颅脑损伤后胶质纤维酸性蛋白表达上调途径及其临床检测的意义
摘要
神经生化标志物的研究已引起许多研究者的注意,目前发现许多神经系统生化标志物可以用来判断神经系统的损害范围和预测预后,并发现一些生化标志物在早期优于CT以及MRI等影像学检查,它们其中包括一些生物酶、神经递质、细胞因子、蛋白质和神经肽类等。脑内星形胶质细胞所表达的血清胶质纤维酸性蛋白是一个较新的神经生物化学标志物,其作用、功能尚未得以完全阐释,特别是在神经系统损伤后其表达及调控的机制目前研究较少。
本研究第一部分将以大鼠为研究对象,通过研究颅脑损伤后大鼠的受损脑组织与其周围脑组织中胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达情况,找到在脑外伤后血清胶质纤维酸性蛋白的变化规律以及表达上调的主要途径;本研究第二部分研究对象为人脑,通过检测脑外伤病人外周血中胶质纤维酸性蛋白浓度变化和检测手术患者脑组织中上述蛋白的表达水平高低,验证外周血血清浓度是否与脑组织蛋白表达水平相一致,并验证其在脑外伤患者诊断及预后判断上的敏感性和特异性,最终揭示该类神经标志物在对脑外伤患者诊断及预后判断上的意义。
实验结果表明受损脑组织从伤后第1天胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶开始上升,持续到第7d开始回落,第7天是表达最高峰。损伤组织胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达具有明显正相关性(r=0.854,P<0.05) ,损伤周围组织胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达无明显相关性(r=0.554,p>0.05)。脑外伤患者GFAP与健康对照组比较差异有显著性(p<0.05),脑外伤患者外周血GFAP水平显著高与健康人群。从非手术脑创伤患者伤后不同时间点外周血GFAP的变化(不包括死亡病例) GFAP最高峰出现在伤后的48小时。可以认为损伤组织胶质纤维酸性蛋白表达上调是通过P38丝裂素活化蛋白激酶介导的,损伤周围组织胶质纤维酸性蛋白上调与P38丝裂素活化蛋白激酶表达无关。人外周血中胶质纤维酸性蛋白水平与脑组织中蛋白表达相一致。颅脑损伤后损伤部位脑组织胶质纤维酸性蛋白表达上调是通过P38丝裂素活化蛋白激酶途径介导,而正常大鼠脑组织中胶质纤维酸性蛋白的表达可能不依赖P38丝裂素活化蛋白激酶。第二部分实验结果显示脑外伤后胶质纤维酸性蛋白与其它神经标志物(如神经元特异性烯醇化酶)比较而言升高缓慢而持久,在预测病情方面具有较高特异性,对于预后判断具有较大意义。然而,胶质纤维酸性蛋白预测病情缺乏敏感性。
Objective: Nerve biochemical detection provide an effective method to judge the clinical prognosis to head injury patiens. The study on the nerve-biochemical marker has appeal to the eye of researchers recently, with the common objective finding the acute.non-invasive index that can reflect the scope and prognosis of central nerve system. Some reachers have found some biochemical markers that can judge the injury limit and prognosis through the study of blood and CSF. At the same time, they found some biochemical markers even surpass CT and MRI et al imageology scopy in earlier period. Such markers include some proteins, enzyme, cytokines and neurotransmitter et al. Glial fibrillary acidic protein (GFAP) is a kind of index needing detedted when the head injury happened. We want to definite the usage of the marker on the diagnosis and prognosis of head injury through detecting the changes of its concentration in peripherial blood of head injury patients. Detecting the expression level of head tissue to those patients needing operation, definite if the expression level of head tissue concord to the level in peripherial blood. At the same time, the sensitivity and specificity to judge the prognosis of head injury (HI)were definited.
Methods: 1. Divided the 80 rats into 10 groups randomly , as the group of nomal control, 1d after injury, 7d after injury, 14d after injury, control with SB203580 (A special inhibitor of P38MAPK), 1d 1d after injury with SB203580, 7d after injury with SB203580, 14d after injury with SB203580. Then detech the content of protein GFAP and P38MAPK by the method of western blot.
