糖皮质激素对放射致海马损伤保护作用的初步研究

发布时间：2018-01-30 09:40

本文关键词： 放射性脑病星形胶质细胞血脑屏障肿瘤坏死因子糖皮质激素海马　出处：《广西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的放射治疗是治疗头颈部等恶性肿瘤的有效手段之一,但是同时也带来了以记忆力减退为主要症状的认知功能障碍。海马在人类的情绪整合、学习、记忆等高级功能中占有重要地位,放射治疗引起海马损伤已经成为日益关注的重要课题。放射治疗引起的脑损伤还涉及到炎性反应,因此采取抗炎手段防治放射性脑病已经成为研究重点,而糖皮质激素已经广泛应用于临床,但是糖皮质激素如何防治放射性脑损伤的作用机制还未完全明了.本实验通过建立低剂量分割照射脑损伤模型,初步探讨糖皮质激素在放射治疗后海马损伤中的作用,为临床防治放射性脑损伤提供理论依据。方法将48只昆明小鼠随机分成空白组、30Gy模型组、模型+地塞米松治疗组、空白+地塞米松治疗组,共4小组,每组12只。在清醒状态下,用自制模具固定小鼠,放置于直线加速器平台上,照射源和平台之间放置带孔铅模,光源透过铅模孔发出光束精确定位照射脑部,2Gy/次,5次/周,连续照射3周,累计剂量30Gy。放射后模型+地塞米松治疗组及空白+地塞米松治疗组.分别给予地塞米松(dexamethasone, DXM)1mg/KG腹腔注射,照射前、地塞米松治疗后通过Morris水迷宫测试小鼠的空间学习和记忆能力。DXM治疗后3周每组随机抽出6只采用石蜡切片免疫组织化学法的方法,观察DXM前、后星形胶质细胞(GFAP+)附性细胞数目,来评价神经干细胞分化和胶质细胞对DXM治疗前、后的反应情况,石蜡切片免疫组织化学的方法检测肿瘤坏死因子(TNF-α)(?)的阳性细胞数目来评价DXM治疗前、后中枢神经系统神经炎性反应的情况,并做海马组织尼氏染色和HE染色等病理切片染色。同时每组随机抽取6只通过尾静脉注射伊文思蓝(EB),通过测定DXM治疗前、后脑组织EB含量和脑组织含水量的改变情况来评价照射后血脑屏障破坏程度。结果 1.一般情况：放射后模型组和模型+DXM治疗组,出现易激惹、相互撕咬等精神症状,模型组出现彗星尾征(见附图)。模型组和模型+DXM治疗组照射后3周出现轻度毛发脱落,经糖皮质激素治疗后模型+DXM治疗组上述症状有所减轻。照射前各组小鼠体重无明显差别,照射后3周,模型组、模型+DXM治疗组体重与空白组相比出现明显差别,治疗后2周,模型+DXM治疗组小鼠体重恢复到空白组水平,模型组小鼠体重虽然出现恢复,但与空白组相比尚未出现明显差别。空白组、空白+DXM治疗组小鼠体重逐渐增加,未见有明显差别。 2.行为学测试：照射前6周,空白组虽然穿越平台次数和第一次穿越平台时间未见有明显差别,但在第一象限和中环区活动时间明显增多,然而采用糖皮质激素干预后,上述的差别恢复到空白组水平。模型组小鼠在放射后6周不仅穿越平台的次数明显下降,第1次找到平台的时间也明显延长,然而采用激素干预后,穿越平台的次数、第1次穿越平台的时间、在各象限和各环寻找平台的时间未见有明显差别。空白+DXM治疗组和模型+DXM治疗组行为学测试未见有明显差异。 3.尼氏染色：放射治疗后模型组与空白组相比在海马CA1区尼氏细胞数目明显减少(P0.01)。地塞米松治疗组与模型组相比在海马CA3区尼氏细胞数目增多(P0.05)。空白+DXM治疗组与空白组相比在海马CA1区细胞数目增多(P0.05)。其余各组未见有明显的差异。 4.苏木精-伊红染色法(hematoxylin-eosin staining)简称HE染色法：放射后模型组与空白组相比细胞数目在海马CA1、CA2、CA4区均没有明显差异(P0.05),模型+DXM治疗组与模型组相比在海马各区也均无显著的统计学意义(P0.05),空白+DXM治疗组与空白组相比未见有显著差异(P0.05)。 5. GFAP免疫组化：放射后模型组与空白组相比在海马CA1、CA2、CA3、CA4各区GFAP阳性细胞表达上调(P0.05),模型+DXM治疗组与模型组相比在海马CA1、CA2、CA3、CA4各区GFAP阳性细胞数目表达增加(P0.05),空白+DXM治疗组与空白组相比在海马CA3区阳性细胞数目表达增加(P0.01)。 6. TNF-α免疫组化：模型组与空白组相比在海马CA2、CA3、CA4区TNF-α阳性细胞数目表达明显上调(P0.05),模型+DXM治疗组与模型组相比在海马CA2、CA3、CA4区TNF-a阳性细胞数目表达显著下降(P0.05)。空白+DXM治疗组与空白组相比在CA4区阳性细胞数目表达明显增加(P0.05)。 7.放射后血脑屏障的改变：放射后模型组与空白组相比脑组织含水量、EB含量显著增加(P0.01)。模型+DXM治疗组与模型组相比脑组织内含水量、EB含量明显降低(P0.05),空白+DXM治疗组与空白组相比没有明显的统计学意义(P0.05)。结论 1.低剂量分割颅脑照射可以引起与海马损伤有关的行为学改变。 2.糖皮质激素治疗后海马区GFAP、TNF-α明显表达下调,脑组织内EB含量、含水量也有明显降低。
