發(fā)布時間：2018-04-29 10:35

  本文選題：彌漫性軸索損傷 + Stat1��； 參考：《河北醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 目的:彌漫性軸索損傷(diffuse axonal injury, DAI)屬于彌漫性腦損傷,指頭部遭受特殊鈍性外力產(chǎn)生加速性運動時,在剪應(yīng)力的作用下,腦內(nèi)發(fā)生的廣泛分布的以神經(jīng)軸索斷裂為特征的一系列病理生理變化。這種改變既可以單獨出現(xiàn)也可以同其他的病理改變相伴隨出現(xiàn)。意識障礙是其典型的表現(xiàn),預(yù)后多差,重型長期處于去腦強直、植物生存狀態(tài)。 以往研究從腦血流改變、缺血再灌注-氧化損傷、鈣離子超載、神經(jīng)遞質(zhì)和細(xì)胞因子表達(dá)改變等方面入手,認(rèn)識到以上病理生理機制均在DAI的發(fā)展過程中起到重要作用,相互作用影響著損傷后神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞的損傷、修復(fù)和再生。Andre Wennersen等應(yīng)用TUNEL的方法觀察到陽性細(xì)胞的總數(shù)在傷后的第一天和第二天達(dá)到最高,并觀察到腦損傷后神經(jīng)細(xì)胞表達(dá)Bax、Bcl-2增高,證實了腦損傷后可以導(dǎo)致神經(jīng)細(xì)胞的凋亡。 JAK-STAT(Janus kinase-signal transducer and activator of transcription)信號轉(zhuǎn)導(dǎo)途徑是近年發(fā)現(xiàn)的一條由細(xì)胞因子刺激的信號轉(zhuǎn)導(dǎo)通路,參與細(xì)胞的增殖、分化、凋亡以及免疫調(diào)節(jié)等過程;并與中樞神經(jīng)系統(tǒng)的發(fā)育及神經(jīng)細(xì)胞的增殖、分化過程,與腦腫瘤、缺血再灌注等病理生理過程密切相關(guān)。Stat1是第一個發(fā)現(xiàn)的Stats成員,可被INF-α、INF-γ、IL-6、IL-10、GM-CSF、FGF和PDGF等細(xì)胞因子激活。Stat1介導(dǎo)Fas、FasL、Bax、Caspases1等細(xì)胞凋亡相關(guān)基因的表達(dá),同時對Bcl-2和Bcl-x等抗凋亡基因的表達(dá)起到負(fù)調(diào)控作用。 神經(jīng)膠質(zhì)細(xì)胞在神經(jīng)系統(tǒng)發(fā)育、突觸傳遞、神經(jīng)免疫、神經(jīng)組織的修復(fù)與再生以及神經(jīng)疾病中的病理機制等方面都起到重要的作用。已有研究發(fā)機械性腦損傷(traumic brain injury, TBI)后中樞神經(jīng)系統(tǒng)中的星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞增生,且隨時間的變化而變化。 TBI后的病理生理改變可造成神經(jīng)細(xì)胞的凋亡,Stat1可參與促進(jìn)細(xì)胞凋亡的發(fā)生發(fā)展。DAI后是否激活了JAK-STAT信號轉(zhuǎn)導(dǎo)途徑,并參與了神經(jīng)細(xì)胞凋亡的病理過程還不清楚。本實驗復(fù)制了Marmarou A等人于1994年首次報道的自由落體撞擊模型,以觀察DAI后是否有Stat1的激活,并初步探討了其與神經(jīng)細(xì)胞凋亡間的相關(guān)關(guān)系,為進(jìn)一步闡明DAI的病理生理學(xué)機制奠定基礎(chǔ)。 方法:DAI模型的制備:稱重麻醉大鼠,切開皮膚,分離各層組織,暴露顱骨頂部位于冠狀縫和人字縫之間的顱骨穹隆處,將一直徑為1cm的不銹鋼墊片固定于該處,縫合多余手術(shù)切口。大鼠清醒后,乙醚麻醉大鼠,將其俯臥固定于致傷裝置的海綿床上。保證大鼠頭部位置端正,顱頂水平,并使墊片的中央正對致傷裝置有機玻璃管的下口。重錘(銅質(zhì),450g)從指定的高處(1.75m)自由下落致圓盤形的墊片上,海綿床連同大鼠要在打擊后迅速撤離有機玻璃管下方以確保是單次打擊。隨后將大鼠轉(zhuǎn)移至操作臺上觀察幾分鐘。檢查顱骨頂部是否有骨折發(fā)生,縫合頭皮。 在撞擊后死亡的大鼠以及顱骨發(fā)生骨折的大鼠都排除出實驗組,選取打擊后動物立即出現(xiàn)昏迷,且昏迷時間在3~5min內(nèi),24h內(nèi)行為學(xué)評分10分以下的大鼠,作為模型列入實驗組。 健康雄性Sprague-Dawley大鼠,體重240~280克,隨機分為正常對照組、假手術(shù)組和打擊后不同時間組(6h、12h、24h、48h、72h、5d和10d)。正常對照組大鼠不作處理;假手術(shù)組大鼠給予手術(shù)及打擊前處理,但不予真正的打擊;實驗組大鼠經(jīng)過手術(shù)后,按上述方法給予打擊,按打擊后按不同時間處死。觀察打擊后大鼠的行為學(xué)表現(xiàn),麻醉灌注后取全腦,制作石蠟切片,采用銀染和免疫組織化學(xué)的方法觀察大鼠腦組織的病理改變。