大鼠弥漫性轴索损伤伴神经细胞凋亡和pStat1表达

发布时间：2018-04-29 10:35

本文选题：弥漫性轴索损伤 + Stat1　；参考：《河北医科大学》2007年硕士论文

【摘要】： 目的:弥漫性轴索损伤(diffuse axonal injury, DAI)属于弥漫性脑损伤,指头部遭受特殊钝性外力产生加速性运动时,在剪应力的作用下,脑内发生的广泛分布的以神经轴索断裂为特征的一系列病理生理变化。这种改变既可以单独出现也可以同其他的病理改变相伴随出现。意识障碍是其典型的表现,预后多差,重型长期处于去脑强直、植物生存状态。以往研究从脑血流改变、缺血再灌注-氧化损伤、钙离子超载、神经递质和细胞因子表达改变等方面入手,认识到以上病理生理机制均在DAI的发展过程中起到重要作用,相互作用影响着损伤后神经元和神经胶质细胞的损伤、修复和再生。Andre Wennersen等应用TUNEL的方法观察到阳性细胞的总数在伤后的第一天和第二天达到最高,并观察到脑损伤后神经细胞表达Bax、Bcl-2增高,证实了脑损伤后可以导致神经细胞的凋亡。 JAK-STAT(Janus kinase-signal transducer and activator of transcription)信号转导途径是近年发现的一条由细胞因子刺激的信号转导通路,参与细胞的增殖、分化、凋亡以及免疫调节等过程;并与中枢神经系统的发育及神经细胞的增殖、分化过程,与脑肿瘤、缺血再灌注等病理生理过程密切相关。Stat1是第一个发现的Stats成员,可被INF-α、INF-γ、IL-6、IL-10、GM-CSF、FGF和PDGF等细胞因子激活。Stat1介导Fas、FasL、Bax、Caspases1等细胞凋亡相关基因的表达,同时对Bcl-2和Bcl-x等抗凋亡基因的表达起到负调控作用。神经胶质细胞在神经系统发育、突触传递、神经免疫、神经组织的修复与再生以及神经疾病中的病理机制等方面都起到重要的作用。已有研究发机械性脑损伤(traumic brain injury, TBI)后中枢神经系统中的星形胶质细胞和小胶质细胞增生,且随时间的变化而变化。 TBI后的病理生理改变可造成神经细胞的凋亡,Stat1可参与促进细胞凋亡的发生发展。DAI后是否激活了JAK-STAT信号转导途径,并参与了神经细胞凋亡的病理过程还不清楚。本实验复制了Marmarou A等人于1994年首次报道的自由落体撞击模型,以观察DAI后是否有Stat1的激活,并初步探讨了其与神经细胞凋亡间的相关关系,为进一步阐明DAI的病理生理学机制奠定基础。方法:DAI模型的制备:称重麻醉大鼠,切开皮肤,分离各层组织,暴露颅骨顶部位于冠状缝和人字缝之间的颅骨穹隆处,将一直径为1cm的不锈钢垫片固定于该处,缝合多余手术切口。大鼠清醒后,乙醚麻醉大鼠,将其俯卧固定于致伤装置的海绵床上。保证大鼠头部位置端正,颅顶水平,并使垫片的中央正对致伤装置有机玻璃管的下口。重锤(铜质,450g)从指定的高处(1.75m)自由下落致圆盘形的垫片上,海绵床连同大鼠要在打击后迅速撤离有机玻璃管下方以确保是单次打击。随后将大鼠转移至操作台上观察几分钟。检查颅骨顶部是否有骨折发生,缝合头皮。在撞击后死亡的大鼠以及颅骨发生骨折的大鼠都排除出实验组,选取打击后动物立即出现昏迷,且昏迷时间在3~5min内,24h内行为学评分10分以下的大鼠,作为模型列入实验组。健康雄性Sprague-Dawley大鼠,体重240~280克,随机分为正常对照组、假手术组和打击后不同时间组(6h、12h、24h、48h、72h、5d和10d)。正常对照组大鼠不作处理;假手术组大鼠给予手术及打击前处理,但不予真正的打击;实验组大鼠经过手术后,按上述方法给予打击,按打击后按不同时间处死。观察打击后大鼠的行为学表现,麻醉灌注后取全脑,制作石蜡切片,采用银染和免疫组织化学的方法观察大鼠脑组织的病理改变。用流式细胞检测脑组织中脑细胞的凋亡情况,用RT-PCR的方法测定脑组织中bax/bcl-2 mRNA的相对表达水平,用免疫组织化学的方法测定脑组织中各个脑区Phospho-Stat1(Ser727)在损伤后不同时间点的表达情况。计量资料数据用均数±标准差(Mean±SD)表示,用SPSS统计分析软件进行统计学分析,各组均数的比较行单因素方差分析(ANOVA),用最小显著差法(LSD)作两两比较,P0.05为有显著性差异。