[Abstract]:Radiotherapy is one of effective methods in the treatment of head and neck malignant tumors, but also brought to memory loss and cognitive dysfunction as the main symptoms. In learning in the emotional integration of human, plays an important role in memory and other advanced features, caused by damage to the hippocampus has become an increasingly important topic concerned in radiotherapy. Radiotherapy for brain it is related to the damage caused by inflammation, so take anti-inflammatory means of prevention and treatment of radiation encephalopathy has become a research focus, and glucocorticoids have been widely used in clinical, but the mechanism of glucocorticoid to prevent radiation-induced brain injury is not entirely clear. The low dose radiation brain injury model segmentation, preliminary study the role of glucocorticoids in hippocampus injury after radiation therapy, to provide a theoretical basis for clinical prevention and treatment of radiation brain injury.
Methods 48 Kunming mice were randomly divided into blank group, 30Gy model group, model + dexamethasone treatment group, blank + dexamethasone treatment group, a total of 4 groups, 12 rats in each group. In the awake state, using self-made mold fixed mice, placed on the linear accelerator platform, with Kong Qianmo placed between the radiation source and platform, through the light source lead a die hole precise positioning beam brain, 2Gy/ time, 5 times / week, 3 weeks of continuous irradiation, total dose of 30Gy. after radiation model + dexamethasone treatment group and blank + dexamethasone treatment group were given dexamethasone (dexamethasone, DXM) 1mg/KG intraperitoneal injection before irradiation after dexamethasone treatment through spatial learning and memory ability.DXM treatment of the Morris water maze test in mice after 3 weeks in each group were randomly selected 6 paraffin sections by immunohistochemistry, DXM was observed before, after astrocytes (GFAP+) the number of cells And to evaluate the differentiation of neural stem cells and glial cells of DXM before treatment, after the reaction, method of paraffin section immunohistochemical detection of tumor necrosis factor alpha (TNF- alpha) (?) the number of positive cells of DXM before treatment, after central nervous system inflammatory response, and Nissl staining and HE staining pathological sections of hippocampus tissue staining. At the same time randomly selected 6 rats by intravenous injection of Evans blue (EB), through the determination of DXM before treatment, the changes of brain tissue EB content and water content of brain tissue to evaluate the damage of blood brain barrier after irradiation.