用流式細(xì)胞檢測腦組織中腦細(xì)胞的凋亡情況,用RT-PCR的方法測定腦組織中bax/bcl-2 mRNA的相對表達(dá)水平,用免疫組織化學(xué)的方法測定腦組織中各個腦區(qū)Phospho-Stat1(Ser727)在損傷后不同時間點的表達(dá)情況。 計量資料數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(Mean±SD)表示,用SPSS統(tǒng)計分析軟件進(jìn)行統(tǒng)計學(xué)分析,各組均數(shù)的比較行單因素方差分析(ANOVA),用最小顯著差法(LSD)作兩兩比較,P0.05為有顯著性差異。 結(jié)果:1組織病理學(xué)變化:Bielschowsky’s鍍銀染色軸索的形態(tài)學(xué)變化:對照組腦組織結(jié)構(gòu)清晰,神經(jīng)元軸突直且長,表面光滑。損傷組大鼠打擊后12h可見皮質(zhì)和小腦等部位的輕度水腫、瘀血,胼胝體、腦干和小腦等部位神經(jīng)軸索扭曲、腫脹,呈串珠狀、波浪狀改變,周圍間質(zhì)水腫,24h及48h上述變化更加明顯。5d上述變化明顯好轉(zhuǎn),10d已不明顯。 酯化銀染色軸索的形態(tài)學(xué)變化:對照組腦組織結(jié)構(gòu)清晰,未見有黑色銀顆粒沉積的變性損傷軸索。損傷組大鼠打擊后6h可見皮質(zhì)、海馬和小腦等部位出現(xiàn)有黑色銀顆粒沉積的變性損傷軸索,走形迂曲,局部擴大如串珠。12h、24h和48h上述改變更加明顯,以24h最為顯著。5d和10d仍能觀察到異常軸索,但數(shù)量明顯減少。 β-APP的免疫組織化學(xué)染色:對照組腦組織中有β-APP的弱表達(dá),但軸突中未見其積聚表達(dá)。實驗組打擊后6h可見有β-APP表達(dá)的升高,但軸突中沒有明顯的積聚現(xiàn)象。12h其表達(dá)量繼續(xù)升高,并開始在軸突中積聚,48h軸突中的積聚最為明顯。而后表達(dá)下降,且在軸突中逐漸減少消失。 2腦組織中腦細(xì)胞凋亡的情況:正常對照組和假手術(shù)對照組的細(xì)胞凋亡率很低,6h組和12h組凋亡率較對照組稍有增高,但沒有明顯的差別;24h組凋亡率較對照組和其他各實驗組有顯著升高(P0.01);48h組凋亡率較24h組有所下降,但較對照組和其他各組均有明顯的差異(P0.01);72h和5d組凋亡率為進(jìn)一步下降;10d組凋亡率明顯降低,但較對照組仍有明顯的差異(P0.05)。 3腦組織中bax及bcl-2 mRNA的比值變化情況:6h bax/bcl-2比值與對照組相比有所升高,但沒有明顯差異。12h bax/bcl-2比值進(jìn)一步升高,與對照組相比有明顯差異(P0.05),24h bax/bcl-2比值達(dá)到最大值。48h bax/bcl-2比值開始下降,72h和5d bax/bcl-2比值較24h有明顯降低,但較對照組仍有明顯的差異(P0.05)。10d bax/bcl-2比值較前幾組繼續(xù)下降,且較對照組已沒有明顯差異(P0.05)。 4大鼠腦組織Ser p-Stat1在不同時間點的表達(dá):對照組大鼠腦組織中Ser p-Stat1有弱的表達(dá),但量很低。實驗組大鼠打擊后6h可見Ser p-Stat1表達(dá)的升高,主要分布于細(xì)胞核中,且與對照組有明顯差異(P0.01)。12h其表達(dá)量繼續(xù)升高,至24h表達(dá)量達(dá)到峰值,48h其表達(dá)開始下降。10d時表達(dá)較24h有非常明顯的降低,但較對照組仍有明顯差異(P0.01)。其中海馬神經(jīng)細(xì)胞Ser p-Stat1的表達(dá)在打擊后6h開始升高,12h表達(dá)量增高達(dá)到峰值,24h開始下降。 實驗組中陽性細(xì)胞主要是神經(jīng)元,在皮層、海馬有少量的神經(jīng)膠質(zhì)細(xì)胞,在腦干和胼胝體有部分神經(jīng)膠質(zhì)細(xì)胞,在小腦髓質(zhì)則主要是神經(jīng)膠質(zhì)細(xì)胞。 5腦組織中Phospho-Stat1(Ser727)的表達(dá)變化與腦細(xì)胞的凋亡率的變化具有正相關(guān)性,相關(guān)系數(shù)r為0.761,具有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論:采用Marmarou A的自由落體撞擊模型復(fù)制了大鼠彌漫性軸索損傷模型;DAI后腦組織中有腦細(xì)胞的凋亡,24h達(dá)到峰值;DAI后大鼠腦組織中Ser p-Stat1的表達(dá)升高,24h達(dá)到峰值;Stat1的激活與腦細(xì)胞的凋亡有正相關(guān)關(guān)系;DAI后大鼠腦組織中Ser p-Stat1不僅在神經(jīng)元中表達(dá),還出現(xiàn)在大量的神經(jīng)膠質(zhì)細(xì)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：D919

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