大鼠脑挫伤后PSD-95表达变化的时间规律性研究
发布时间：2018-07-21 09:57
【摘要】： 脑是人体的生命中枢器官,也是暴力损伤中极易受累的器官,因而脑挫伤是法医学鉴定工作中常见的损伤类型。脑挫伤形成机制、脑挫伤时间推断、脑挫伤程度判断一直受法医学工作者重视。由于国民经济高速发展,建筑业和交通运输业的兴起,创伤性脑损伤的发生率在近几年逐年增高,居创伤首位,是青壮年人群首要死亡原因。目前,关于脑挫伤转归机制尚不完全清楚,有许多理论学说,包括钙离子超载、微循环障碍等学说,然而依据相应的理论学说研究出的治疗方法鲜有可通过Ⅲ期临床实验,应用于临床治疗。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,至今尚未圆满解决因此,有必要进一步研究脑挫伤的分子机理及找出相应的指标,为临床诊治和法医鉴定提供理论依据。 材料与方法 一、动物模型的制作,分组与对照 成年雄性SD大鼠50只,体重200-250g,由中国医科大学实验动物部提供。随机分为9组,每组5只,其中8组为实验组,1组为对照组,其余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。正中切开大鼠项部头皮,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3h、6h、12h、1d、3d、5d、7d和10d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,一部分用于免疫组化染色,另一部分用于Western blot检测。 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。PSD-95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HE染色。山羊抗大鼠PSD-95多克隆抗体购自北京博奥森公司,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,湿转法转印,5%脱脂牛奶封闭,一抗(1:200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 对照组神经细胞胞浆中可见少量PSD-95阳性染色。实验组中,脑挫伤后3h,挫伤周边区神经细胞中阳性细胞数增多;伤后6h,阳性细胞数继续升高;伤后12h组阳性细胞数与伤后6h组相似;伤后1d,PSD-95阳性细胞数下降;伤后3d,PSD-95阳性细胞数降至正常水平;伤后5d,阳性细胞数再次达到高峰;伤后7d,SD-95阳性细胞数开始减少;伤后10d,阳性细胞继续减少。 二、Western blot结果 对照组仅见少量PSD-95的表达;挫伤后3h-6h,出现PSD-95表达缓慢升高,12h达高峰,1d后下降,3d降至约正常水平,5d时又再次升高并达到高峰,以后逐渐下降。应用Fluorchem V 2.0 Stand Alone软件获取感光条带的平均光密度值,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PSD-95表达情况,结果表明: 1、正常大鼠脑组织有少量PSD-95的表达。 2、PSD-95在脑挫伤后3-12h在挫伤周边区表达开始增多,3d后降至约正常水平,5d再次升高,后缓慢下降,呈双峰改变。 3、大鼠脑挫伤后PSD-95表达呈时间规律性变化。
[Abstract]:Brain is the central organ of human life, and it is also a very vulnerable organ in the violent injury, so the brain contusion is a common type of damage in forensic identification work. The formation mechanism of the brain contusion, the time of brain contusion inference, the degree of brain contusion has been valued by the forensic workers. The incidence of traumatic brain injury has increased year by year in recent years, and it is the first cause of death. It is the primary cause of death in young and middle-aged people. At present, the mechanism of cerebral contusion transfer is not completely clear. There are many theories, including the theory of calcium overload, microcirculation obstacle and so on. However, the treatment method based on the corresponding theory is based on the theory. There are few clinical trials that can be used in stage III clinical trials. 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 science to infer the time of cerebral contusion accurately. Therefore, it is necessary to further study the brain contusion time. To explore the molecular mechanism of brain contusion and find out the corresponding indicators, so as to provide theoretical basis for clinical diagnosis and forensic identification.
Materials and methods
The production of animal models, grouping and comparison
50 adult male SD rats, weighing 200-250g, were provided by the experimental animal Department of China Medical University. They were randomly divided into 9 groups, with 5 rats in each group, of which 8 were the experimental group and the 1 group was the control group. The rest of the experimental groups were made by Wu Xu's brain contusion model and made the rat brain contusion model. After weighing, ether inhalation preanesthesia, 2% amyl Intraperitoneal injection of sodium bital (30mg/kg) was intraperitoneally anaesthetized. The scalp was cut in the midterm of the rat, 3mm in front of the seams and a circular bone window with a diameter of 5mm near the sagittal seam to keep the dura mater 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 clean and air after the operation. After the injury, 3h, 6h, 12h, 1D, 3D, 5D, 7d and 10d removed the rats after anesthesia and removed the cervical spine and removed the brain tissue. The contusion area was divided into two parts along the coronal direction. Some were used in immunohistochemical staining and the other part was used for Western blot detection.
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. Using streptomycin and biotin (SP), the immuno histochemical staining was performed and the nucleus was restained with hematoxylin. The specific steps were diluted with the reagent box.PSD-95 antibody 1:400 and incubated for the night at 4. A negative control was replaced by PBS as a negative control. Routine HE staining was performed at the same time. The PSD-95 polyclonal antibody of Goat anti rat PSD-95 was bought from boorson company in Beijing, and the SP immuno kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
Three, Western blot detection
Extraction of brain tissue protein and protein quantitative, polyacrylamide gel electrophoresis, wet transfer printing, 5% skim milk closed, one anti (1:200 dilution), two anti (1:2500 dilution) incubation, ECL color. In the experiment, GAPDH was used as the internal reference.
experimental result
First, immunohistochemical staining results
In the control group, a small amount of PSD-95 positive staining was found in the cytoplasm of the nerve cells. In the experimental group, the number of positive cells in the peripheral nerve cells in the peripheral area increased after the brain contusion, and the number of positive cells in 6h after injury continued to rise. The number of positive cells in group 12h after injury was similar to that of the 6h group after injury; the number of positive cells in 1D, PSD-95 after injury decreased; 3D, PSD-95 positive cells after injury decreased after injury. At normal level, the number of positive cells reached a peak again after 5D, and the number of positive cells of 7D and SD-95 began to decrease after injury, and the positive cells continued to decrease after 10d.