Result
1. general situation: model group and +DXM treatment group after radiation model, irritability, mental symptoms such as biting each other, model group, comet tail sign (see photo). Mild hair loss appeared in model group and +DXM treatment group 3 weeks after irradiation, after the treatment with corticosteroids in the treatment group symptom model +DXM reduced. Body weight of mice in each group had no significant difference before irradiation, irradiation after 3 weeks, the model group, +DXM treatment group weight model compared with the blank group appeared obvious difference, after 2 weeks of treatment, the treatment group +DXM model mice weight recovery to the level of the blank control group, body weight of mice in the model group while the recovery, but not yet compared with the control group there was significantly difference. The blank group, blank +DXM treatment group mice body weight increased gradually, there are obvious differences between the No.
Test 2. behavior: 6 weeks before irradiation, the blank group although the number of cross platform and cross platform of the first time there was no significant difference, but significantly increased in the first quadrant and the central area of activity time, however with glucocorticoid intervention, the difference back to the level of the blank group. The mice in the model group after radiation 6 not only the number of weeks of crossing the platform was significantly decreased, the first time to find the platform is also significantly prolonged, but the use of hormone intervention, times of passing through the platform, the first cross platform time in each quadrant and the ring of the time to find the platform did not have significant difference. The blank +DXM treatment group and +DXM treatment group behavior model the test did not have obvious difference.
3. Nissl staining: after radiotherapy in model group compared with blank group decreased significantly in the hippocampal CA1 region of Nissl cell number (P0.01). Dexamethasone treatment group compared with the model group in the CA3 region of the hippocampus Nissl cells (P0.05). The blank +DXM treatment group compared with the control group in the hippocampus CA1 number (P0.05). While there was no significant difference.
4. hematoxylin eosin staining (hematoxylin-eosin staining) HE staining: after radiation model group compared to control group the number of cells in the hippocampus CA1, CA2, there are no significant differences between the CA4 region (P0.05), model +DXM treatment group compared with model group, statistically significant in the hippocampus also were not significant (P0.05) blank, +DXM treatment group compared with the control group there was no significant difference (P0.05).
5. GFAP immunohistochemical staining: after radiation model group compared with blank group in hippocampus CA1, CA2, CA3, CA4 expression of the GFAP positive cells (P0.05), model +DXM treatment group compared with model group in hippocampus CA1, CA2, CA3, CA4 the number of GFAP positive cells increased expression of +DXM (P0.05), blank treatment group compared with the control group the number of positive cells in the hippocampus CA3 expression increased (P0.01).
6. alpha TNF- immunohistochemistry: model group in hippocampus CA2, CA3 compared with the control group, CA4 TNF- alpha positive cells were significantly up-regulated (P0.05) model, +DXM treatment group compared with model group in hippocampus CA2, CA3, CA4, TNF-a positive cell number was significantly decreased (P0.05). The blank treatment +DXM group compared with the control group the number of positive cells in CA4 area was significantly increased (P0.05).
Changes of blood brain barrier after radiotherapy: 7. after radiotherapy of brain tissue compared to the blank group and the model group water content, EB content increased significantly (P0.01). Model +DXM treatment group compared with model group, the brain water content, EB content decreased significantly (P0.05), blank +DXM treatment group and blank group compared with no obvious statistical significance the (P0.05).
conclusion
1. low dose fractionated craniocerebral irradiation can cause behavioral changes associated with hippocampal damage.
After 2. glucocorticoid treatment, the expression of GFAP, TNF- - alpha in the hippocampus, the content of EB in the brain tissue and the water content were also decreased.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R818

【参考文献】