發(fā)布時間：2018-06-04 06:27

  本文選題：腦損傷 + 炎性細(xì)胞因子��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:復(fù)制大鼠Marmarou’s閉合性腦損傷模型,研究大鼠創(chuàng)傷性腦損傷后血清及腦組織中炎性細(xì)胞因子的表達(dá)變化,探尋腦損傷生物標(biāo)志物,為創(chuàng)傷性腦損傷臨床診斷、治療及預(yù)后評估提供理論基礎(chǔ)。方法:選雄性SD大鼠隨機(jī)分為手術(shù)對照組及損傷后1h、4h、8h、12h、24h、48h、72h組,復(fù)制Marmarou’s分級腦損傷模型。分別進(jìn)行神經(jīng)行為學(xué)評價、HE染色、焦油紫染色和酯化銀染色進(jìn)行病理學(xué)觀察,測定腦組織及血清中MDA含量及MPO、SOD活性,檢測血腦屏障通透性的改變;采用QAR-CYT-3芯片和ELISA技術(shù)檢測炎性細(xì)胞因子在閉合性腦損傷中的變化。結(jié)果:1臨床表現(xiàn):輕度損傷組大鼠于打擊后出現(xiàn)短暫呼吸暫停、心跳加速,但迅速恢復(fù);中度損傷組大鼠95只,其中6只(6.31%)于打擊后死亡,11只(11.58%)于打擊后出現(xiàn)抽搐并伴有呼吸暫停,持續(xù)約30s,無驚厥;重度損傷組大鼠125只,其中41只(32.8%)于打擊后全身抽搐、心跳停止死亡,68只(80.95%)出現(xiàn)抽搐并伴有呼吸暫停,持續(xù)約5min,28只(33.33%)出現(xiàn)驚厥,持續(xù)7-8s。2神經(jīng)行為學(xué)評價:損傷后各組大鼠平衡能力、行走、逃避和四肢功能均較損傷前有所減弱,mNSS評分結(jié)果顯示,重度損傷組評分最高,中度損傷組次之,輕度損傷組最低,隨時間延長,神經(jīng)行為學(xué)功能逐漸恢復(fù),評分下降,至48h,輕度損傷組基本恢復(fù)正常。3病理學(xué)改變:輕度損傷組大鼠大體病理學(xué)無明顯改變,腦干偶見少量蛛網(wǎng)膜下腔出血;中度損傷組蛛網(wǎng)膜下腔出血明顯,主要分布在腦底及腦干,無挫傷,切面偶見雙側(cè)腦室少量出血;重度損傷組可見大量蛛網(wǎng)膜下腔出血,主要分布在腦底、小腦及腦干,切面可見胼胝體點狀出血及雙側(cè)腦室出血,頂葉偶見輕微挫傷。HE染色可見手術(shù)對照組大鼠海馬區(qū)神經(jīng)元排列整齊,胞核清晰呈淡紫色,包漿呈淡粉色。輕度損傷組大鼠在損傷后1~4h時,CA1區(qū)錐形細(xì)胞腫脹變圓,排列疏松,包漿淡染;損傷后8h,海馬ca1區(qū)出現(xiàn)散在分布的紅色神經(jīng)元;損傷后12h,壞死神經(jīng)元細(xì)胞增多,胞核固縮細(xì)胞固縮成三角形,呈強(qiáng)嗜酸性。中、重度損傷組病理學(xué)改變較輕度損傷組明顯,壞死神經(jīng)元數(shù)量增多。焦油紫染色可見壞死神經(jīng)元呈深紫色,損傷后各組壞死神經(jīng)元分布趨勢同he染色。在酯化銀染色中,損傷神經(jīng)元呈黑色,神經(jīng)纖維呈波浪狀改變。4生化指標(biāo)改變:(1)tbi大鼠腦組織mpo活性變化:輕、中、重度損傷組大鼠腦組織mpo活性分別于損傷后12h、8h和4h開始升高,至24h達(dá)高峰,然后逐漸下降,損傷程度越重,升高幅度越明顯,與對照組相比有顯著性差異(p0.05或p0.01)。(2)tbi大鼠腦組織及血清sod活性變化:損傷后各組大鼠腦組織sod活性于8h開始降低,損傷后12h達(dá)低谷,然后逐漸恢復(fù),損傷程度越重,降幅越明顯。損傷后8~48h,損傷各組大鼠腦組織sod活性均低于對照組,有顯著性差異(p0.05或p0.01)。損傷后72h,損傷各組腦組織sod活性恢復(fù)正常,與對照組相比無顯著性差異(p0.05)。損傷后各組大鼠血清sod活性逐步降低,至12h達(dá)低谷,然后逐漸恢復(fù),變化大體同腦組織sod活性改變一致。(3)tbi大鼠腦組織及血清中mda含量的變化:損傷后各組大鼠腦組織mda含量逐漸上升,至12h達(dá)高峰,然后逐漸下降。與手術(shù)對照組相比,損傷后8h,輕度損傷組腦組織mda含量升高;損傷后1~12h,中度損傷組腦組織mda含量逐步上升;損傷后1~72h,重度損傷組腦組織mda含量均升高,與手術(shù)對照組相比差別均有有顯著性差異(p0.05或p0.01)。損傷后各組大鼠血清mda含量逐漸上升,至8~12h達(dá)高峰,然后逐漸下降。損傷后12~24h,輕度損傷組血清mda含量升高;損傷后12~48h,中度損傷組血清mda含量升高;損傷后1~72h,重度損傷組血清mda含量升高,與手術(shù)對照組相比,差別均有顯著性差異(p0.05或p0.01)5血腦屏障的改變:腦皮質(zhì)eb含量檢測結(jié)果顯示,大鼠血腦屏障通透性在損傷后迅速增加,至4h達(dá)高峰,然后逐漸下降。損傷后4~48h,輕度損傷組大鼠血腦屏障通透性均高于手術(shù)對照組,差別有顯著性差異(p0.01)。中、重度損傷組大鼠血腦屏障通透性均高于手術(shù)對照組和輕度損傷組(p0.05或p0.01)。重度損傷組大鼠各組血腦屏障通透性均高于中度損傷組,差別有顯著性差異(p0.05或p0.01)。6細(xì)胞因子的檢測:(1)il-10:腦損傷組大鼠血清il-10含量呈先上升后下降而后逐漸恢復(fù)趨勢。損傷后8h,中度損傷組大鼠血清il-10含量升高,與手術(shù)對照組相比有顯著性差異(p0.05);損傷后24h,輕、中度損傷組血清il-10含量下降,與手術(shù)對照組相比有顯著性差異(p0.05);至72h,輕、中度損傷組血清il-10含量逐漸恢復(fù)正常。損傷組大鼠海馬組織il-10表達(dá)先升高后下降。損傷后24h,輕度損傷組il-10含量升高,與手術(shù)對照組相比有顯著性差異(p0.05),而后逐漸下降。損傷后8~12h,中度損傷組il-10含量升高,與手術(shù)對照組、輕度損傷組相比均有顯著性差異(p0.05),而后逐漸下降。損傷后4h,重度損傷組il-10含量升高,與手術(shù)對照組相比有顯著性差異(p0.05),然后逐漸下降,于12h到達(dá)最低點,與手術(shù)對照組相比有顯著性差異(p0.05或p0.01),損傷后24~48h,il-10含量逐漸恢復(fù)正常。(2)fractalkine:腦損傷后,輕度損傷組大鼠血清fractalkine含量逐漸下降,至24h達(dá)到低谷,與手術(shù)對照組相比有顯著性差異(p0.05),然后逐漸升高;中度損傷組大鼠血清fractalkine含量逐漸升高,至24h達(dá)到高峰,與手術(shù)對照組、輕度損傷組相比有顯著性差異(p0.05)。大鼠海馬組織fractalkine的表達(dá)呈先升高后下降趨勢。損傷后12~24h,輕度及中度損傷組fractalkine含量升高,與手術(shù)對照組相比有顯著性差異(p0.05或p0.01)。損傷后12h,重度損傷組fractalkine含量升高,與手術(shù)對照組、輕度損傷組相比均有顯著性差異(p0.01);損傷后24h,fractalkine含量下降,與中度損傷組相比有顯著性差異(p0.05)。(3)prolactinr(prlr):腦損傷后,輕度損傷組血清中prlr含量呈先升高后下降、隨后又升高趨勢;中度損傷組血清中prlr含量先下降,而后逐漸恢復(fù)正常。