TAK1信号通路在创伤性脑损伤中的作用机制研究

发布时间：2018-06-01 15:25

本文选题：TBI + TAK1　；参考：《南京大学》2013年硕士论文

【摘要】：第一部分题目：创伤性脑损伤后TAKl的表达时相和定位目的：研究TAK1在TBI后动态表达和分布情况,探讨TAK1通路和TBI的相互关系,以期为TBI的治疗提供一新的治疗靶点。方法：1、实验模型建立和分组：选择健康成年SD雄性大鼠60只,体重为250-300g,采用改良的Feeney法于右侧开颅制作TBI模型。将动物按损伤不同时间点分为6组,分别为假手术组、伤后1h组、伤后3h组、伤后6h组、伤后12h组、伤后24h组,各组动物均为10只。 2、TBI后TAK1及p-TAK1蛋白表达测定：按不同时间点分别提取大鼠挫伤灶周围皮层脑组织,采用western blot方法检测TAK1和p-TAK1蛋白的动态表达。 3、采用免疫组化法观察：TAK1在伤灶周围脑组织的表达和分布情况。 4、采用免疫荧光技术观察：TAK1在TBI后损伤灶周围皮层中不同细胞表达和分布情况。结果：1、TBI组术后各时间点TAK1和p-TAK1的蛋白水平24h内持续升高,24h明显升高(p0.05)； 2、免疫组化结果显示：假手术组中,TAK1在正常脑组织中有表达,主要分布于核周,外伤后胞浆表达量明显升高；荧光显微镜结果显示,在正常脑组织中,TAK1的表达主要位于神经元中,在星形胶质细胞中微量表达,损伤后TAK1在伤灶周围皮层的神经元和胶质细胞的胞浆中的表达量明显升高。结论：1、TAK1在TBI早期即可被激活。 2、TBI后TAK1表达主要位于神经元和胶质细胞胞浆中,且其激活的区域与脑外伤后损伤区域相一致。第二部分题目：OZ对TBI的神经保护作用及机制研究目的：通过研究OZ对TBI后TAK1通路的调控,探讨OZ对TBI的神经保护作用及其具体分子调控机制,以期为临床治疗TBI提供新的策略,同时也为OZ临床应用提供理论基础和实验依据方法：1、实验动物模型和分组：采用健康SD大鼠,运用改良的Feeney法于右侧开颅制作TBI模型。实验动物分组：大鼠分为五组：假手术组、外伤组、外伤+溶剂组、外伤+药物组(2μg,10μg和20μg)。溶剂和药物在损伤后10mim或3h通过侧脑室注射5μ1剂量。 2、神经功能评分：采用平衡木实验评价实验动物TBI后7天内神经功能缺损状况。 3、神经元凋亡检测：取各组实验动物大脑组织,石蜡固定,切片,TUNEL法检测神经元凋亡情况。 4、炎性因子检测：取各组伤灶周围脑组织,采用ELISA法检测炎性因子的表达量； 5、TAK1活性及通路下游NF-κB和AP-1的活性：取各组实验动物伤灶周围皮层组织,采用western blot方法检测TAK1和p-TAK1的蛋白含量,EMSA方法检测NF-κB和AP。1的活性。 6、尼氏染色法检测：外伤后7天神经元的存活情况。结果：1、与假手术组相比,TBI后大鼠表现出明显的神经功能缺损、神经元凋亡,两组间有显著性差异(P0.05)；而OZ治疗能明显改善大鼠神经功能评分、减轻神经元的凋亡,两组间有显著性差异(P0.05)。 2、TBI后损伤灶周围脑组织中TAK1蛋白表达增强；0Z明显下调TAK1蛋白表达。 3、与TBI组相比,OZ明显降低了NF-kB和AP-1的活性。 4、假手术组大鼠脑组织中TNF-α和IL-1p蛋白水平较低；脑损伤组中TNF-α和几.1p蛋白水平明显上调,与TBI组相比,TNF-α和IL.1p蛋白水平明显下降。 5、与假手术组相比,TBI后7天尼氏染色损伤神经元个数明显增加,而OZ治疗能明显改善神经元的损伤。结论：1、OZ能减轻TBI后继发性损伤,对TBI有神经保护作用。 2、OZ神经保护作用是通过抑制NF-κB和AP.1通路,抑制炎性因子的产生从而减轻炎性损伤来实现。 3、以TAK1为靶点的药物治疗对临床TBI的防治具有良好的应用前景。第三部分题目：TAKl在TBI患者挫伤区皮层的表达和定位目的：研究TAK1在TBI患者挫伤区皮层的表达及分布。方法挫伤区皮层标本来自5例TBI患者,伤后取标本时间24h,阴性对照组为3例肿瘤患者的正常脑组织(由于手术入路要求切除的),通过免疫组织化学和免疫荧光方法检测TAK1在挫伤皮层的表达和定位。结果：与阴性对照组相比,免疫组化结果显示挫伤区皮层TAK1的表达量明显升高；免疫荧光结果显示TAK1在TBI患者挫伤区皮层的神经元和胶质细胞中均有表达。结论：TAK1在挫伤区皮层的表达量升高,提示针对TAK1的靶点治疗在TBI的临床治疗中具有重要的临床应用价值。
[Abstract]:Part one
Title: expression and location of TAKl after traumatic brain injury
Objective: To study the dynamic expression and distribution of TAK1 after TBI, and to explore the relationship between TAK1 pathway and TBI, in order to provide a new therapeutic target for TBI treatment.
Methods: 1, the establishment and grouping of the experimental model: 60 healthy adult SD male rats were selected and the weight was 250-300g. The modified Feeney method was used to make the TBI model on the right craniotomy. The animals were divided into 6 groups according to the different time points of injury. They were sham operation group, 1H group after injury, 3h group after injury, 6h group after injury, 12h group after injury, group 24h after injury, all the animals of each group were 1. 0.
2, after TBI, the expression of TAK1 and p-TAK1 protein was measured. The brain tissue around the rat contusion was extracted at different time points, and the dynamic expression of TAK1 and p-TAK1 protein was detected by Western blot.
3, immunohistochemical staining was used to observe the expression and distribution of TAK1 in the brain tissue around the lesion.
4, immunofluorescence technique was used to observe the expression and distribution of TAK1 cells in the cortex around the lesion after TBI.
Results: 1. The protein levels of TAK1 and p-TAK1 in group TBI increased continuously during 24h, and 24h increased significantly (P0.05).
2, the results of immunohistochemistry showed that in the sham operation group, TAK1 was expressed in normal brain tissue, mainly distributed in the perinuclear tissue, and the expression of cytoplasm was significantly increased after trauma. The results of fluorescence microscopy showed that in normal brain tissue, the expression of TAK1 was mainly in the neurons, and in the astrocytes, the TAK1 was in the surrounding skin after injury. The expression levels of neurons and glial cells in the cytoplasm increased significantly.
Conclusion: 1, TAK1 can be activated in the early stage of TBI.
2, the expression of TAK1 was mainly located in cytoplasm of neurons and glial cells after TBI, and the region of activation was consistent with that of post traumatic brain injury.
The second part