结果:1组织病理学变化:Bielschowsky’s镀银染色轴索的形态学变化:对照组脑组织结构清晰,神经元轴突直且长,表面光滑。损伤组大鼠打击后12h可见皮质和小脑等部位的轻度水肿、瘀血,胼胝体、脑干和小脑等部位神经轴索扭曲、肿胀,呈串珠状、波浪状改变,周围间质水肿,24h及48h上述变化更加明显。5d上述变化明显好转,10d已不明显。酯化银染色轴索的形态学变化:对照组脑组织结构清晰,未见有黑色银颗粒沉积的变性损伤轴索。损伤组大鼠打击后6h可见皮质、海马和小脑等部位出现有黑色银颗粒沉积的变性损伤轴索,走形迂曲,局部扩大如串珠。12h、24h和48h上述改变更加明显,以24h最为显著。5d和10d仍能观察到异常轴索,但数量明显减少。 β-APP的免疫组织化学染色:对照组脑组织中有β-APP的弱表达,但轴突中未见其积聚表达。实验组打击后6h可见有β-APP表达的升高,但轴突中没有明显的积聚现象。12h其表达量继续升高,并开始在轴突中积聚,48h轴突中的积聚最为明显。而后表达下降,且在轴突中逐渐减少消失。 2脑组织中脑细胞凋亡的情况:正常对照组和假手术对照组的细胞凋亡率很低,6h组和12h组凋亡率较对照组稍有增高,但没有明显的差别;24h组凋亡率较对照组和其他各实验组有显著升高(P0.01);48h组凋亡率较24h组有所下降,但较对照组和其他各组均有明显的差异(P0.01);72h和5d组凋亡率为进一步下降;10d组凋亡率明显降低,但较对照组仍有明显的差异(P0.05)。 3脑组织中bax及bcl-2 mRNA的比值变化情况:6h bax/bcl-2比值与对照组相比有所升高,但没有明显差异。12h bax/bcl-2比值进一步升高,与对照组相比有明显差异(P0.05),24h bax/bcl-2比值达到最大值。48h bax/bcl-2比值开始下降,72h和5d bax/bcl-2比值较24h有明显降低,但较对照组仍有明显的差异(P0.05)。10d bax/bcl-2比值较前几组继续下降,且较对照组已没有明显差异(P0.05)。 4大鼠脑组织Ser p-Stat1在不同时间点的表达:对照组大鼠脑组织中Ser p-Stat1有弱的表达,但量很低。实验组大鼠打击后6h可见Ser p-Stat1表达的升高,主要分布于细胞核中,且与对照组有明显差异(P0.01)。12h其表达量继续升高,至24h表达量达到峰值,48h其表达开始下降。10d时表达较24h有非常明显的降低,但较对照组仍有明显差异(P0.01)。其中海马神经细胞Ser p-Stat1的表达在打击后6h开始升高,12h表达量增高达到峰值,24h开始下降。实验组中阳性细胞主要是神经元,在皮层、海马有少量的神经胶质细胞,在脑干和胼胝体有部分神经胶质细胞,在小脑髓质则主要是神经胶质细胞。 5脑组织中Phospho-Stat1(Ser727)的表达变化与脑细胞的凋亡率的变化具有正相关性,相关系数r为0.761,具有统计学意义(P0.05)。结论:采用Marmarou A的自由落体撞击模型复制了大鼠弥漫性轴索损伤模型;DAI后脑组织中有脑细胞的凋亡,24h达到峰值;DAI后大鼠脑组织中Ser p-Stat1的表达升高,24h达到峰值;Stat1的激活与脑细胞的凋亡有正相关关系;DAI后大鼠脑组织中Ser p-Stat1不仅在神经元中表达,还出现在大量的神经胶质细胞中。
[Abstract]:Objective: diffuse axonal injury (DAI) is a series of pathophysiological changes in the brain, which is characterized by the fracture of the nerve axon that occurs in the brain, when the finger is subjected to a special blunt external force to produce accelerated motion. Disturbance of consciousness is a typical manifestation of many other pathological changes. The prognosis is poor.
