋白酶家族的重要成員,參與組織形態(tài)發(fā)生、損傷修復(fù)、炎癥反應(yīng)等一系列的生理、病理過程,在腦挫傷修復(fù)過程中具有重要的調(diào)節(jié)作用。本實(shí)驗(yàn)通過建立大鼠腦挫傷模型,研究基質(zhì)金屬蛋白酶3在腦挫傷修復(fù)過程中的表達(dá)變化規(guī)律,以期為法醫(yī)學(xué)實(shí)踐中準(zhǔn)確推斷腦挫傷時(shí)間提供一種新的方法。 材料與方法 一、動(dòng)物模型的制作,分組與對(duì)照 雄性SD大鼠50只,體重在200-250g之間,由中國(guó)醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物部提供。隨機(jī)分為10組,每組5只,其中8組為實(shí)驗(yàn)組,1組為對(duì)照組,1組為假手術(shù)組。實(shí)驗(yàn)組采用吳旭等研制的大鼠腦挫傷模型制作方法,制造大鼠腦挫傷模型。大鼠稱重后,乙醚吸入預(yù)麻醉,2%戊巴比妥鈉(30mg/kg)腹腔注射麻醉。正中切開大鼠頂部頭皮,在人字縫前3mm,矢狀縫旁3mm處鉆直徑為5mm的圓形骨窗,保持硬腦膜完好。采用自由落體打擊裝置,以30g重錘從25cm高處落下,打擊暴露的腦組織。術(shù)后動(dòng)物分籠飼養(yǎng),保持墊料清潔及空氣通暢。于傷后6h、12h、24h、3d、5d、7d、10d、14d將大鼠麻醉后脫頸椎處死,手術(shù)取出腦組織,沿冠狀方向?qū)⒋靷麉^(qū)平均分為兩部分,一份用于免疫組化染色,另一份用于Western blot檢測(cè)。假手術(shù)組僅行顱骨鉆孔。對(duì)照組及假手術(shù)組,以同樣方法取相同部位的腦組織作為對(duì)照。 二、免疫組織化學(xué)染色 腦組織經(jīng)4%多聚甲醛固定后,水洗,梯度酒精脫水,二甲苯透明,石蠟包埋,制作5μm厚度切片。采用鏈霉素-生物素法(SP法)進(jìn)行免疫組化染色,并用蘇木素復(fù)染細(xì)胞核,具體步驟同試劑盒說明書。MMP-3抗體1:400稀釋,4℃孵育過夜。染色過程中另以PBS替代一抗,作為陰性對(duì)照。同時(shí)進(jìn)行常規(guī)H.E.染色。山羊抗大鼠MMP-3多克隆抗體購(gòu)自美國(guó)Santa Cruz公司,SP免疫組織化學(xué)試劑盒購(gòu)自北京中杉金橋生物技術(shù)有限公司。 三、Western blot檢測(cè) 提取腦組織蛋白并進(jìn)行蛋白定量,聚丙烯酰胺凝膠電泳,半干法轉(zhuǎn)印,5%脫脂牛奶封閉,一抗(1:1000稀釋)、二抗(1:2500稀釋)孵育后,ECL顯色。實(shí)驗(yàn)中以GAPDH為內(nèi)參。 實(shí)驗(yàn)結(jié)果 一、免疫組織化學(xué)染色結(jié)果 對(duì)照組及假手術(shù)組腦組織未見MMP-3陽(yáng)性染色。實(shí)驗(yàn)組中,傷后6h組腦組織開始出現(xiàn)陽(yáng)性染色,胞漿著色,程度較弱,陽(yáng)性細(xì)胞主要為神經(jīng)元,12h組染色程度增強(qiáng)。傷后24h組表達(dá)MMP-3的神經(jīng)元顯著增多,并可見少量膠質(zhì)細(xì)胞出現(xiàn)MMP-3陽(yáng)性。3d組MMP-3陽(yáng)性染色進(jìn)一步增強(qiáng),傷后5d組陽(yáng)性細(xì)胞數(shù)及染色強(qiáng)度都達(dá)到高峰,呈深棕黃色,彌漫分布于神經(jīng)元及膠質(zhì)細(xì)胞的胞漿和胞核中。傷后7d組MMP-3陽(yáng)性染色開始減弱,10d組陽(yáng)性細(xì)胞減少,至傷后14d仍有少量MMP-3表達(dá)。使用Motic Images Advanced 3.2軟件對(duì)各組MMP-3免疫組化染色陽(yáng)性反應(yīng)物進(jìn)行檢測(cè),統(tǒng)計(jì)分析結(jié)果顯示,陽(yáng)性反應(yīng)物面積百分比及陽(yáng)性反應(yīng)物平均光密度值差異均具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 二、Westerna blot結(jié)果 對(duì)照組及假手術(shù)組腦組織未見MMP-3表達(dá),挫傷后6h開始出現(xiàn)MMP-3表達(dá),隨后表達(dá)量逐漸上升,5d達(dá)到高峰,以后逐漸下降,14d時(shí)仍有表達(dá)。應(yīng)用Fluorchem V 2.0 Stand Alone軟件獲取感光條帶的平均灰度值,經(jīng)統(tǒng)計(jì)分析,差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。 結(jié)論 本實(shí)驗(yàn)在建立大鼠腦挫傷模型的基礎(chǔ)上,應(yīng)用免疫組織化學(xué)染色、Western blot方法檢測(cè)大鼠腦挫傷后MMP-3表達(dá)情況,結(jié)果表明: 1、正常大鼠腦組織無(wú)MMP-3表達(dá): 2、大鼠腦挫傷后6h即可見MMP-3表達(dá); 3、大鼠腦挫傷后MMP-3表達(dá)情況呈規(guī)律性變化,提示MMP-3可作為腦挫傷時(shí)間推斷的指標(biāo)之一。
[Abstract]:Preface
Cerebral contusion is a common type of injury in forensic work. It is inferred that the time of brain contusion is of great significance to the investigation and trial of criminal cases. However, it is a difficult problem in forensic medicine to accurately infer the time of brain contusion, and it has not been satisfactorily solved. Matrix metalloproteinase 3 (matrix metaUoproteinase-3, MMP-3) is a matrix metalloproteinase family. The important members, participating in a series of physiological and pathological processes, such as tissue morphogenesis, injury repair and inflammatory reaction, have an important regulatory role in the process of brain contusion repair. In this experiment, the expression of matrix metalloproteinase 3 in the process of cerebral contusion was studied by establishing a rat brain contusion model, so as to be a forensic practice. It provides a new way to accurately infer the time of brain contusion.
