ApoE在小鼠脊髓损伤修复过程中的表达变化及其对星型胶质细胞的作用研究

发布时间：2018-06-27 11:04

本文选题：脊髓损伤 + 载脂蛋白E类　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]本研究通过建立C57/BL6野生型小鼠和ApoE基因敲除小鼠脊髓损伤模型,从动物整体水平探讨ApoE对小鼠脊髓损伤后运动功能活动和星形胶质细胞影响,以期为ApoE对中枢神经损伤后的作用机制的研究提供理论依据。[方法]本研究采用脑立体定位仪自制脊髓打击器制作C57/BL6小鼠脊髓损伤动物模型,探究ApoE在脊髓损伤恢复进程的影响及其可能的作用机制。所有动物分为三组:假手术组、野生型小鼠组以及ApoE-/-组。对野生型小鼠运用免疫印迹分析和RT-PCR,定量测定ApoE蛋白和mRNA在脊髓损伤恢复过程中的表达变化。建模后每天行BBB功能评分直至实验结束,表示脊髓损伤后小鼠运动功能恢复状态。对脊髓损伤处组织行苏木精-伊红染色,观察损伤节段细胞水肿及炎症反应情况。通过Western-Blot和免疫荧光染色观察损伤节段星形胶质细胞特异性标记物GFAP的表达变化及神经纤维的生长情况。[结果]1.C57/BL6野生型小鼠SCI后,ApoE最早从第3天开始升高,第7d和14d时,ApoE的表达量显著增加(P0.01)。其中第7d时,ApoE mRNA含量达到最高,第14d时ApoE蛋白含量达到最高,28天时含量有所下降,但是仍然高于损伤后第1天和假手术组(P0.05)。2.BBB评分结果显示,SCI后第7d开始,C57/BL6野生型小鼠得分优于ApoE-/-小鼠,第14d时差异最明显;第28d时,两组评分差异减小,但野生型小鼠BBB评分仍高于ApoE-/-(P0.05),说明ApoE存在的状态下,有利于小鼠脊髓损伤后的运动功能恢复。3.小鼠脊髓损伤节段HE染色结果显示,SCI后第14d,野生型小鼠脊髓组织结构较ApoE-/-小鼠完整,脊髓灰、白质组织结构可辨认,炎性细胞较少,细胞核固缩较轻,可见清晰胞质胞核,形态较正常。ApoE-/-小鼠组织结构紊乱,广泛水肿,有较多炎性细胞浸润,细胞核呈游散状态,固缩严重。4.Western blot结果显示各组脊髓损伤后均有GFAP蛋白表达。且在第7d,14d,28d,ApoE-/-小鼠的GFAP表达量高于野生型小鼠。5.与野生型小鼠相比,ApoE-/-组小鼠损伤节段的GFAP阳性染色细胞较多,轴突数目相对较多,这提示星形胶质细胞活化程度相对较高。[结论]1.小鼠脊髓损伤后,ApoE表达量随着病程进展呈进行性增加;2.ApoE能加快脊髓损伤小鼠后肢运动功能恢复进程,对脊髓损伤后的神经恢复有正向作用;3.ApoE对脊髓损伤的修复作用可能与下调GFAP的含量,抑制损伤周围星形胶质瘢痕的形成有关。
[Abstract]:[objective] to investigate the effects of ApoE on motor function and astrocytes after spinal cord injury in C57 / BL6 wild-type mice and ApoE knockout mice. To provide a theoretical basis for the study of the mechanism of ApoE after central nerve injury. [methods] the spinal cord injury model of C57 / BL6 mice was made by using the brain stereotactic locator to investigate the effect of ApoE on the recovery process of spinal cord injury and its possible mechanism. All animals were divided into three groups: sham operation group, wild type mice group and ApoE-P-group. The expression of ApoE protein and mRNA during the recovery of spinal cord injury in wild type mice was determined by Western blot and RT-PCR. After modeling, BBB scores were performed every day until the end of the experiment, indicating the recovery of motor function after spinal cord injury. Hematoxylin-eosin staining was used to observe the edema and inflammatory reaction in the injured spinal cord tissue. The expression of GFAP and the growth of nerve fibers were observed by Western-Blot and immunofluorescence staining. [results] 1. The expression of ApoE in C57 / BL6 wild-type mice increased at the earliest from the 3rd day after sci, and the expression of ApoE increased significantly on the 7th and 14th days (P0.01). On the 7th day, the ApoE mRNA content reached the highest level, and the ApoE protein content reached the highest on the 14th day, and decreased at 28 days. But it was still higher than that of the first day after injury and the sham-operation group (P0.05). 2. The results of BBB score showed that the score of C57 / BL6 wild-type mice was better than that of ApoE-r-mice on the 7th day after sci, and the difference was the most obvious at the 14th day, and the difference between the two groups decreased on the 28th day. But the BBB score of wild type mice was still higher than that of ApoE-r- (P0.05), which indicated that the presence of ApoE was beneficial to the recovery of motor function after spinal cord injury. 3. The results of HE staining showed that the structure of spinal cord of wild type mice was more intact than that of ApoE-r-mice on the 14th day after sci, the structure of gray and white matter tissue was recognizable, the inflammatory cells were less, the nucleus was less pyknosis, and the cytoplasmic nucleus was clear. Compared with the normal. ApoE-r- mice, the tissue structure was disordered, extensive edema, more inflammatory cells infiltrated, and the nucleus was swimming away. 4. The results of Western blot showed that GFAP protein was expressed in all groups after spinal cord injury. The expression of GFAP in ApoE-r-mice was higher than that in wild-type mice. Compared with the wild type mice, the GFAP positive staining cells and the number of axons in the injured segments of the ApoE-r- group were more than those in the wild type mice, which suggested that the astrocytes were more activated than the wild type mice. [conclusion] 1. The expression of ApoE in mice after spinal cord injury increased progressively with the progression of the disease. 2. ApoE could accelerate the recovery of motor function in the hind limbs of mice with spinal cord injury. The effect of ApoE on the repair of spinal cord injury may be related to the down-regulation of GFAP content and the inhibition of astrocyte scar formation around the injury.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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