The previous studies have learned from the changes in cerebral blood flow, ischemia-reperfusion oxidative damage, calcium overload, neurotransmitter and cytokine expression. It is recognized that the above pathophysiological mechanism plays an important role in the development of DAI. The interaction affects the damage of the deity and glial cells after injury, and the repair and regeneration of.And Re Wennersen and other methods of TUNEL observed that the number of positive cells reached the highest level in the first and second days after injury, and the expression of Bax and Bcl-2 after brain injury showed that the apoptosis of nerve cells could be caused by brain injury.
JAK-STAT (Janus kinase-signal transducer and activator of transcription) signal transduction pathway is a signal transduction pathway stimulated by cytokines in recent years, and participates in the process of cell proliferation, differentiation, apoptosis and immunomodulation, and the development of the central nervous system and the proliferation of nerve cells, differentiation process, and brain swelling. The pathophysiological processes such as tumor, ischemia-reperfusion and other pathophysiological processes are closely related to.Stat1, the first Stats member of the discovery, which can be mediated by INF- alpha, INF- gamma, IL-6, IL-10, GM-CSF, FGF and PDGF to mediate the expression of apoptotic genes, such as Fas, FasL, FGF, and other apoptotic genes. Use.
Neuroglia plays an important role in the development of nervous system, synaptic transmission, neuroimmunology, repair and regeneration of nerve tissue, and the pathological mechanism of neural diseases. Astrocytes and microglia in the central nervous system have been studied after traumic brain injury (TBI), and the proliferation of astrocytes and microglia has been studied. Change with time.
The pathophysiological changes after TBI can cause the apoptosis of nerve cells. Stat1 can participate in the activation of JAK-STAT signal transduction pathway after.DAI, and it is not clear to participate in the pathological process of neuronal apoptosis. This experiment replicates the first free falling body impact model, which was first reported by Marmarou A et al in 1994. The activation of Stat1 after DAI was observed and the correlation between the apoptosis and the neuron apoptosis was preliminarily discussed, which lay the foundation for further clarifying the pathophysiological mechanism of DAI.
Methods: DAI model: weigh anaesthetized rats, cut the skin, separate layers of tissue, expose the top of the skull to the skull dome between the coronal seams and the human suture, fixed a stainless steel gasket with a diameter of 1cm, and suture the extra surgical incision. After the rat was awake, the ether was anesthetized and fixed to the sea of the injury device. On the bed. Ensure the head position of the rat head, the level of the cranial top, and the center of the gasket to the lower port of the injured device. The heavy hammer (copper, 450g) is free from the designated height (1.75m) on the disc shaped pad, the sponge bed and the rat will quickly evacuate under the plexiglass tube to ensure that it is a single blow. The rats were transferred to the operation table for a few minutes to check whether there was any fracture at the top of the skull and suture the scalp.
Rats who died after the impact and the rats with fracture of the skull were excluded from the experimental group, and the rats after the attack were immediately comatose, and the time of coma was within 3~5min, and the rats with a behavioral score of less than 10 points in 24h were included in the experimental group.
Healthy male Sprague-Dawley rats, weight 240~280 grams, were randomly divided into normal control group, sham operation group and different time group after attack (6h, 12h, 24h, 48h, 72h, 5D and 10d). The rats in the normal control group were not treated; the sham operation group was given the operation and pre treatment, but not the real blow; the experimental group was operated on the above parties after the operation. The behavior of the rats was executed at different time after the attack. The behavior of the rats after the attack was observed. After the anesthesia, the whole brain was taken and the paraffin section was made. The pathological changes of the brain tissue were observed by the method of silver staining and immunohistochemistry. The apoptosis of brain cells in the brain tissue was detected by flow cytometry, and the brain was measured by RT-PCR method. The relative expression level of bax/bcl-2 mRNA in the tissue was used to determine the expression of Phospho-Stat1 (Ser727) in different brain regions of the brain tissues at different time points in the brain tissue.
The measured data data were expressed with mean mean standard deviation (Mean + SD). Statistical analysis was performed with SPSS statistical analysis software. The comparison of the average number of each group was compared with the single factor variance analysis (ANOVA), and the minimum significant difference (LSD) was used as the 22 comparison, and the P0.05 was significantly different.