損傷后8h,輕度損傷組血清中prlr含量升高,與手術(shù)對照組相比有顯著性差異(p0.05);中度損傷組prlr含量下降,與手術(shù)對照組及輕度損傷組相比有顯著性差異(p0.05)。損傷后24h,輕度損傷組prlr含量下降,與手術(shù)對照組及中度損傷組相比有顯著性差異(P0.05)。損傷后72h,輕度損傷組PRLR含量升高,與手術(shù)對照組相比有顯著性差異(P0.05)。各損傷組海馬組織中PRLR含量升高,4h達(dá)高峰,隨后逐漸下降,恢復(fù)至對照組水平。損傷后4~12h,輕度損傷組PRLR含量升高,與手術(shù)對照組相比,均有顯著性差異(P0.05)。損傷后4h~8h,中度損傷組PRLR含量升高,與手術(shù)對照組相比有顯著性差異(P0.05),損傷后12~24h,PRLR含量逐漸下降,與手術(shù)對照組相比無顯著性差異(P0.05),損傷后48~72h,PRLR含量進(jìn)一步下降,與手術(shù)對照組相比有顯著性差異(P0.05或P0.01)。損傷后4~8h,重度損傷組PRLR含量升高,與手術(shù)對照組相比有顯著性差異(P0.05),然后逐漸降低,至72h,PRLR含量降低,與手術(shù)對照組、輕度損傷組、中度損傷組相比均有顯著性差異(P0.05)。結(jié)論:1成功復(fù)制大鼠分級TBI動物模型,損傷越重,病理學(xué)改變、急性炎癥反應(yīng)、氧化應(yīng)激反應(yīng)及血腦屏障損害越嚴(yán)重。2腦損傷可引起血清及海馬組織中炎性細(xì)胞因子表達(dá)變化。血清和海馬中檢測到的炎性細(xì)胞因子表達(dá)存在差異,提示腦損傷后引發(fā)的免疫應(yīng)答存在延遲反應(yīng)。3在血清和海馬中檢測到的差異表達(dá)炎性細(xì)胞因子可能是介導(dǎo)繼發(fā)性腦損傷的關(guān)鍵靶點,對其深入研究探索,有助于了解腦損傷的分子生物學(xué)機(jī)制,為腦損傷的診斷和治療提供理論依據(jù)。
[Abstract]:Objective: to reproduce the model of Marmarou 's closed brain injury in rats, to study the expression of inflammatory cytokines in the serum and brain tissue of the rat after traumatic brain injury, to explore the biomarkers of brain injury, and to provide a theoretical basis for the clinical diagnosis, treatment and prognosis evaluation of traumatic brain injury. Methods: the male SD rats were randomly divided into surgical control. Group and after injury 1H, 4h, 8h, 12h, 24h, 48h, 72h, replicate Marmarou 's classification brain damage model. Neurobehavioral Evaluation, HE staining, tar purple staining and esterified silver staining were performed to observe the pathological changes of MDA content in brain tissue and serum, MPO, activity and detection of blood brain barrier permeability. The changes of inflammatory cytokines in closed brain injury were detected by A. Results: 1 clinical manifestations: mild injury group had transient apnea, rapid heartbeat, but rapid recovery; 95 rats in moderate injury group, of which 6 (6.31%) died after the attack, 11 (11.58%) had convulsions and apnea after the attack. There was no convulsion in 30s, and 125 rats in severe injury group, of which 41 (32.8%) were convulsive after the attack, the heart beat was stopped, 68 (80.95%) had convulsions with apnea, and continued about 5min, 28 (33.33%) had convulsions and continued 7-8s.2 Neurobehavioral Evaluation: the balance ability, walking, escape and limb function of all rats after injury were compared. The mNSS score showed that the score of the severe injury group was the highest, the moderate injury group was the highest, the mild injury group was the lowest, the neurobehavioral function gradually resumed with the time, the score decreased, to 48h, and the mild injury group basically recovered the normal.3 pathological changes: the mild injury group had no obvious changes in general pathology, brain stem pairs. A small amount of subarachnoid hemorrhage was seen. The subarachnoid hemorrhage in the moderate injury group was obvious, mainly in the brain and brain stem, without contusion, and a small amount of hemorrhage in the bilateral ventricle, and a large number of subarachnoid hemorrhage in the severe injury group, mainly in the brain fundus, cerebellum and brain stem, and the punctate hemorrhage of the corpus callosum