大鼠脑挫伤后基质金属蛋白酶3表达变化的研究

发布时间：2018-05-08 17:55

本文选题：法医病理学 + 脑挫伤　；参考：《中国医科大学》2008年硕士论文

【摘要】： 前言脑挫伤是法医学工作中常见的损伤类型,推断脑挫伤时间对刑事案件的侦查及审判具有重要意义,然而准确推断脑挫伤时间一直是法医学中的难题,至今尚未圆满解决。基质金属蛋白酶3(matrix metaUoproteinase-3,MMP-3)是基质金属蛋白酶家族的重要成员,参与组织形态发生、损伤修复、炎症反应等一系列的生理、病理过程,在脑挫伤修复过程中具有重要的调节作用。本实验通过建立大鼠脑挫伤模型,研究基质金属蛋白酶3在脑挫伤修复过程中的表达变化规律,以期为法医学实践中准确推断脑挫伤时间提供一种新的方法。材料与方法一、动物模型的制作,分组与对照雄性SD大鼠50只,体重在200-250g之间,由中国医科大学实验动物部提供。随机分为10组,每组5只,其中8组为实验组,1组为对照组,1组为假手术组。实验组采用吴旭等研制的大鼠脑挫伤模型制作方法,制造大鼠脑挫伤模型。大鼠称重后,乙醚吸入预麻醉,2%戊巴比妥钠(30mg/kg)腹腔注射麻醉。正中切开大鼠顶部头皮,在人字缝前3mm,矢状缝旁3mm处钻直径为5mm的圆形骨窗,保持硬脑膜完好。采用自由落体打击装置,以30g重锤从25cm高处落下,打击暴露的脑组织。术后动物分笼饲养,保持垫料清洁及空气通畅。于伤后6h、12h、24h、3d、5d、7d、10d、14d将大鼠麻醉后脱颈椎处死,手术取出脑组织,沿冠状方向将挫伤区平均分为两部分,一份用于免疫组化染色,另一份用于Western blot检测。假手术组仅行颅骨钻孔。对照组及假手术组,以同样方法取相同部位的脑组织作为对照。二、免疫组织化学染色脑组织经4%多聚甲醛固定后,水洗,梯度酒精脱水,二甲苯透明,石蜡包埋,制作5μm厚度切片。采用链霉素-生物素法(SP法)进行免疫组化染色,并用苏木素复染细胞核,具体步骤同试剂盒说明书。MMP-3抗体1:400稀释,4℃孵育过夜。染色过程中另以PBS替代一抗,作为阴性对照。同时进行常规H.E.染色。山羊抗大鼠MMP-3多克隆抗体购自美国Santa Cruz公司,SP免疫组织化学试剂盒购自北京中杉金桥生物技术有限公司。三、Western blot检测提取脑组织蛋白并进行蛋白定量,聚丙烯酰胺凝胶电泳,半干法转印,5%脱脂牛奶封闭,一抗(1:1000稀释)、二抗(1:2500稀释)孵育后,ECL显色。实验中以GAPDH为内参。实验结果一、免疫组织化学染色结果对照组及假手术组脑组织未见MMP-3阳性染色。实验组中,伤后6h组脑组织开始出现阳性染色,胞浆着色,程度较弱,阳性细胞主要为神经元,12h组染色程度增强。伤后24h组表达MMP-3的神经元显著增多,并可见少量胶质细胞出现MMP-3阳性。3d组MMP-3阳性染色进一步增强,伤后5d组阳性细胞数及染色强度都达到高峰,呈深棕黄色,弥漫分布于神经元及胶质细胞的胞浆和胞核中。伤后7d组MMP-3阳性染色开始减弱,10d组阳性细胞减少,至伤后14d仍有少量MMP-3表达。使用Motic Images Advanced 3.2软件对各组MMP-3免疫组化染色阳性反应物进行检测,统计分析结果显示,阳性反应物面积百分比及阳性反应物平均光密度值差异均具有统计学意义(P＜0.05)。二、Westerna blot结果对照组及假手术组脑组织未见MMP-3表达,挫伤后6h开始出现MMP-3表达,随后表达量逐渐上升,5d达到高峰,以后逐渐下降,14d时仍有表达。应用Fluorchem V 2.0 Stand Alone软件获取感光条带的平均灰度值,经统计分析,差异有统计学意义(P＜0.05)。结论本实验在建立大鼠脑挫伤模型的基础上,应用免疫组织化学染色、Western blot方法检测大鼠脑挫伤后MMP-3表达情况,结果表明: 1、正常大鼠脑组织无MMP-3表达: 2、大鼠脑挫伤后6h即可见MMP-3表达; 3、大鼠脑挫伤后MMP-3表达情况呈规律性变化,提示MMP-3可作为脑挫伤时间推断的指标之一。
[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.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R741;D919

【参考文献】