2. All objects recruited were examed by head CT to testify contusion and laceration of brain, scored with GCS system, all those severe combined injury ; auto-immune disease; malignant tumor; heametology disease and severe infection were removed. Operation group and non-operation group were divided according if operation was undertaked, dividing the patients into gently; meddle and severe groups according the head injury extent. The peripherial blood was detected in the patients without operation at 4h,6h,8h,12h,16h,24h,48h after in-patient,but at 2h,4h,8h,24h before and after operation to those patients undertaking the operation. Other detection such as the expression of GFAP in cerebral tissue was executed to those operation patients. At the same time, MAP、ICP、CCP need to be monitored to severe head injury. Calculate the relationship between GFAP and the MAP、ICP、CCP,prognosis. hyperthermy ,acute cardiogenic oedema pulmonum, hyperglykemia originated from diabetes. gastrorrhagia from ulcer, senile dementia, inflammation of the central nervous system, epilepsy, migraine and dementia praecox patients were recruited as pathology control group. Healthy objects recruited as health control group.
Resluts: GFAP concentration in peripherial blood after head injury show significant difference with health control group (p<0.05). The peak concentration of GFAP is 48h after head injury to those no-operation patients. The concentration of GFAP decreases gradually after operation. Those patients who decreasing of such indexes is not obviously indicate the bad prognosis. the concentration of GFAP in peripherial blood show significant difference with fever,edema of lung,high hyperglycaemia, gastrorrhagia patients(p<0.05). The concentration of GFAP in peripherial blood of senile dementia , inflammation of the central nervous system, epilepsy, migraine and dementia praecox patients show no difference with health objects (p>0.05). The concentraion of peripherial blood, the expression of brain tissue of GFAP is higher in ICP<25 mmHg, MAP<60 mmHg, CCP<60 mmHg patients than ICP≥25 mmHg, MAP≥60 mmHg, CCP≥60 mmHg patients. The concentration GFAP show obviously relationship with prognosis(r=0.915, p<0.05),the more high of The concentration of GFAP when hospital admission, the more bad of the prognosis.
conclusions: The concentration of GFAP increased both in peripherial blood and injury tissues in patients and experimental rats afer head injury. For patients, the more high of the concentration of GFAP when hospital admission, the more bad of the prognosis, and the concentration GFAP show obviously relationship with prognosis. GFAP up-regulation after HI may mainly through the pathway of P38MAPK signal. Comparing with other nerve-biochemical markers, GFAP increasing slowly and persistently. The sensitivity for deteching may inferior to other markers as GFAP up-regulation was not so fast as other matkers (for example NES) in early period of HI. If combine with other nerve-biochemical markers, GFAP level will assit to diagnosis head injury and judge the condition of prognosis.
本研究第一部分将以大鼠为研究对象,通过研究颅脑损伤后大鼠的受损脑组织与其周围脑组织中胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达情况,找到在脑外伤后血清胶质纤维酸性蛋白的变化规律以及表达上调的主要途径;本研究第二部分研究对象为人脑,通过检测脑外伤病人外周血中胶质纤维酸性蛋白浓度变化和检测手术患者脑组织中上述蛋白的表达水平高低,验证外周血血清浓度是否与脑组织蛋白表达水平相一致,并验证其在脑外伤患者诊断及预后判断上的敏感性和特异性,最终揭示该类神经标志物在对脑外伤患者诊断及预后判断上的意义。
实验结果表明受损脑组织从伤后第1天胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶开始上升,持续到第7d开始回落,第7天是表达最高峰。损伤组织胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达具有明显正相关性(r=0.854,P<0.05) ,损伤周围组织胶质纤维酸性蛋白和P38丝裂素活化蛋白激酶的表达无明显相关性(r=0.554,p>0.05)。脑外伤患者GFAP与健康对照组比较差异有显著性(p<0.05),脑外伤患者外周血GFAP水平显著高与健康人群。从非手术脑创伤患者伤后不同时间点外周血GFAP的变化(不包括死亡病例) GFAP最高峰出现在伤后的48小时。可以认为损伤组织胶质纤维酸性蛋白表达上调是通过P38丝裂素活化蛋白激酶介导的,损伤周围组织胶质纤维酸性蛋白上调与P38丝裂素活化蛋白激酶表达无关。人外周血中胶质纤维酸性蛋白水平与脑组织中蛋白表达相一致。颅脑损伤后损伤部位脑组织胶质纤维酸性蛋白表达上调是通过P38丝裂素活化蛋白激酶途径介导,而正常大鼠脑组织中胶质纤维酸性蛋白的表达可能不依赖P38丝裂素活化蛋白激酶。第二部分实验结果显示脑外伤后胶质纤维酸性蛋白与其它神经标志物(如神经元特异性烯醇化酶)比较而言升高缓慢而持久,在预测病情方面具有较高特异性,对于预后判断具有较大意义。然而,胶质纤维酸性蛋白预测病情缺乏敏感性。
Objective: Nerve biochemical detection provide an effective method to judge the clinical prognosis to head injury patiens. The study on the nerve-biochemical marker has appeal to the eye of researchers recently, with the common objective finding the acute.non-invasive index that can reflect the scope and prognosis of central nerve system. Some reachers have found some biochemical markers that can judge the injury limit and prognosis through the study of blood and CSF. At the same time, they found some biochemical markers even surpass CT and MRI et al imageology scopy in earlier period. Such markers include some proteins, enzyme, cytokines and neurotransmitter et al. Glial fibrillary acidic protein (GFAP) is a kind of index needing detedted when the head injury happened. We want to definite the usage of the marker on the diagnosis and prognosis of head injury through detecting the changes of its concentration in peripherial blood of head injury patients. Detecting the expression level of head tissue to those patients needing operation, definite if the expression level of head tissue concord to the level in peripherial blood. At the same time, the sensitivity and specificity to judge the prognosis of head injury (HI)were definited.
