N-乙酰基-L-半胱氨酸（NAC）对椎间盘退变影响的体外实验研究

发布时间：2018-06-21 04:17

本文选题：椎间盘退变模型 + 体外培养　；参考：《南京医科大学》2015年硕士论文

【摘要】：[目的]:建立小鼠椎间盘体外培养退变模型,分析抗氧化剂N-乙酰基-L-半胱氨酸(NAC)对椎间盘退变的影响。[方法]:选取8周龄ICR小鼠作为实验对象,无菌条件下取出小鼠腰段椎间盘,随机分为三组:对照组、退变模型组及NAC处理组,在退变模型组中的培养基加入白细胞介素(IL)1β和肿瘤坏死因子(TNF)α,NAC处理组的培养基中除了加入上述炎症因子外,再加入配制好的NAC溶液。在培养液中经过不同时段培养后,分别于3天、7天、10天取出培养液中的椎间盘。通过组织学和免疫组织化学等方法分析椎间盘形态学特征,并提取椎间盘组织中的蛋白和RNA,分别通过Western-blot和RT-PCR方法检测椎间盘退变过程中的相关蛋白表达和基因转录水平。[结果]:与退变模型组相比,培养3、7、10天的NAC处理组椎间盘经HE-阿尔新蓝复合染色显示其结构逐渐分界清楚,蛋白多糖含量丰富;二型胶原(Col-Ⅱ)、十型胶原(Col-X)及基质金属蛋白酶-3(MMP-3)表达均降低;抗氧化蛋白SOD-1表达升高,抗氧化相关基因GSR、Gpx4表达水平升高;增殖相关蛋白Cyclin D1表达升高;促凋亡蛋白Caspase-3表达降低,促凋亡相关基因nox A、Perp表达水平降低;NAC处理组上述各项指标均接近对照组水平。[结论]:通过在培养基中加入炎症因子IL-1β和TNF-α,可以成功建立椎间盘体外培养退变模型。抗氧化剂NAC补充可以通过降低氧化应激,促进细胞增殖,减少细胞凋亡延缓椎间盘退变。
[Abstract]:[Objective] to establish an in vitro culture degenerative model of the mouse intervertebral disc and analyze the effect of antioxidant N- acetyl -L- cysteine (NAC) on the degeneration of intervertebral disc. [Methods] the 8 week old ICR mice were selected as the experimental subjects, and the lumbar intervertebral discs were taken out under aseptic conditions and divided into three groups randomly: the control group, the degeneration model group and the NAC treatment group, in the degenerative model. The medium in the group was added to interleukin (IL) 1 beta and tumor necrosis factor (TNF) alpha. The medium of NAC treatment group was added to the prepared NAC solution in addition to the above inflammatory factors. After different periods of culture in the culture medium, the intervertebral discs were removed from the culture solution at 3 days, 7 days and 10 days respectively. Chemical and other methods were used to analyze the morphological characteristics of intervertebral disc, and extract the protein and RNA in the intervertebral disc tissue. The protein expression and gene transcription level in the process of intervertebral disc degeneration were detected by Western-blot and RT-PCR. [results]: compared with the degenerative model group, the NAC treatment group of NAC treatment group with 3,7,10 days was mixed with alnew blue. The staining showed that the structure gradually became clear and the content of proteoglycan was rich; the expression of type two collagen (Col- II), type ten collagen (Col-X) and matrix metalloproteinase -3 (MMP-3) decreased, the expression of antioxidant protein SOD-1 increased, the expression level of antioxidant related gene GSR, Gpx4 increased, the D1 expression of proliferation related protein Cyclin increased, and apoptotic protein Caspase- was increased. The expression of 3 was reduced, the expression of apoptosis related genes NOx A and Perp decreased, and the above indexes in the NAC treatment group were close to the control level. [Conclusion]: by adding the inflammatory factor IL-1 beta and TNF- alpha in the medium, the model of the intervertebral disc in vitro culture degenerative model can be successfully established. The antioxidant NAC supplement can promote the cell by reducing oxidative stress. Proliferation, reducing apoptosis and retarding intervertebral disc degeneration.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.53

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