Title: the neuroprotective effect and mechanism of OZ on TBI
Objective: To explore the neuroprotective effect of OZ on TBI and its specific molecular regulation mechanism by studying the regulation of OZ on the TAK1 pathway after TBI, so as to provide a new strategy for the clinical treatment of TBI, and to provide theoretical basis and experimental basis for the clinical application of OZ.
Methods: 1, experimental animal models and groups: using healthy SD rats, using the modified Feeney method to make the TBI model on the right craniotomy. Experimental animals were divided into five groups: sham operation group, trauma group, trauma + solvent group, trauma + drug group (2 mu g, 10 mu g and 20 micron g). The solution and drug were injected 5 mu through the lateral ventricle of 10mim or 3h after injury. Dose.
2, neurological function score: the balance function test was used to evaluate the neurological deficit in 7 days after TBI.
3, neuron apoptosis detection: take brain tissues of experimental animals, paraffin fixed and slice, and detect apoptosis of neurons by TUNEL.
4, inflammatory factors were detected: the brain tissue around each lesion was collected and the expression of inflammatory factors was detected by ELISA.
5, the activity of TAK1 and the activity of NF- kappa B and AP-1 downstream of the pathway: the protein content of TAK1 and p-TAK1 was detected by Western blot method, and the activity of NF- kappa B and AP.1 were detected by Western blot method.
6, Nissl staining was used to detect the survival of neurons on the 7 day after trauma.
Results: 1, compared with the sham operation group, the rats showed obvious nerve function defect and neuron apoptosis after TBI, and there was significant difference between the two groups (P0.05), and OZ treatment could obviously improve the neural function score of rats and reduce the apoptosis of the neurons. There was significant difference between the two groups (P0.05).
2, after TBI, the expression of TAK1 protein was increased in the brain tissue around the lesion. 0Z significantly decreased the expression of TAK1 protein.
3, compared with group TBI, OZ significantly reduced the activity of NF-kB and AP-1.
4, the level of TNF- alpha and IL-1p protein in the rat brain tissue of the sham operation group was low, and the level of TNF- alpha and several.1p protein in the brain injury group was obviously up, and the level of TNF- alpha and IL.1p protein decreased significantly compared with the TBI group.
5, compared with the sham operation group, the number of Nissl stained neurons increased significantly on the 7 day after TBI, while OZ treatment significantly improved neuronal damage.
Conclusion: 1, OZ can alleviate secondary injury after TBI and has neuroprotective effect on TBI.
2, the neuroprotective effect of OZ is achieved by inhibiting the NF- kappa B and AP.1 pathway, inhibiting the production of inflammatory factors and reducing inflammatory injury.
3, TAK1 targeted drug therapy has a good application prospect in the prevention and treatment of clinical TBI.
The third part
Title: expression and localization of TAKl in the contusion cortex of TBI patients
Objective: To study the expression and distribution of TAK1 in the contusion area of TBI patients.
Methods the cortical specimens from the contusion area were from 5 patients with TBI. The time was 24h after injury, and the normal brain tissue of the negative control group was 3 cases of the tumor patients (due to the surgical procedure). The expression and location of TAK1 in the contusion cortex were detected by immunohistochemistry and immunofluorescence.
Results: compared with the negative control group, the immunohistochemical results showed that the expression of TAK1 in the contusion area was significantly increased, and the results of immunofluorescence showed that TAK1 was expressed in the neurons and glia cells in the contusion area of TBI patients.
Conclusion: the expression level of TAK1 in the cortex of the contusion area is higher, suggesting that targeted therapy for TAK1 has important clinical value in the clinical treatment of TBI.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R651.15

【共引文献】