自噬在急性期脊髓损伤大鼠膀胱平滑肌表达的实验研究

发布时间：2018-05-20 02:09

本文选题：自噬 + 急性期脊髓损伤　；参考：《山东大学》2017年硕士论文

【摘要】：研究背景脊髓损伤(Spinal cord injury,SCI)是一种可造成中枢神经系统的重度创伤并可引起多种并发症的疾病,脊髓损伤后其中常见的并发症之一即是神经源性膀胱。因为低级的排尿中枢存在于脊髓腰骶段,所以当损伤腰骶或以上时就会出现排尿障碍,表现为膀胱逼尿肌过度活动症、膀胱逼尿肌收缩无力或者逼尿肌与尿道括约肌协同失调等,严重影响患者生活质量。已有研究证实自噬在大鼠脊髓损伤模型中脊髓损伤处活跃,其参与神经损伤修复,促进自噬表达也有助于恢复小鼠后肢的运动功能,此外,在气管平滑肌、心肌及血管平滑肌中已证实自噬现象存在,其异常表达的水平,在慢性阻塞性肺部疾病、心脏疾病及动脉粥样硬化形成过程中发挥作用。自噬是一种选择性降解胞质蛋白、蛋白堆积物及细胞器的胞内代谢机制,首先形成自噬小体,然后运至溶酶体并与其融合形成自噬溶酶体,最终由自噬溶酶体降解胞质成分。目的观察自噬相关基因、蛋白在急性期脊髓损伤后大鼠膀胱平滑肌组织的表达,以探讨其在神经源性膀胱发病机制可能存在作用。方法24只雄性成年Wistar大鼠随机分为两组即模型组(12只)和对照组(12只)。模型组应用改良Allens'法构造急性期脊髓损伤模型,对照组仅予以椎板切除术。术后6h对大鼠下肢运动功能进行BBB(Basso Beattle Bresnahan)运动评分,尼氏染色观察两组大鼠脊髓横断面形态学变化,Western blotting检测膀胱平滑肌细胞微管相关蛋白 1 轻链 3(microtubule associated protein 1 light chain 3,LC3)和 P62 蛋白相对表达量,免疫荧光染色法检测LC3和P62蛋白在平滑肌细胞位置及阳性细胞率,RT-PCR检测膀胱平滑肌细胞中基因LC3、P62、Beclin1mRNA相对水平。结果1.术后6h模型组大鼠下肢运动功能BBB评分较对照组低(P0.01)2.尼氏染色观察镜下观察对照组脊髓灰质神经元细胞尼氏小体结构完整,神经元胞体呈多角形,模型组可观察到损伤处脊髓横断面神经元数量减少,胞体肿胀变为圆形,尼氏小体减少。3.Western blotting提示模型组膀胱平滑肌细胞蛋白LC3-II表达比对照组平滑肌细胞蛋白高(P0.05),模型组膀胱平滑肌细胞蛋白P62表达比对照组膀胱平滑肌细胞蛋白降低(P0.05)。4.免疫荧光染色法提示LC3蛋白位于膀胱平滑肌细胞质内,阳性细胞率比对照组高(P0.05),P62蛋白亦位于膀胱平滑肌细胞质内,阳性细胞率比对照组低(P0.05)。5.RT-PCR检测模型组LC3、P62、Beclin1 mRNA水平比对照组升高(P0.05)。结论在脊髓损伤急性期大鼠膀胱平滑肌中自噬表达活跃,可能在脊髓损伤所致神经源性膀胱发病机制中起作用。
[Abstract]:Background Spinal cord injury (sci) is a kind of disease which can cause severe injury to the central nervous system and cause many complications. One of the common complications after spinal cord injury is neurogenic bladder. Because the lower voiding center is present in the lumbosacral segment of the spinal cord, dysuria occurs when the lumbosacral area is injured or above, which is characterized by bladder detrusor hyperactivity. Bladder detrusor contraction weakness or detrusor and urethral sphincter coordination, seriously affect the quality of life of patients. It has been proved that autophagy is active in the spinal cord injury model of rats, which is involved in the repair of nerve injury, promotes autophagy expression and helps to restore the motor function of the hind limbs of mice, in addition, in the tracheal smooth muscle, Autophagy has been confirmed in myocardium and vascular smooth muscle. The abnormal expression of autophagy plays an important role in the development of chronic obstructive pulmonary disease, heart disease and atherosclerosis. Autophagy is a mechanism of selective degradation of cytoplasmic proteins, protein accumulations and organelles. Firstly, autophagy bodies are formed, then transported to lysosomes and fused to form autophagy lysosomes, and the cytoplasmic components are degraded by autophagy lysosomes. Objective to investigate the expression of autophagy related genes and proteins in the smooth muscle of bladder after acute spinal cord injury (sci) in order to explore the possible role of autophagy gene and protein in the pathogenesis of neurogenic bladder. Methods Twenty-four adult male Wistar rats were randomly divided into two groups: model group (n = 12) and control group (n = 12). The model group used improved Allens' method to construct acute spinal cord injury model, while the control group was treated with laminectomy only. The lower extremity motor function of rats was evaluated with BBB(Basso Beattle Bresnahan motor score at 6 hours after operation. Morphological changes of spinal cord in the two groups were observed by Nissl staining. The relative expression of microtubule-associated protein 1 (3(microtubule associated protein 1 light chain 3) and P62 protein in bladder smooth muscle cells was detected by Western blotting. The location and positive rate of LC3 and P62 protein in smooth muscle cells were detected by immunofluorescence staining. RT-PCR was used to detect the relative level of gene LC3P62 / Beclin1 mRNA in bladder smooth muscle cells. Result 1. The BBB score of lower extremity motor function in the model group was lower than that in the control group at 6 hours postoperatively (P < 0.01). The structure of the neurons in the control group was intact and the neuronal bodies were polygonal. In the model group, the number of neurons in the cross section of the spinal cord was decreased, and the swelling of the neurons became round. The expression of bladder smooth muscle cell protein (LC3-II) in the model group was higher than that in the control group (P 0.05), and the expression of P62 in the model group was lower than that in the control group (P 0.05 路4). Immunofluorescence staining showed that the LC3 protein was located in the cytoplasm of the bladder smooth muscle, and the positive cell rate was higher than that in the control group. The positive cell rate was lower than that in the control group, and the positive cell rate was lower than that in the control group (P 0.05). 5. The level of LC3 protein in the model group was higher than that in the control group (P 0.05). Conclusion the active expression of autophagy in the bladder smooth muscle of rats with acute spinal cord injury may play an important role in the pathogenesis of neurogenic bladder induced by spinal cord injury.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【参考文献】