HMGB1在人脑挫裂伤组织中的表达及作用
发布时间：2018-08-15 17:31
【摘要】：背景创伤性脑损伤包括原发性损伤和继发性损伤,而继发性损伤是目前研究的热点及难点。继发性损伤由多种因素参与:兴奋性毒性、氧自由基、钙超载、水肿、炎症反应等,而水肿、炎症反应在其中发挥着重要的作用,但具体机制目前还不十分清楚。研究报道高迁移率族蛋白B1(high-mobility group box 1,HMGB1)在众多损伤的急性、慢性炎症及水肿的调控中发挥着关键的作用。那么HMGB1在人脑挫裂伤后的继发性损伤中发挥着怎样的作用值得深入的研究。目的探讨HMGB1在人脑挫裂伤组织中的表达及作用。方法通过收集32例脑挫裂伤病人手术切除的脑组织标本作为实验组,根据伤后时间不同,分为伤后6 h组(n=7)、12 h组(n=9)、1 d组(n=10)、3 d组(n=6),同时搜集正常脑组织作为对照组(n=5)。所有新鲜标本用4%多聚甲醛固定,然后用30%蔗糖溶液脱水处理,最后制作冰冻切片。然后进行苏木精-伊红染色(H.E.染色)、免疫荧光染色。H.E.染色:取实验组和对照组各标本进行染色,观察每张视野下淋巴细胞和中性粒细胞数目,统计分析各时间点炎性细胞浸润情况。免疫荧光染色:取实验组和对照组各标本进行染色,将HMGB1与DAPI(细胞核染液)共标,荧光显微镜视野下观察人脑挫裂伤后组织中各个时间点HMGB1在胞核内及胞浆中的数目,进一步了解HMGB1从细胞核内释放到核外的情况;将HMGB1与神经元标志物NeuN进行共标,观察统计人脑挫裂伤后早期神经元数目、神经元中HMGB1数目及神经元外的HMGB1数目,进一步了解损伤后神经元与HMGB1的转移情况;将水通道蛋白4(AQP4)与星形胶质细胞标记物(GFAP)进行共标,计算各时间点水通道蛋白4(AQP4)表达的荧光强度,分析水通道蛋白4在星形胶质细胞表达的情况。采用SPSS 12.0统计软件,计量资料以x±s表示,多组间比较采用OnewayANOVA分析,以P0.05为差异具有统计学意义。结果人脑挫裂伤后,浸润的淋巴细胞细胞于1d开始明显增加(P0.05);浸润的中性粒细胞于12h开始明显增加(P0.05)。随时间延长,炎性细胞数均逐渐升高。正常人脑组织的HMGB1表达主要集中在细胞核内,人脑挫裂伤组HMGB1在细胞核内的细胞数于12 h开始下降(P0.05),并持续下降至72 h;而HMGB1从核内转移至胞浆内的数目从6 h即开始增加(P0.05),随时间增加HMGB1也随之大量转移到胞浆中,在人脑挫裂伤后24 h达到高峰(P0.05)。人脑挫裂伤后早期,HMGB1主要表达在神经元中(P0.05),脑挫裂伤后神经元的数目逐渐减少(P0.05),而HMGB1从神经元核内转移至胞浆的比例在6 h明显增加(P0.05),于24 h-72 h达到高峰(P0.05);同时研究发现AQP4主要表达在星形胶质细胞,并且随着损伤时间的延长,表达量逐渐增加,于伤后3 d达到高峰。结论明确人脑挫裂伤后早期炎症细胞浸润情况;HMGB1在人脑挫裂伤后早期迅速从细胞核内释放,参与炎症反应;HMGB1可能参与了人脑挫裂伤后神经元的坏死或凋亡;HMGB1可能参与了人脑挫裂伤后脑水肿的病理生理过程。
[Abstract]:Background traumatic brain injury includes primary injury and secondary injury. Secondary injury is involved by many factors: excitatory toxicity, oxygen free radical, calcium overload, edema, inflammatory reaction, etc. Edema and inflammatory reaction play an important role, but the specific mechanism is not very clear. It is reported that high mobility group protein B1 (high-mobility group box 1 HMGB1) plays a key role in the regulation of acute, chronic inflammation and edema. So what role HMGB1 plays in the secondary injury after human brain contusion is worth further study. Objective to investigate the expression and role of HMGB1 in human brain contusion and laceration. Methods 32 brain tissue specimens were collected from 32 patients with cerebral contusion and laceration as experimental group. According to the different time after injury, they were divided into two groups: 6 h group (n = 7), 12 h group (n = 9), 1 d group (n = 10) and 3 d group (n = 6), and normal brain tissue was collected as control group (n 5). All fresh specimens were fixed with 4% paraformaldehyde and then dehydrated with 30% sucrose solution. Then hematoxylin-eosin staining (H. E. Immunofluorescence staining. Staining: the numbers of lymphocytes and neutrophils in each field of vision were observed and the infiltration of inflammatory cells was analyzed at each time point. Immunofluorescence staining: the samples of experimental group and control group were stained with HMGB1 and DAPI. The number of HMGB1 in nucleus and cytoplasm of human brain contusion and laceration was observed under fluorescence microscope. To further understand the release of HMGB1 from the nucleus to the nucleus, the HMGB1 was colabeled with the neuron marker NeuN, and the number of neurons, the number of HMGB1 in neurons and the number of HMGB1 outside of neurons were observed and counted after the contusion and laceration of human brain. To further understand the metastasis between neurons and HMGB1 after injury, we colabeled aquaporin-4 (AQP4) with astrocyte marker (GFAP) to calculate the fluorescence intensity of aquaporin-4 (AQP4) expression at different time points. The expression of aquaporin 4 in astrocytes was analyzed. The statistical software SPSS 12.0 was used, the measurement data was expressed as x 卤s, and the OnewayANOVA analysis was used to compare the data among groups. The difference was statistically significant with P0.05. Results after contusion and laceration of human brain, the number of infiltrating lymphocytes increased significantly at 1 day (P0.05), and the infiltration of neutrophils increased significantly at 12 hours (P0.05). As time went on, the number of inflammatory cells increased gradually. The expression of HMGB1 in normal human brain tissue is mainly concentrated in the nucleus. In the contusion and laceration group, the number of HMGB1 cells in the nucleus began to decrease at 12 h (P0.05) and continued to decrease to 72 h, while the number of HMGB1 transferred from the nucleus to the cytoplasm began to increase from 6 h (P0.05), and the number of HMGB1 transferred to the cytoplasm increased with time. The peak was reached 24 hours after contusion and laceration of human brain (P0.05). HMGB1 was mainly expressed in neurons at the early stage of contusion and laceration (P0.05), and the number of neurons decreased gradually after contusion (P0.05), while the proportion of HMGB1 transferred from the nucleus of neurons to cytoplasm increased significantly at 6 h (P0.05), and reached its peak at 24h-72 h (P0.05). It was found that AQP4 was mainly expressed in astrocytes. With the extension of injury time, the expression level increased gradually and reached the peak 3 days after injury. Conclusion HMGB1 was rapidly released from the nucleus at the early stage of contusion and laceration of human brain and the infiltration of inflammatory cells in the early stage of contusion and laceration of human brain. HMGB1 may be involved in neuronal necrosis or apoptosis after human brain contusion and laceration. HMGB1 may be involved in the pathophysiological process of brain edema after contusion and laceration.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
