【作者】 皮斌；

【导师】 李康华；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 目的研究三七总皂苷对大鼠脊髓损伤(spinal cord injury, SCI)模型的脊髓神经功能的保护作用及对损伤段脊髓组织胞浆型磷脂酶A2(cytosolic phospholipase A2, cPLA2)和胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)表达的影响。方法SD成年大鼠55只(雌雄不计),随机分成三组：(1)假手术组(Sham operation group, n=5);(2)脊髓损伤组(Control group, n=25);(3)PNS治疗组(PNS experimental group, n=25)。脊髓损伤组和PNS治疗组大鼠按术后存活时间再细分为五个亚组,即1天组,3天组,7天组,14天组和21天组,每时间点5只大鼠。采用脊髓半横断法制作脊髓损伤模型,假手术组只行椎板切除,不行脊髓半横断操作。PNS治疗组大鼠在造模成功后15分钟开始予三七总皂苷注射液经腹腔内注射20mg/kg,每天给药一次。脊髓损伤组大鼠则在造模成功后给予等量的生理盐水经腹腔内注射,给药次数和PNS治疗组相同。所有大鼠在处死前于平坦处行BBB评分,评估脊髓损伤后大鼠脊髓运动神经功能,统计学分析两组间是否有差异。脊髓损伤组及PNS治疗组大鼠在造模后的相应时间点分别处死行主动脉插管灌注固定并取脊髓组织标本,每只大鼠取以损伤段为中心的长约1cm的脊髓组织。在每个脊髓标本纵轴中心取横行切片,每个检测指标2张,分别作cPLA2和GFAP的免疫组化染色与尼氏染色,观察各自的表达情况并计数。SPSS13.0统计分析软件对比两组动物的脊髓标本不同时间点cPLA2、GFAP的表达是否有统计学差异,并分析其相关性。结果脊髓损伤后两组大鼠其BBB评分值逐渐升高,PNS治疗组均较脊髓损伤组评分值高,除术后第一天BBB评分与脊髓损伤组相比无明显统计学意义,其余各时间点PNS治疗组行为学评分均显著高于脊髓损伤组,第三天、第七天比较其P值<0.05,第十四天、第二十一天P值均小于0.01。尼氏染色病理学检测显示假手术组大鼠脊髓组织神经元形态完整,胞浆内尼式小体清晰可见。脊髓损伤组光镜下可见脊髓组织正常结构丧失,灰质形成较大空腔,少见完整的细胞结构,胞浆混浊,尼氏体显示不清。PNS治疗组脊髓组织结构比较清晰,损伤侧的灰质内,偶见小空腔,有较多的正常神经元,神经元细胞核稍浓聚,核膜完整,胞浆内尼氏体清晰可见,轴突清晰完整。cPLA2免疫组化结果示脊髓损伤后1d起cPLA2表达增加,免疫阳性细胞数增多,神经元肿胀,胞体增大,而PNS干预组cPLA2阳性细胞数较少,且cPLA2染色较浅,神经元肿胀程度较轻,形态更为完整,周围神经组织破坏程度较轻。在7d,14d,21d损伤组cPLA2表达均显著强于PNS干预组(P<0.01),且在各时间点内PNS组cPLA2表达均弱于损伤组。GFAP免疫组化结果显示,脊髓损伤后1d起GFAP表达增加,免疫阳性细胞数增多,神经元肿胀,胞体增大,而PNS干预组GFAP阳性细胞数较少,且染色较浅,神经元肿胀程度较轻,形态更为完整,周围神经组织破坏程度较轻。在7d,14d,21d, SCI组GFAP表达均显著强于PNS干预组(P<0.05),且在各时间点内PNS组GFAP表达均弱于SCI组。对两指标光密度值应用Pearson相关性分析,脊髓损伤后GFAP与cPLA2表达有显著相关性(r=0.90,P=0.04),说明在脊髓损伤过程中,星形胶质细胞激活与cPLA2表达有一定联系。结论三七总皂苷对脊髓继发性损伤有神经保护作用。三七总皂苷能抑制大鼠脊髓损伤模型损伤段脊髓cPLA2及GFAP表达,且其神经保护机制可能与抑制cPLA2表达,调控星形胶质细胞活化有关。更多还原

【Abstract】 OBJECTIVE This study was to investigate the expression of cytosolic phospholipase A2(cPLA2) and glial fibrillary acidic protein (GFAP) in spinal tissues of rats with spinal cord injury after panax notoginseng saponins (PNS) treatment.METHODS A total of55healthy adult Sprague Dawley rats were divided randomly into three groups without discriminating of gender (1)Sham operation group (group S, n=5);(2) spinal cord injury group (group C, n=25);(3) PNS treatment group (group T, n=25).According to the survival time, SCI group and PNS group are divided in to5groups, which include survive for1day,3days,7days,14days and21days.The spinal cord injury models were made by T10hemisection while the Sham group just receive laminectomy without injurying the spinal cord.20mg/kg of PNS was injected to the T group intraperitoneally and the C group was treated with the same volume of saline vehicle (NS)15minutes after injury. Each model was given the same dosage once a day. Animal behavioral scores—modified by BBB score on spinal cord functional deficits were performed to the rats following SCI to analyze the difference between the two groups.At the corresponding time points following the injury, the rats of two groups were sacrificed and the spinal cord tissue samples were collected from the injured segment with the length of1cm. Each sample got2continuous histological sections of10um thickness through microtomy in the middle of the sample to test cPLA2and GFAP by immunohisto-chemistry method separately and Nissle staining.The number of positive cells in every section was counted,and the average positive cells of each group at the same time point were calculated. Software SPSS13.0was applied to analyze if there was significant difference between the two related groups at each time point and correlationship between the cPLA2and GFAP.RESULTS The BBB scores of the two groups are increased after the spinal cord injury, and the PNS group gets higher scores than the SCI group. All the time points show significant difference except the1day, and the P value for3d and7day is less than0.05with the value for14d and21d less than0.01.The Nissle staining for pathology test shows sham group is integrated in morphology, and the Nissle body is clear in cytoplasm. The SCI group shows damaged structures with cavities in grey matter and the Nissle body is fuzzy. The neural structure is relatively integrated and Nissle body can be seen clearly with more normal neurons in which the nucleolus is a little dense with complete karyotheca.The expression of cPLA2is increased after the first day of spinal cord injury with increased positive cells, swollen neurons and neural bodies, while the positive cells in PNS group is less, with lighter staining, less swollen neurons and more integrated tissue. At7d,14d and21d, the expression of cPLA2in SCI group is significantly increased than the PNS group(P<0.01), and at all time points the expression of cPLA2in PNS group is decreased than the SCI group. The expression of GFAP is increased after the first day in both groups with increased positive cells, swollen neurons and enlarged neural bodies, while the positive cells is fewer in PNS group with lighter staining, less swollen neurons and more integrated structure. At7d,14d and21d, the GFAP expression in SCI group is significantly increased than the PNS group(P<0.05), and at all the time points, GFAP in PNS group is more decreased than the SCI group. The cPLA2is significantly correlated with GFAP under Pearson correlation analysis (r=0.90, P=0.04), which explains the activation of astrocyte is associated with cPLA2.CONCULUSION The PNS can inhabit the expressions of the cPLA2and GFAP of the SD rats after SCI. It has neuroprotective effects on the second injury mechanism of the SCI, and it is associated with the activation of astrocyte.更多还原