丙戊酸对大鼠颅脑损伤后神经保护作用的实验研究

发布时间：2018-05-02 18:58

本文选题：创伤性颅脑损伤 + 丙戊酸　；参考：《新乡医学院》2013年硕士论文

【摘要】：背景创伤性颅脑损伤(Traumatic Brain Injury, TBI)是神经外科常见急症,TBI后由于神经细胞的凋亡、坏死及炎症反应等继发性损伤,造成运动和感觉功能障碍,严重影响患者预后。目前,对神经细胞的继发损伤尚无切实有效的治疗方法。因此TBI后早期进行神经细胞的保护及炎症反应的控制是神经外科研究的一个热点。近年的研究发现丙戊酸(valproic acid, VPA)具有多方面的神经营养和神经保护作用。本研究在建立闭合性颅脑损伤动物模型的基础上,早期应用丙戊酸,通过行为学评分和脑组织内神经细胞凋亡情况、HSP70和caspase-3、TNF-α和IL-1β蛋白表达的变化,探讨VPA对TBI大鼠神经功能恢复的影响及其机制,为临床应用提供理论基础。目的通过建立大鼠闭合性颅脑损伤模型,观察丙戊酸干预后,不同时间点大鼠脑组织内神经细胞凋亡情况、HSP70和caspase-3、TNF-α和IL-1β蛋白表达的影响,及脑组织中TNF-α和IL-1β的动态变化,探讨丙戊酸对实验性颅脑损伤大鼠的抗细胞凋亡、神经保护作用和减轻炎性反应的作用。方法将健康SD雄性大鼠随机分成正常对照组、单纯损伤组、VPA治疗组。建立大鼠闭合性颅脑损伤模型,采用神经系统疾病严重程度评分(Neurological Severity Scores, NSS)来评价各组大鼠不同时间点的神经功能；运用光学显微镜观察脑组织形态学的改变,应用原位末端转移酶标记技术(TUNEL)检测大鼠脑组织内细胞凋亡情况；运用免疫组织化学染色法(SP法)对各组不同时间点内脑细胞中的HSP70和caspase-3蛋白进行检测；利用免疫荧光技术检测各组不同时间点内脑细胞中的TNF-α和IL-1β蛋白表达情况；采用酶联免疫吸附试验(ELISA)法测定各组不同时间点内脑组织中TNF-α和IL-1β的动态变化,并作统计学分析。结果 1.行为学测定显示：大鼠行为学评分结果VPA治疗后大鼠神经功能有不同程度的恢复,在创伤后3d内3组间差异无显著性意义(P0.05); VPA治疗后7d、14d、21d、28d, VPA治疗组神经功能恢复程度优于单纯损伤组,两组间比较差异有显著性意义(P0.05)。 2.原位末端标记法(TUNEL)检测显示：单纯损伤组与VPA治疗组大鼠损伤区皮质各时间点凋亡阳性细胞数均高于正常对照组,差异均有统计学意义(P0.05)。VPA治疗组各时间点凋亡阳性细胞数均低于单纯损伤组,差异均有统计学意义(P0.05)。 3.免疫组织化学检测显示：VPA治疗组相同时间点HSP70平均灰度值明显低于单纯损伤组,两者差异有统计学意义(P0.05)。VPA治疗组相同时间点caspase-3平均灰度值明显高于单纯损伤组,两者差异有统计学意义(P0.05)。 4.免疫荧光技术检测：VPA治疗组相同时间点脑组织TNF-α和IL-1β阳性细胞数明显低于单纯损伤组,两者差异有统计学意义(P0.05)。 5.酶联免疫吸附试验(ELISA)检测：造模后单纯损伤组和VPA治疗组相同时间点脑组织中TNF-α和IL-1β蛋白浓度均高于正常对照组,差异均有统计学意义(P0.05); VPA治疗组相同时间点脑组织中TNF-α和IL-1β蛋白浓度均低于单纯损伤组,差异均有统计学意义(P0.05)。结论 VPA治疗后能够改善创伤性颅脑损伤大鼠的神经功能；VPA有抗细胞凋亡、保护神经细胞和减轻炎性反应的作用。
[Abstract]:background
Traumatic brain injury (Traumatic Brain Injury, TBI) is a common emergency in the Department of neurosurgery. After TBI, the secondary injury of nerve cell apoptosis, necrosis and inflammatory reaction causes motor and sensory dysfunction, which seriously affects the prognosis of the patients. At present, there is no effective and effective treatment for the injury of nerve cells. So TBI early after the injury. The protection of the nerve cells and the control of the inflammatory reaction are a hot spot in the Department of neurosurgery. In recent years, the study found that valproic acid (VPA) has many aspects of neurotrophic and neuroprotective effects. The changes of neuronal apoptosis and the expression of HSP70 and Caspase-3, TNF- alpha and IL-1 beta protein in the brain tissue, and to explore the effect and mechanism of VPA on the recovery of neural function in TBI rats, provide a theoretical basis for clinical application.
objective
By establishing a rat model of closed craniocerebral injury, the effects of apoptosis, the expression of HSP70 and Caspase-3, TNF- - A and IL-1 beta in the brain tissue of rats at different time points, the dynamic changes of TNF- - A and IL-1 beta in brain tissue were observed, and the anti apoptosis of the rats with experimental craniocerebral injury was investigated. The protective effect and alleviating the effect of inflammatory reaction.
Method