and bilateral ventricle hemorrhage were seen in the tangent surface. Top of the subarachnoid hemorrhage and the top of the ventricle were seen in the cutting surface. .HE staining of slight contusion showed that the neurons in the hippocampus of the operation control group were arranged neatly, the nucleus of the cell was clear in the light purple, and the pulp was pale pink. In the mild injury group, the conical cells in the CA1 area were swollen and round when the injury was 1~4h, and the cells were loosely arranged and the pulp was pale dye; after the injury, the red neurons scattered in the hippocampus CA1 area appeared in the red neurons; injury was damaged in the hippocampus CA1 area. After 12h, the necrosis neuron cells increased and the nucleus retraction cells of the nucleus became triangular and strongly eosinophilic. In the severe injury group, the pathological changes were more obvious than those in the mild injury group, and the number of necrotic neurons increased. The necrotic neurons were dark purple in the tar purple staining. The injured neurons were black and the nerve fibers changed the biochemical index of.4 in wave form: (1) the activity of MPO in the brain tissue of TBI rats was changed: the activity of MPO in the brain tissue of the rats of the moderate and severe injury groups began to rise after the injury, respectively, 12h, 8h and 4h, and then to the peak of 24h, then gradually decreased, the more significant the damage was, the more obvious the increase was, compared with the control group. There were significant differences (P0.05 or P0.01). (2) the changes in the activity of brain tissue and serum SOD in TBI rats: the activity of SOD in the brain tissue of the rats after injury was reduced after injury, and the 12h reached the low valley after injury, and then gradually recovered, the more serious the damage was, the more obvious the decrease was. The activity of SOD in the brain tissue of rats after injury was lower than that of the control group, which was significantly lower than that of the control group. There was a significant difference. Different (P0.05 or P0.01). After 72h injury, the activity of SOD in the brain tissue of each group recovered to normal, and there was no significant difference compared with the control group (P0.05). The serum SOD activity decreased gradually after the injury, to the valley of 12h, and then gradually recovered. (3) the changes of MDA content in the brain tissue and serum of the rats were changed. After injury, the content of MDA in brain tissue increased gradually after injury to the peak of 12h, and then decreased gradually. Compared with the operation control group, the content of MDA in the brain tissue of the mild injury group increased after the injury, 8h and the mild injury group. After the injury, the content of MDA in the brain tissue of the moderate injury group was gradually increased, and the level of MDA in the brain tissue of the severe injury group increased after 1 ~72h, and the operation was the same as that in the severe injury group. There were significant differences between the groups (P0.05 or P0.01). After the injury, the serum MDA content increased gradually, to the peak of 8~12h, and then