大鼠腦挫傷后PSD-95表達(dá)變化的時間規(guī)律性研究
發(fā)布時間：2018-07-21 09:57
【摘要】： 腦是人體的生命中樞器官,也是暴力損傷中極易受累的器官,因而腦挫傷是法醫(yī)學(xué)鑒定工作中常見的損傷類型。腦挫傷形成機(jī)制、腦挫傷時間推斷、腦挫傷程度判斷一直受法醫(yī)學(xué)工作者重視。由于國民經(jīng)濟(jì)高速發(fā)展,建筑業(yè)和交通運(yùn)輸業(yè)的興起,創(chuàng)傷性腦損傷的發(fā)生率在近幾年逐年增高,居創(chuàng)傷首位,是青壯年人群首要死亡原因。目前,關(guān)于腦挫傷轉(zhuǎn)歸機(jī)制尚不完全清楚,有許多理論學(xué)說,包括鈣離子超載、微循環(huán)障礙等學(xué)說,然而依據(jù)相應(yīng)的理論學(xué)說研究出的治療方法鮮有可通過Ⅲ期臨床實驗,應(yīng)用于臨床治療。腦挫傷是法醫(yī)學(xué)工作中常見的損傷類型,推斷腦挫傷時間對刑事案件的偵查及審判具有重要意義,然而準(zhǔn)確推斷腦挫傷時間一直是法醫(yī)學(xué)中的難題,至今尚未圓滿解決因此,有必要進(jìn)一步研究腦挫傷的分子機(jī)理及找出相應(yīng)的指標(biāo),為臨床診治和法醫(yī)鑒定提供理論依據(jù)。 材料與方法 一、動物模型的制作,分組與對照 成年雄性SD大鼠50只,體重200-250g,由中國醫(yī)科大學(xué)實驗動物部提供。隨機(jī)分為9組,每組5只,其中8組為實驗組,1組為對照組,其余實驗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。正中切開大鼠項部頭皮,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)后動物分籠飼養(yǎng),保持墊料清潔及空氣通暢。于傷后3h、6h、12h、1d、3d、5d、7d和10d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,一部分用于免疫組化染色,另一部分用于Western blot檢測。 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。PSD-95抗體1:400稀釋,4℃孵育過夜。染色過程中另以PBS替代一抗,作為陰性對照。同時進(jìn)行常規(guī)HE染色。山羊抗大鼠PSD-95多克隆抗體購自北京博奧森公司,SP免疫組織化學(xué)試劑盒購自北京中杉金橋生物技術(shù)有限公司。 三、Western blot檢測 提取腦組織蛋白并進(jìn)行蛋白定量,聚丙烯酰胺凝膠電泳,濕轉(zhuǎn)法轉(zhuǎn)印,5%脫脂牛奶封閉,一抗(1:200稀釋)、二抗(1:2500稀釋)孵育后,ECL顯色。實驗中以GAPDH為內(nèi)參。 實驗結(jié)果 一、免疫組織化學(xué)染色結(jié)果 對照組神經(jīng)細(xì)胞胞漿中可見少量PSD-95陽性染色。實驗組中,腦挫傷后3h,挫傷周邊區(qū)神經(jīng)細(xì)胞中陽性細(xì)胞數(shù)增多;傷后6h,陽性細(xì)胞數(shù)繼續(xù)升高;傷后12h組陽性細(xì)胞數(shù)與傷后6h組相似;傷后1d,PSD-95陽性細(xì)胞數(shù)下降;傷后3d,PSD-95陽性細(xì)胞數(shù)降至正常水平;傷后5d,陽性細(xì)胞數(shù)再次達(dá)到高峰;傷后7d,SD-95陽性細(xì)胞數(shù)開始減少;傷后10d,陽性細(xì)胞繼續(xù)減少。 二、Western blot結(jié)果 對照組僅見少量PSD-95的表達(dá);挫傷后3h-6h,出現(xiàn)PSD-95表達(dá)緩慢升高,12h達(dá)高峰,1d后下降,3d降至約正常水平,5d時又再次升高并達(dá)到高峰,以后逐漸下降。應(yīng)用Fluorchem V 2.0 Stand Alone軟件獲取感光條帶的平均光密度值,經(jīng)統(tǒng)計分析,差異有統(tǒng)計學(xué)意義(P＜0.05)。 結(jié)論 本實驗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方法檢測大鼠腦挫傷后PSD-95表達(dá)情況,結(jié)果表明: 1、正常大鼠腦組織有少量PSD-95的表達(dá)。 2、PSD-95在腦挫傷后3-12h在挫傷周邊區(qū)表達(dá)開始增多,3d后降至約正常水平,5d再次升高,后緩慢下降,呈雙峰改變。 3、大鼠腦挫傷后PSD-95表達(dá)呈時間規(guī)律性變化。
[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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：D919
本文編號：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.
【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：D919
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 張佳娟;幼年和成年單眼形覺剝奪性弱視大鼠視皮質(zhì)PSD-95表達(dá)的研究[D];新鄉(xiāng)醫(yī)學(xué)院;2012年
，本文編號：2135154
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