Results: 1 histopathological changes: the morphological changes of Bielschowsky 's Silver Plated axons: the brain tissue of the control group is clear, the neuron axon is straight and long, the surface is smooth. After the injury group, 12h can see the mild edema in the cortex and cerebellum, the blood stasis, the corpus callosum, the brainstem and cerebellum. Beads, wave like changes, interstitial edema, 24h and 48h above changes more obvious.5d above changes significantly improved, 10d has not been obvious.
The morphological changes of axons stained with silver esterified: the brain tissue of the control group was clear, and no denatured injury axons were found with black silver particles. After the attack, the 6h visible cortex, the hippocampus and the cerebellum were found to have denatured damage axons with black silver particles deposited in the hippocampus, and a local expansion such as beads.12h, 24h and 48h. It is more obvious that the abnormal axons are still observed in.5d and 10d, but the number is significantly reduced by 24h.
The immunohistochemical staining of beta -APP showed that there was a weak expression of beta -APP in the brain tissue of the control group, but there was no accumulation in the axon. The expression of beta -APP was found in the experimental group after the attack, but there was no obvious accumulation in the axon, but the expression of.12h continued to rise and began to accumulate in the axon, and the accumulation of 48h axons was the most obvious. Then, the accumulation of the axon was the most obvious. Then the accumulation of 6h in axon was the most obvious. The expression decreased and decreased gradually in the axon.
2 the apoptosis of brain cells in the 2 brain tissue: the apoptosis rate of the normal control group and the sham control group was very low. The apoptosis rate in the group 6h and the 12h group was slightly higher than that in the control group, but there was no significant difference. The apoptosis rate in the group 24h was significantly higher than that in the control group and the other experimental groups (P0.01); the apoptosis rate in the 48h group was lower than that in the 24h group, but compared with the control group and the control group, the apoptosis rate was lower than that of the control group. There were significant differences in the other groups (P0.01), and the apoptosis rate in 72h and 5D groups was further decreased, and the apoptosis rate in the 10d group decreased obviously, but there was a significant difference compared with the control group (P0.05).
The ratio of Bax and Bcl-2 mRNA in 3 brain tissues: the ratio of 6h bax/bcl-2 to the control group was higher than that of the control group, but there was no significant difference between the.12h bax/bcl-2 ratio and the control group. The ratio of 24h bax/bcl-2 to the control group was significantly different (P0.05), and the ratio of 24h bax/bcl-2 to maximum.48h bax/ began to decline. It was significantly lower than that of the control group (P0.05), but the ratio of.10d bax/bcl-2 decreased significantly compared with that of the control group, and there was no significant difference between the two groups (P0.05).
The expression of Ser p-Stat1 in the brain tissue of 4 rats at different time points: the expression of Ser p-Stat1 in the brain tissue of the control group was weak, but the amount of Ser p-Stat1 expression was very low in the experimental group. The expression of Ser p-Stat1 in the experimental group was mainly distributed in the nucleus, and there was a significant difference between the control group and the control group (P0.01).12h its expression increased and the expression of 24h reached the peak. Value, when the expression of 48h began to decrease.10d, the expression was significantly lower than that of 24h, but there was a significant difference compared with the control group (P0.01). The expression of Ser p-Stat1 in the hippocampus of the hippocampus began to rise after the attack, and the expression of 12h increased to the peak, and 24h began to decline.
In the experimental group, the positive cells are mainly neurons, in the cortex, the hippocampus has a small number of glial cells, and there are some glial cells in the brain stem and the corpus callosum, and the medulla in the cerebellum is mainly glial cells.
The changes of Phospho-Stat1 (Ser727) expression in 5 brain tissues were positively correlated with the changes in the apoptosis rate of brain cells, and the correlation coefficient r was 0.761, which was statistically significant (P0.05).
Conclusion: the model of diffuse axonal injury in rats was replicated by the free falling body impact model of Marmarou A. After DAI, the apoptosis of brain cells and the peak value of 24h were found in the brain tissue after DAI. The expression of Ser p-Stat1 in the brain tissue of rats increased and the 24h reached the peak value. The activation of Stat1 was positively related to the apoptosis of brain cells; Ser P in the brain tissue of rats after DAI was Ser P. -Stat1 is expressed not only in neurons but also in a large number of glial cells.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：D919

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