decreased gradually. After the injury, the serum MDA content in the mild injury group increased after 12~24h injury, and the serum MDA content in the moderate injury group increased after the injury, and the serum MDA content of the severe injury group was raised after injury. Higher, compared with the operation control group, the difference was significant (P0.05 or P0.01) 5 blood brain barrier change: the cerebral cortex EB content detection results showed that the blood brain barrier permeability of rats increased rapidly after injury, to the peak of 4h, and then gradually decreased. After injury, the permeability of blood brain barrier in the mild injury group was higher than that of the operation control group. The difference was significant (P0.01). In the severe injury group, the permeability of blood brain barrier in the rats was higher than that of the operation control group and the mild injury group (P0.05 or P0.01). The blood brain barrier permeability in the severe injury group was higher than that of the moderate injury group. There was a significant difference (P0.05 or P0.01).6 cytokine detection: (1) the rats of il-10: brain injury group Serum IL-10 content increased first and then decreased and then gradually recovered. After 8h injury, the serum IL-10 content in the moderate injury group increased significantly (P0.05) compared with the operation control group (P0.05). The serum level of 24h, light and moderate injury group decreased after injury (P0.05) compared with the operation group (P0.05); to 72h, light, moderate injury. The content of serum IL-10 in the group of the group gradually recovered to normal. The expression of IL-10 in the hippocampus of the injured group first increased and then decreased. The content of IL-10 in the mild injury group increased after 24h injury, and there was a significant difference compared with the operation control group (P0.05), and then gradually decreased. The content of IL-10 in the moderate injury group was higher than that in the moderate injury group, compared with the operation control group and the mild injury group. There were significant differences (P0.05), and then decreased gradually. The content of IL-10 in 4H and severe injury group increased significantly after injury (P0.05), and then decreased gradually and reached the lowest point at 12h (P0.05 or P0.01) compared with the operation control group (P0.05 or P0.01), and the IL-10 content gradually returned to normal after injury (2) fractalk. After ine: brain injury, the content of serum fractalkine decreased gradually in the mild injury group, and reached the low valley to 24h. Compared with the operation control group, there was a significant difference (P0.05), and then increased gradually. The serum fractalkine content in the moderate injury group increased gradually and reached the peak to 24h (P0). Compared with the operation control group, the mild injury group had a significant difference (P0 .05). The expression of fractalkine in the hippocampus of rats was increased first and then decreased. The content of fractalkine in 12~24h, mild and moderate injury group was significantly higher than that in the control group (P0.05 or P0.01). The content of fractalkine