兔脊髓缺血再灌注后胶质细胞活化及JAK-STAT通路、炎症因子表达规律的实验研究

发布时间：2018-07-16 23:53

【摘要】：第一部分星形胶质细胞与JAK-STAT通路在兔脊髓缺血再灌注损伤后变化规律的研究目的:观察兔脊髓缺血再灌注后星形胶质细胞(AS)、胶质细胞源性神经营养因子(GDNF)、脑源性神经营养因子(BDNF)以及Janus激酶/信号转导和转录激活子(JAK-STAT)的表达规律。方法:采用胸主动脉阻断法建立兔脊髓缺血(22min)/再灌注损伤模型。健康成年(36周)雄性新西兰大白兔54只,采用计算机随机数法随机分为缺血再灌注Oh、1h、2h、3h、8h、24h、48h、72h组和假手术组(每组6只)共9组。缺血再灌注组分别于缺血再灌注后Oh、1h、2h、3h、8h、24h、48h和72h检测缺血段脊髓组织中正常神经元、凋亡神经元以及GFAP(星形胶质细胞特征性标记物)、GDNF、BDNF、p-STAT3的表达水平。结果:正常神经元数量随着再灌注时间延长而减少;TUNEL染色结果显示,脊髓缺血再灌注损伤后8h TUNEL阳性神经元开始增多,24hTUNEL阳性神经元最多。再灌注后3hGFAP表达水平开始增多,48h达到高峰;GDNF在再灌注后3h和72h各达到一次高峰;再灌注后24h BDNF表达水平最高;p-STAT3表达水平分别在再灌注后1h和48h到达高峰。假手术组:少见TUNEL阳性细胞,GFAP、GDNF、BDNF仅有少量表达,p-STAT3几乎无表达。结论:脊髓缺血再灌注损伤可以诱发星形胶质细胞激活,神经营养因子GDNF、BDNF水平升高,JAK-STAT信号通路激活并呈现不同的表达规律。第二部分兔脊髓缺血再灌注后小胶质细胞活化和炎性细胞因子变化的实验研究目的:观察兔脊髓缺血再灌注后小胶质细胞活化及炎性因子IL-6、IL-10、核转录因子NF-κB的变化规律,为后处理干预时机提供理论依据。方法:采用胸主动脉球囊阻断法建立兔脊髓缺血(22min)/再灌注损伤模型。健康成年(36周)雄性新西兰大白兔54只,随机分为假手术组S和缺血再灌注组C0、C1、C2、C3、C8、C24、C48、C72(每组6只)。缺血再灌注组分别于再灌注后Oh、1h、2h、3h、8h、24h、48h和72h应用形态学、分子生物学等检测方法检测缺血段脊髓组织中正常神经元、凋亡神经元以及Iba-1(小胶质细胞特征性标记物)、IL-6、IL-10、NF-κB的表达水平。结果:正常神经元数量随再灌注时间延长而减少;脊髓缺血再灌注损伤后8h TUNEL阳性神经元开始增多,24hTUNEL阳性神经元达高峰。再灌注2hIba-1表达开始增多,8h达到高峰;NF-κB于再灌注3h开始增高,8h为表达高峰;IL-6和IL-10均在再灌注24h达到高峰。NF-κB、IL-6、IL-10的表达水平与Iba-1显著正相关。结论:脊髓缺血再灌注后小胶质细胞激活呈动态变化。NF-κB、IL-6、IL-10的表达水平与小胶质细胞激活显著正相关,在小胶质细胞激活前给予后处理可降低神经元损伤。
[Abstract]:The study of changes of astrocytes and JAK-STAT Pathway after Spinal Cord Ischemia-reperfusion injury in Rabbits objective: to observe astrocytes (as), glial cell derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) after spinal cord ischemia-reperfusion injury in rabbits Expression of neurotrophic factor (BDNF) and Janus kinase / signal transduction and activator of transcription (JAK-STAT). Methods: rabbit spinal cord ischemia (22min) / reperfusion injury model was established by thoracic aortic occlusion. Fifty-four male New Zealand white rabbits (36 weeks old) were randomly divided into 9 groups (n = 6 in each group). The expressions of normal neurons, apoptotic neurons and GFAP (astrocytic characteristic marker) BDNF p-STAT3 in ischemic spinal cord were detected in the ischemia reperfusion group at 1 h, 3 h, 8 h, 24 h and 72 h after ischemia and reperfusion, respectively. Results: the number of normal neurons decreased with the prolongation of reperfusion time. The results of Tunel staining showed that the number of Tunel positive neurons began to increase at 8 h after spinal cord ischemia reperfusion injury, and the number of Tunel positive neurons increased most at 24 h after spinal cord ischemia reperfusion injury. The expression level of GFAP began to increase at 3 h after reperfusion and reached the peak at 48 h and reached the peak at 3 h and 72 h after reperfusion respectively, and the highest level of BDNF at 24 h after reperfusion reached the peak at 1 h and 48 h after reperfusion, respectively. In sham-operation group, few Tunel positive cells expressed BDNF and p-STAT3. Conclusion: spinal cord ischemia-reperfusion injury can induce astrocyte activation, and the level of neurotrophic factor GDNFN BDNF increases the activation of JAK-STAT signal pathway and presents different expression patterns. The second part of the experimental study on microglia activation and inflammatory cytokines after spinal cord ischemia reperfusion in rabbits objective: to observe the changes of microglia activation and inflammatory cytokine IL-10 and nuclear transcription factor NF- 魏 B after spinal cord ischemia reperfusion in rabbits. To provide theoretical basis for post-processing intervention timing. Methods: spinal cord ischemia (22min) / reperfusion injury model was established by balloon occlusion of thoracic aorta. Fifty-four male New Zealand white rabbits (36 weeks old) were randomly divided into sham-operated group (S) and ischemia reperfusion group (C0C _ 1C _ (2) C _ (2) C _ (3) C _ (3) C _ (24) C _ (48) C _ (72) (n = 6 in each group). The expressions of normal neurons, apoptotic neurons and Iba-1 (microglial characteristic marker) IL-10NF- 魏 B in ischemic spinal cord tissues were detected by morphological and molecular biology methods in the ischemia reperfusion group at 1 h, 2 h, 8 h and 24 h after reperfusion, respectively. Results: the number of normal neurons decreased with the prolongation of reperfusion time, and the number of Tunel positive neurons began to increase at 8 h after spinal cord ischemia-reperfusion injury and reached the peak at 24 h. The expression of Iba-1 began to increase at 2 h after reperfusion and reached the peak at 8 h. NF- 魏 B increased at 3 h after reperfusion. The expression peak of IL-6 and IL-10 reached the peak at 24 h after reperfusion. The expression level of IL-6 and IL-10 was positively correlated with Iba-1. Conclusion: the dynamic changes of microglia activation after spinal cord ischemia-reperfusion. The expression level of IL-6 / IL-10 is positively correlated with microglial activation, and post treatment before and after microglia activation can reduce neuronal injury.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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