The healthy SD male rats were randomly divided into normal control group, simple injury group and VPA treatment group. The rat model of closed craniocerebral injury was established, and the neurological disease severity score (Neurological Severity Scores, NSS) was used to evaluate the neural function of the rats at different time points in each group; the optical microscope was used to observe the morphology of the brain tissue. Change, in situ TDT labeling technique (TUNEL) was used to detect the cell apoptosis in the brain tissue of rats. The HSP70 and caspase-3 protein in the brain cells were detected by immunohistochemistry (SP method) and the TNF- alpha and IL- in the brain cells of each group were detected by immunofluorescence technique. The expression of 1 beta protein was detected by enzyme linked immunosorbent assay (ELISA), and the dynamic changes of TNF- alpha and IL-1 beta in the brain tissues were measured at different time points, and the statistical analysis was made.
Result
1. behavior test showed that the rats' behavioral score results were recovered in different degrees after VPA treatment, and there was no significant difference between the 3 groups after the trauma (P0.05). After VPA treatment, 7d, 14d, 21d, 28d, and VPA treatment group were better than the single pure injury group, and there was significant difference between the two groups (P0.05).
2. in situ end labeling (TUNEL) detection showed that the number of apoptotic cells in the cortex at all time points in the injured area of the injured group and the VPA group were higher than that in the normal control group, the difference was statistically significant (P0.05), the number of apoptotic cells at each time point in the.VPA treatment group was lower than that in the simple injury group, the difference was statistically significant (P0.05).
3. the immunohistochemical test showed that the mean gray value of HSP70 in the VPA treatment group was significantly lower than that of the simple injury group. The difference was statistically significant (P0.05) the mean gray value of Caspase-3 in the same time point of the.VPA treatment group was significantly higher than that of the simple injury group, and the difference had the significance of the unified planning (P0.05).
4. immunofluorescence assay: the number of TNF- alpha and IL-1 beta positive cells in the brain tissue at the same time point in the VPA treatment group was significantly lower than that in the simple injury group, and the difference was statistically significant (P0.05).
5. enzyme linked immunosorbent assay (ELISA) test: the concentration of TNF- alpha and IL-1 beta protein in the brain tissue at the same time point in the same time point after the model building and the VPA treatment group were higher than the normal control group, the difference was statistically significant (P0.05). The concentration of TNF- a and IL-1 beta protein in the brain tissue at the same time point in the VPA treatment group was lower than that of the simple injury group. Statistical significance (P0.05).
conclusion
VPA can improve the neurological function of rats with traumatic brain injury. VPA can resist apoptosis, protect nerve cells and reduce inflammatory reaction.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R651.15

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