in the severe injury group was higher than that in the severe injury group, and the level of fractalkine in the severe injury group was significantly higher than that in the operation control group and the mild injury group. The sex difference (P0.01); the content of 24h and fractalkine decreased after injury (P0.05). (3) prolactinr (PRLR): after brain injury, the serum level of PRLR in the mild injury group increased first and then decreased, then increased, and the serum PRLR content in the moderate injury group decreased first and then gradually returned to normal. 8h, light after injury, was light. The level of PRLR in the serum of the degree injury group was significantly higher than that in the operation control group (P0.05). The PRLR content in the moderate injury group decreased significantly (P0.05) compared with the operation control group and the mild injury group (P0.05). After the injury, the content of the mild injury group decreased, and there was a significant difference compared with the operation control group and the moderate injury group (P0.05 After the injury, the content of PRLR in the mild injury group increased, and compared with the operation control group (P0.05). The content of PRLR in the hippocampus of the injured group increased, the 4H reached the peak, and then decreased gradually to the control level. After the injury, the PRLR content in the mild injury group increased, and there were significant differences (P0.05) compared with the operation control group (P0.05). The content of PRLR in 4h~8h and moderate injury group was higher than that of the control group (P0.05). The content of 12~24h and PRLR decreased gradually after the injury, and there was no significant difference compared with the operation control group (P0.05). The content of 48~72h and PRLR decreased further after the injury. There was a significant difference (P0.05 or P0.01) compared with the operation control group (P0.05 or P0.01). 4~8h after injury. The content of PRLR in severe injury group was significantly higher than that in the operation control group (P0.05), and then decreased gradually, to 72h, and PRLR content decreased. Compared with the operation control group, the mild injury group and the moderate injury group, there were significant differences (P0.05). Conclusion: 1 the successful reproduced rat classification TBI animal model, the heavier the injury, the pathological changes, acute inflammation. The expression of inflammatory cytokines in serum and hippocampal tissues can be induced by.2 injury, the more severe the oxidative stress reaction and the damage of the blood brain barrier, the difference in the expression of inflammatory cytokines in the serum and hippocampus suggests the difference in the detection of delayed response.3 in the serum and hippocampus after the brain injury. The expression of inflammatory cytokines may be the key target to mediate secondary brain injury. It is helpful to understand the molecular biological mechanism of brain injury and provide a theoretical basis for the diagnosis and treatment of brain injury.
【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】：2017
【分類號】：R651.15

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