Two, Western blot results
In the control group, only a small amount of PSD-95 expression was found. After the contusion, the expression of PSD-95 increased slowly, 12h reached its peak, 1D decreased, and 3D dropped to a normal level. 5D was raised again and reached its peak again, and then decreased gradually. The average optical density value of the photosensitive strip was obtained with Fluorchem V 2 Stand Alone software. Statistical analysis showed that the difference was statistically significant. Learning significance (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 PSD-95 after cerebral contusion in rats. The results showed that:
1, there was a small amount of PSD-95 expression in the brain tissue of normal rats.
2, the expression of 3-12h in peripheral area of PSD-95 after cerebral contusion began to increase. After 3D, it decreased to about normal level after 3D, and 5D increased again, then slowly decreased, showing a change in Shuangfeng.
3, the expression of PSD-95 in rats after brain contusion was time regular.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919
本文编号：2135154
[Abstract]:Brain is the central organ of human life, and it is also a very vulnerable organ in the violent injury, so the brain contusion is a common type of damage in forensic identification work. The formation mechanism of the brain contusion, the time of brain contusion inference, the degree of brain contusion has been valued by the forensic workers. The incidence of traumatic brain injury has increased year by year in recent years, and it is the first cause of death. It is the primary cause of death in young and middle-aged people. At present, the mechanism of cerebral contusion transfer is not completely clear. There are many theories, including the theory of calcium overload, microcirculation obstacle and so on. However, the treatment method based on the corresponding theory is based on the theory. There are few clinical trials that can be used in stage III clinical trials. 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 science to infer the time of cerebral contusion accurately. Therefore, it is necessary to further study the brain contusion time. To explore the molecular mechanism of brain contusion and find out the corresponding indicators, so as to provide theoretical basis for clinical diagnosis and forensic identification.
Materials and methods
The production of animal models, grouping and comparison
50 adult male SD rats, weighing 200-250g, were provided by the experimental animal Department of China Medical University. They were randomly divided into 9 groups, with 5 rats in each group, of which 8 were the experimental group and the 1 group was the control group. The rest of the experimental groups were made by Wu Xu's brain contusion model and made the rat brain contusion model. After weighing, ether inhalation preanesthesia, 2% amyl Intraperitoneal injection of sodium bital (30mg/kg) was intraperitoneally anaesthetized. The scalp was cut in the midterm of the rat, 3mm in front of the seams and a circular bone window with a diameter of 5mm near the sagittal seam to keep the dura mater 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 clean and air after the operation. After the injury, 3h, 6h, 12h, 1D, 3D, 5D, 7d and 10d removed the rats after anesthesia and removed the cervical spine and removed the brain tissue. The contusion area was divided into two parts along the coronal direction. Some were used in immunohistochemical staining and the other part was used for Western blot detection.
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. Using streptomycin and biotin (SP), the immuno histochemical staining was performed and the nucleus was restained with hematoxylin. The specific steps were diluted with the reagent box.PSD-95 antibody 1:400 and incubated for the night at 4. A negative control was replaced by PBS as a negative control. Routine HE staining was performed at the same time. The PSD-95 polyclonal antibody of Goat anti rat PSD-95 was bought from boorson company in Beijing, and the SP immuno kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
Three, Western blot detection
Extraction of brain tissue protein and protein quantitative, polyacrylamide gel electrophoresis, wet transfer printing, 5% skim milk closed, one anti (1:200 dilution), two anti (1:2500 dilution) incubation, ECL color. In the experiment, GAPDH was used as the internal reference.
experimental result
First, immunohistochemical staining results
In the control group, a small amount of PSD-95 positive staining was found in the cytoplasm of the nerve cells. In the experimental group, the number of positive cells in the peripheral nerve cells in the peripheral area increased after the brain contusion, and the number of positive cells in 6h after injury continued to rise. The number of positive cells in group 12h after injury was similar to that of the 6h group after injury; the number of positive cells in 1D, PSD-95 after injury decreased; 3D, PSD-95 positive cells after injury decreased after injury. At normal level, the number of positive cells reached a peak again after 5D, and the number of positive cells of 7D and SD-95 began to decrease after injury, and the positive cells continued to decrease after 10d.
Two, Western blot results
In the control group, only a small amount of PSD-95 expression was found. After the contusion, the expression of PSD-95 increased slowly, 12h reached its peak, 1D decreased, and 3D dropped to a normal level. 5D was raised again and reached its peak again, and then decreased gradually. The average optical density value of the photosensitive strip was obtained with Fluorchem V 2 Stand Alone software. Statistical analysis showed that the difference was statistically significant. Learning significance (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 PSD-95 after cerebral contusion in rats. The results showed that:
1, there was a small amount of PSD-95 expression in the brain tissue of normal rats.
2, the expression of 3-12h in peripheral area of PSD-95 after cerebral contusion began to increase. After 3D, it decreased to about normal level after 3D, and 5D increased again, then slowly decreased, showing a change in Shuangfeng.
3, the expression of PSD-95 in rats after brain contusion was time regular.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919
【引证文献】
相关硕士学位论文 前1条
1 张佳娟;幼年和成年单眼形觉剥夺性弱视大鼠视皮质PSD-95表达的研究[D];新乡医学院;2012年
，本文编号：2135154
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