本文编号：2184917
[Abstract]:Background traumatic brain injury includes primary injury and secondary injury. Secondary injury is involved by many factors: excitatory toxicity, oxygen free radical, calcium overload, edema, inflammatory reaction, etc. Edema and inflammatory reaction play an important role, but the specific mechanism is not very clear. It is reported that high mobility group protein B1 (high-mobility group box 1 HMGB1) plays a key role in the regulation of acute, chronic inflammation and edema. So what role HMGB1 plays in the secondary injury after human brain contusion is worth further study. Objective to investigate the expression and role of HMGB1 in human brain contusion and laceration. Methods 32 brain tissue specimens were collected from 32 patients with cerebral contusion and laceration as experimental group. According to the different time after injury, they were divided into two groups: 6 h group (n = 7), 12 h group (n = 9), 1 d group (n = 10) and 3 d group (n = 6), and normal brain tissue was collected as control group (n 5). All fresh specimens were fixed with 4% paraformaldehyde and then dehydrated with 30% sucrose solution. Then hematoxylin-eosin staining (H. E. Immunofluorescence staining. Staining: the numbers of lymphocytes and neutrophils in each field of vision were observed and the infiltration of inflammatory cells was analyzed at each time point. Immunofluorescence staining: the samples of experimental group and control group were stained with HMGB1 and DAPI. The number of HMGB1 in nucleus and cytoplasm of human brain contusion and laceration was observed under fluorescence microscope. To further understand the release of HMGB1 from the nucleus to the nucleus, the HMGB1 was colabeled with the neuron marker NeuN, and the number of neurons, the number of HMGB1 in neurons and the number of HMGB1 outside of neurons were observed and counted after the contusion and laceration of human brain. To further understand the metastasis between neurons and HMGB1 after injury, we colabeled aquaporin-4 (AQP4) with astrocyte marker (GFAP) to calculate the fluorescence intensity of aquaporin-4 (AQP4) expression at different time points. The expression of aquaporin 4 in astrocytes was analyzed. The statistical software SPSS 12.0 was used, the measurement data was expressed as x 卤s, and the OnewayANOVA analysis was used to compare the data among groups. The difference was statistically significant with P0.05. Results after contusion and laceration of human brain, the number of infiltrating lymphocytes increased significantly at 1 day (P0.05), and the infiltration of neutrophils increased significantly at 12 hours (P0.05). As time went on, the number of inflammatory cells increased gradually. The expression of HMGB1 in normal human brain tissue is mainly concentrated in the nucleus. In the contusion and laceration group, the number of HMGB1 cells in the nucleus began to decrease at 12 h (P0.05) and continued to decrease to 72 h, while the number of HMGB1 transferred from the nucleus to the cytoplasm began to increase from 6 h (P0.05), and the number of HMGB1 transferred to the cytoplasm increased with time. The peak was reached 24 hours after contusion and laceration of human brain (P0.05). HMGB1 was mainly expressed in neurons at the early stage of contusion and laceration (P0.05), and the number of neurons decreased gradually after contusion (P0.05), while the proportion of HMGB1 transferred from the nucleus of neurons to cytoplasm increased significantly at 6 h (P0.05), and reached its peak at 24h-72 h (P0.05). It was found that AQP4 was mainly expressed in astrocytes. With the extension of injury time, the expression level increased gradually and reached the peak 3 days after injury. Conclusion HMGB1 was rapidly released from the nucleus at the early stage of contusion and laceration of human brain and the infiltration of inflammatory cells in the early stage of contusion and laceration of human brain. HMGB1 may be involved in neuronal necrosis or apoptosis after human brain contusion and laceration. HMGB1 may be involved in the pathophysiological process of brain edema after contusion and laceration.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
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