牛磺熊去氧胆酸对大鼠急性脊髓损伤神经细胞自噬的影响及对其保护作用

发布时间：2018-02-09 16:09

本文关键词： 脊髓损伤牛磺熊去氧胆酸自噬神经保护　出处：《宁夏医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨牛磺熊去氧胆酸对改良Allen’s重物打击法构建大鼠急性脊髓损伤模型神经细胞自噬的影响。方法选取6-8周雄性SD大鼠,体重250-300g共计72只。随机分为三组:假手术组、手术组对照组(生理盐水组)、牛磺熊去氧胆酸(TUDCA)组(分高低剂量两个亚组)。分别于脊髓损伤后24小时、5天、10天处死实验动物。参照Basso Beattie Bresnahan(BBB)运动功能评分,对各组大鼠损伤后和猝死前作后肢运动功能评分,比较对照组与TUDCA组大鼠在运动功能方面的差异性;采用电子显微镜,观察神经细胞中自噬现象的发生发展及TUDCA干预后受损脊髓神经细胞的生物学特性;HE染色对比脊髓组织形态在光镜下的差异;采用Western blotting方法,检测自噬相关因子(Beclin-1)在各组的表达情况;检测凋亡相关因子caspass-3在各组的表达情况;并采用RT-PCR方法分别检测上述蛋白的mRNA表达。结果BBB运动功能评分中假手术组大鼠的活动不受限。对照组与实验组比较,对照组的BBB评分值明显低于实验组,反映到大鼠的活动上则可见活动性略差于实验组。实验组中高低药物剂量对大鼠活动的影响无明显差异,即生理盐水组的运动功能评分明显减弱。采用电子显微镜,观察神经细胞中自噬现象的发生发展及TUDCA干预后受损脊髓神经细胞的生物学特性;HE染色对比脊髓组织形态在光镜下的差异。正常脊髓形态组织,脊髓神经元及胶质细胞完整,未见明显坏死细胞。对照组损伤处的脊髓组织,可以见到细胞呈空泡状变性,其间大片的出血、水肿,细胞组织结构疏松明显,灰质组织被破坏,伴有大量神经元细胞坏死,而存活的神经元细胞中,部分可见核固缩损伤处的脊髓组织。TUDCA处理的实验组组,与对照组相比标本上同样以出血、水肿和坏死为主要表现,但对照组最大的特点表现在,损伤中心附近的节段,同样出现损伤,可以看到损伤范围扩大,与其相邻的神经细胞破坏明显,部分细胞形态有了较明显的改变,实验组中,虽然细胞损伤的范围较之前同样有所扩大,但是和对照组相比,波及的范围明显较小,并且相邻神经细胞的破坏程度较轻。采用Western blotting方法,检测自噬相关因子(Beclin-1)在各组的表达情况。实验组Beclin-1的表达均高于对照组,并且有统计学差异,同样TUDCA的给药剂量的高低之间比较无明显统计学差异。采用Western blotting方法,检测凋亡相关因子(caspass-3)在各组的表达情况。SCI早期对照组大鼠各时间点caspase-3的表达均较假手术组明显增高,而实验组在给予TUDCA干预后caspase-3的表达均低于对照组,并且有统计学差异。而实验组中TUDCA给药高低剂量的比较中无明显统计学差异。结论TUDCA的干预可显著激活大鼠急性脊髓损伤神经细胞自噬的表达,抑制神经细胞的凋亡对神经元细胞有保护作用。
[Abstract]:Objective to investigate the effect of taurine ursodeoxycholic acid on neuronal autophagy in acute spinal cord injury model of rats established by modified Allen's heavy body attack. Methods 72 male Sprague-Dawley rats (6-8 weeks old) weighing 250-300g were randomly divided into three groups: sham operation group. The control group (normal saline group, TUDCA group) was divided into two subgroups. The experimental animals were killed 24 hours after spinal cord injury and 10 days after spinal cord injury. The motor function score of Basso Beattie Bresnahan was compared. The motor function of hind limbs was scored after injury and before sudden death in each group, and the difference of motor function between the control group and the TUDCA group was compared. To observe the occurrence and development of autophagy in nerve cells and the biological characteristics of injured spinal cord neurons after TUDCA intervention and to compare the difference of morphology of spinal cord with HE staining under light microscope, Western blotting method was used. The expression of autophagy related factor (Beclin-1) and apoptosis-related factor (caspass-3) in each group were detected. RT-PCR method was used to detect the mRNA expression of the above proteins. Results in the BBB motor function score, the activity of rats in the sham operation group was not restricted. Compared with the experimental group, the BBB score of the control group was significantly lower than that of the experimental group. In the experimental group, there was no significant difference in the effect of high and low dose of drugs on the activity of rats, that is, the score of motor function in the saline group was obviously weakened. To observe the occurrence and development of autophagy in neurons and the biological characteristics of injured spinal cord neurons after TUDCA intervention and to compare the difference of morphology of spinal cord with HE staining under light microscope, the morphology of normal spinal cord, spinal cord neurons and glial cells were intact. There were no obvious necrotic cells. In the control group, the injured spinal cord tissue showed vacuolar degeneration, during which large areas of hemorrhage, edema, loose tissue structure, destruction of gray matter and necrosis of a large number of neuronal cells were observed. But in the surviving neuron cells, some of the spinal cord tissue. TUDCA treatment group showed hemorrhage, edema and necrosis as compared with the control group, but the characteristics of the control group were as follows:. In the region near the center of the injury, the damage also occurred, and the range of injury was enlarged, the damage of the adjacent nerve cells was obvious, and the morphology of some of the cells was obviously changed, in the experimental group, Although the extent of cell damage was also enlarged, the range of damage was significantly smaller than that of the control group, and the extent of damage to adjacent nerve cells was lighter. Western blotting method was used. The expression of Beclin-1 in the experimental group was higher than that in the control group, and there was statistical difference. There was no significant difference between the dosage of TUDCA and the dosage of the same TUDCA. Western blotting method was used to detect the expression of the autophagy related factor Beclin-1. The expression of caspase-3 in early sci group was significantly higher than that in sham operation group, and the expression of caspase-3 in experimental group was lower than that in control group after TUDCA intervention. There was no significant difference between the high and low doses of TUDCA in the experimental group. Conclusion the intervention of TUDCA can significantly activate the expression of autophagy in rats with acute spinal cord injury. Inhibition of neuronal apoptosis has a protective effect on neuronal cells.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【参考文献】