HtrA1促进髓核细胞基质金属蛋白酶的表达及其机制

发布时间：2018-04-05 06:33

  本文选题：椎间盘退变(IDD)　切入点：高温必需因子A1(Htr　出处：《江苏大学》2017年硕士论文

【摘要】：目的:椎间盘退变(intervertebral disc degeneration,IDD)是导致颈肩腰腿痛的一个重要原因。IDD的发病原因及其机制十分复杂,其影响因素之间又相互联系,目前尚未完全阐明。高温必需因子A1(High temperature requirement A1,Htr A1)属于丝氨酸蛋白酶(Htr A)家族,已经有越来越多的证据,暗示Htr A1作为一个分泌型蛋白酶在骨、关节、和肌肉等疾病中发挥作用。基质金属蛋白酶(MMPs)是细胞外基质降解过程中所不可或缺的蛋白水解酶之一,其的生物学作用是参与ECM的代谢,目前已经发现的MMPs有28多种,而在IVD过程中发挥作用的研究较多的主要有MMP1、MMP3、MMP7、MMP9、MMP13。而对于在IDD中Htr A1以及MMPs的内在机制尚不清楚,故本研究旨在检测椎间盘髓核组织中Htr A1及MMPs的表达,分析Htr A1与MMPs在髓核中的表达有无相关性。并进一步地于人椎间盘髓核细胞探讨Htr A1MMPs的可能存在的内在机制。方法:(1)临床病例分析:男14例,女18例,年龄58~73岁,平均年龄67.7岁;对照组18例,均为脊柱外伤患者,男12例、女6例,年龄45~62岁,平均年龄56.3岁。分离髓核组织,RT-q PCR方法检测髓核组织中Htr A1、MMP-1、MMP-3、MMP-7、MMP-9、MMP-13的m RNA表达水平;Western-blot法检测髓核组织中的Htr A1、MMP-1、MMP-3、MMP-7、MMP-9、MMP-13的蛋白含量;直线回归法分析Htr A1的m RNA表达水平与MMP-1、MMP-3、MMP-7、MMP-13之间的相关性。(2)r Htr A1刺激HNPCs及抑制剂的干预实验:1)r Htr A1刺激HNPCs后Htr A1及MMPs的表达:用r Htr A1刺激HNPCs,RT-q PCR方法检测HNPCs中Htr A1、MMP-1、MMP-3、MMMP-13的m RNA表达水平;Western-blot法检测HNPCs中的Htr A1、MMP-1、MMP-3、MMP-13的蛋白含量,ELISA检测HNPCs上清中MMP-1、MMP-3、MMP-13的含量。2)抑制剂的干预:用r Htr A1刺激HNPCs并以ERK1/2和ROCK的抑制剂进行干预,RT-q PCR方法检测HNPCs中Htr A1、MMP-1、MMP-3、MMP-13的m RNA表达水平;Western-blot法检测HNPCs中的Htr A1、MMP-1、MMP-3、MMP-13的蛋白含量,ELISA检测HNPCs上清中MMP-1、MMP-3、MMP-13的含量。结果:(1)椎间盘退变患者髓核组织中Htr A1的表达相对于对照者明显升高,且MMP-1、MMP-3、MMP-13的表达也随之升高;同时,相关性分析结果显示,Htr A1与MMP-1、MMP-3、MMP-13呈正相关。(2)r Htr A1刺激HNPCs后,MMP-1、MMP-3、MMP-13的m RNA以及蛋白的表达均上调,并且r Htr A1可引起ERK1/2和ROCK的磷酸化。(3)ERK1/2和ROCK的抑制剂干预之后,MMP-1、MMP-3、MMP-13的m RNA以及蛋白的表达均下调,同时,细胞上清中的MMP-1、MMP-3、MMP-13亦下调。
[Abstract]:Objective: intervertebral disc degeneration intervertebral disc degeneration.IDD is an important cause of neck, shoulder, waist and leg pain. The pathogenesis and mechanism of IDD are very complicated, and the influencing factors are related to each other, which has not been fully elucidated at present.A1(High temperature requirement A1 (Htr A1) belongs to the serine protease Htr A) family, and there is increasing evidence that Htr A1, as a secretory protease, plays a role in bone, joint, and muscle diseases.Matrix metalloproteinase (MMPs) is one of the indispensable proteolytic enzymes in the degradation of extracellular matrix. Its biological function is to participate in the metabolism of ECM. At present, more than 28 kinds of MMPs have been found.MMP1 / MMP3 / MMP7MMP9 / MMP13 are the main ones that play an important role in the IVD process.However, the intrinsic mechanism of Htr A1 and MMPs in IDD is not clear, so the purpose of this study is to detect the expression of Htr A1 and MMPs in nucleus pulposus of intervertebral disc, and to analyze the correlation between Htr A1 and MMPs in nucleus pulposus.The possible mechanism of Htr A1MMPs was further explored in human disc nucleus pulposus cells.Methods Clinical case analysis: male 14, female 18, age 5873 years, mean age 67.7 years, control group 18 cases, male 12 cases, female 6 cases, age 4562 years, mean age 56.3 years.The level of m RNA expression of Htr A 1, MMP-1, MMP-3, MMP-7, MMP-9, MMP-13 in nucleus pulposus was detected by RT-Q PCR. Western-blot method was used to detect the protein content of Htr A 1, MMP-1, MMP-3, MMP-7, MMP-9 and MMP-13 in nucleus pulposus.鐩寸嚎鍥炲綊娉曞垎鏋怘tr A1鐨刴 RNA琛ㄨ揪姘村钩涓嶮MP-1,MMP-3,MMP-7,MMP-13涔嬮棿鐨勭浉鍏虫，

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