Methods: 1. Divided the 80 rats into 10 groups randomly , as the group of nomal control, 1d after injury, 7d after injury, 14d after injury, control with SB203580 (A special inhibitor of P38MAPK), 1d 1d after injury with SB203580, 7d after injury with SB203580, 14d after injury with SB203580. Then detech the content of protein GFAP and P38MAPK by the method of western blot.
2. All objects recruited were examed by head CT to testify contusion and laceration of brain, scored with GCS system, all those severe combined injury ; auto-immune disease; malignant tumor; heametology disease and severe infection were removed. Operation group and non-operation group were divided according if operation was undertaked, dividing the patients into gently; meddle and severe groups according the head injury extent. The peripherial blood was detected in the patients without operation at 4h,6h,8h,12h,16h,24h,48h after in-patient,but at 2h,4h,8h,24h before and after operation to those patients undertaking the operation. Other detection such as the expression of GFAP in cerebral tissue was executed to those operation patients. At the same time, MAP、ICP、CCP need to be monitored to severe head injury. Calculate the relationship between GFAP and the MAP、ICP、CCP,prognosis. hyperthermy ,acute cardiogenic oedema pulmonum, hyperglykemia originated from diabetes. gastrorrhagia from ulcer, senile dementia, inflammation of the central nervous system, epilepsy, migraine and dementia praecox patients were recruited as pathology control group. Healthy objects recruited as health control group.
Resluts: GFAP concentration in peripherial blood after head injury show significant difference with health control group (p<0.05). The peak concentration of GFAP is 48h after head injury to those no-operation patients. The concentration of GFAP decreases gradually after operation. Those patients who decreasing of such indexes is not obviously indicate the bad prognosis. the concentration of GFAP in peripherial blood show significant difference with fever,edema of lung,high hyperglycaemia, gastrorrhagia patients(p<0.05). The concentration of GFAP in peripherial blood of senile dementia , inflammation of the central nervous system, epilepsy, migraine and dementia praecox patients show no difference with health objects (p>0.05). The concentraion of peripherial blood, the expression of brain tissue of GFAP is higher in ICP<25 mmHg, MAP<60 mmHg, CCP<60 mmHg patients than ICP≥25 mmHg, MAP≥60 mmHg, CCP≥60 mmHg patients. The concentration GFAP show obviously relationship with prognosis(r=0.915, p<0.05),the more high of The concentration of GFAP when hospital admission, the more bad of the prognosis.
conclusions: The concentration of GFAP increased both in peripherial blood and injury tissues in patients and experimental rats afer head injury. For patients, the more high of the concentration of GFAP when hospital admission, the more bad of the prognosis, and the concentration GFAP show obviously relationship with prognosis. GFAP up-regulation after HI may mainly through the pathway of P38MAPK signal. Comparing with other nerve-biochemical markers, GFAP increasing slowly and persistently. The sensitivity for deteching may inferior to other markers as GFAP up-regulation was not so fast as other matkers (for example NES) in early period of HI. If combine with other nerve-biochemical markers, GFAP level will assit to diagnosis head injury and judge the condition of prognosis.
引文
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