Materials and methods
The production of animal models, grouping and comparison
50 male SD rats, with a weight of 200-250g, were provided by the experimental animal Department of China Medical University. They were randomly divided into 10 groups, with 5 rats in each group, of which 8 were the experimental group, the 1 group was the control group and the 1 group was the sham operation group. The experimental group was made by Wu Xu and other rats' brain contusion model. The rat model of brain contusion was made. Rats were weighed and ether inhaled. Preanesthesia and intraperitoneal injection of 2% pentobarbital sodium (30mg/kg) were intraperitoneally injected. The scalp on the top of the rat was cut into the top of the scalp. A circular bone window with a diameter of 5mm was drilled before the seams of the human character, and the dural was intact. The free falling body was used to drop the 30g weight from the 25cm height to strike the exposed brain tissue. The animals were kept in cage and kept after the operation. The animals were kept in cage and kept pads after the operation. After the injury, 6h, 12h, 24h, 3D, 5D, 7d, 10d, 14d put the rats off the cervical spine and removed the cervical vertebra after the injury. The brain tissue was removed and the contusion area was divided into two parts along the coronal direction. One was used in immunohistochemical staining and the other was used in the Western blot test. The sham operation group was only bored with the skull. The control group and the sham operation group were the same. The same part of the brain tissue was taken as the control.
Two, immunohistochemical staining
After 4% paraformaldehyde was fixed, the brain tissue was washed, dehydrated with gradient alcohol, dimethylbenzene was transparent and paraffin was embedded, and the thickness of 5 m was made. It was immunohistochemical staining with streptomycin biotin (SP), and the cell nucleus was restained with hematoxylin. The specific steps were diluted with the reagent box.MMP-3 antibody 1:400 and incubated for the night at 4. A negative control was replaced by PBS as a negative control. Routine H.E. staining was performed at the same time. The MMP-3 polyclonal antibody of Goat anti rat MMP-3 was purchased from the American Santa Cruz company, and the SP immuno kit was bought from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
Three, Western blot detection
Brain tissue protein was extracted and protein quantitative, polyacrylamide gel electrophoresis, semi dry transfer, 5% skimmed milk closed, one anti (1:1000 dilution), and two anti (1:2500 dilution) incubated, ECL was coloured. In the experiment, GAPDH was used as the internal reference.
experimental result
First, immunohistochemical staining results
In the experimental group, there was no MMP-3 positive staining in the brain tissue of the control group and the sham operation group. In the experimental group, the positive staining of the brain tissue began to appear in group 6h after injury, and the degree of cytoplasm coloring was weak. The positive cells were mainly neurons, and the staining degree of the 12h group was enhanced. The number of MMP-3 neurons in the group 24h after injury increased significantly, and a small amount of glial cells appeared to have a MMP-3 positive.3d group MMP-. 3 positive staining was further enhanced. The number and intensity of positive cells and staining intensity in 5D group reached the peak, which showed deep brown yellow and diffuse in the cytoplasm and nucleus of neurons and glial cells. After injury, the positive staining of MMP-3 in group 7d began to weaken, and the positive cells in group 10D decreased, and there were still a small amount of MMP-3 expression after the injury. Motic Images Advanced 3.2 soft was used. The positive reaction of MMP-3 immunohistochemical staining was detected in each group. The statistical analysis showed that the difference of the percentage of positive reactant and the mean light density of the positive reactant had statistical significance (P < 0.05).
Two, Westerna blot results
There was no MMP-3 expression in the brain tissue of the control group and the sham operation group. The expression of 6h began to appear MMP-3 expression after the contusion, then the expression amount increased gradually, the 5D reached the peak, and then gradually declined, and the expression was still expressed in 14d. The average gray value of the photosensitive strip was obtained by Fluorchem V 2 Stand Alone software. The statistical analysis showed that the difference was statistically significant (P < 0.05).
conclusion
On the basis of establishing rat brain contusion model in this experiment, immunohistochemical staining and Western blot method were used to detect the expression of MMP-3 after cerebral contusion in rats. The results showed that:
1, there was no MMP-3 expression in the brain tissue of normal rats.
2, MMP-3 expression was seen in 6h after cerebral contusion in rats.
3, the expression of MMP-3 in rats after brain contusion is regular, suggesting that MMP-3 can be used as an indicator of brain contusion time.

【學(xué)位授予單位】：中國(guó)醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R741;D919

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