本文選題：椎間盤退變(IDD)　切入點：高溫必需因子A1(Htr　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:椎間盤退變(intervertebral disc degeneration,IDD)是導(dǎo)致頸肩腰腿痛的一個重要原因。IDD的發(fā)病原因及其機制十分復(fù)雜,其影響因素之間又相互聯(lián)系,目前尚未完全闡明。高溫必需因子A1(High temperature requirement A1,Htr A1)屬于絲氨酸蛋白酶(Htr A)家族,已經(jīng)有越來越多的證據(jù),暗示Htr A1作為一個分泌型蛋白酶在骨、關(guān)節(jié)、和肌肉等疾病中發(fā)揮作用�；|(zhì)金屬蛋白酶(MMPs)是細胞外基質(zhì)降解過程中所不可或缺的蛋白水解酶之一,其的生物學(xué)作用是參與ECM的代謝,目前已經(jīng)發(fā)現(xiàn)的MMPs有28多種,而在IVD過程中發(fā)揮作用的研究較多的主要有MMP1、MMP3、MMP7、MMP9、MMP13。而對于在IDD中Htr A1以及MMPs的內(nèi)在機制尚不清楚,故本研究旨在檢測椎間盤髓核組織中Htr A1及MMPs的表達,分析Htr A1與MMPs在髓核中的表達有無相關(guān)性。并進一步地于人椎間盤髓核細胞探討Htr A1MMPs的可能存在的內(nèi)在機制。方法:(1)臨床病例分析:男14例,女18例,年齡58~73歲,平均年齡67.7歲;對照組18例,均為脊柱外傷患者,男12例、女6例,年齡45~62歲,平均年齡56.3歲。分離髓核組織,RT-q PCR方法檢測髓核組織中Htr A1、MMP-1、MMP-3、MMP-7、MMP-9、MMP-13的m RNA表達水平;Western-blot法檢測髓核組織中的Htr A1、MMP-1、MMP-3、MMP-7、MMP-9、MMP-13的蛋白含量;直線回歸法分析Htr A1的m RNA表達水平與MMP-1、MMP-3、MMP-7、MMP-13之間的相關(guān)性。(2)r Htr A1刺激HNPCs及抑制劑的干預(yù)實驗:1)r Htr A1刺激HNPCs后Htr A1及MMPs的表達:用r Htr A1刺激HNPCs,RT-q PCR方法檢測HNPCs中Htr A1、MMP-1、MMP-3、MMMP-13的m RNA表達水平;Western-blot法檢測HNPCs中的Htr A1、MMP-1、MMP-3、MMP-13的蛋白含量,ELISA檢測HNPCs上清中MMP-1、MMP-3、MMP-13的含量。2)抑制劑的干預(yù):用r Htr A1刺激HNPCs并以ERK1/2和ROCK的抑制劑進行干預(yù),RT-q PCR方法檢測HNPCs中Htr A1、MMP-1、MMP-3、MMP-13的m RNA表達水平;Western-blot法檢測HNPCs中的Htr A1、MMP-1、MMP-3、MMP-13的蛋白含量,ELISA檢測HNPCs上清中MMP-1、MMP-3、MMP-13的含量。結(jié)果:(1)椎間盤退變患者髓核組織中Htr A1的表達相對于對照者明顯升高,且MMP-1、MMP-3、MMP-13的表達也隨之升高;同時,相關(guān)性分析結(jié)果顯示,Htr A1與MMP-1、MMP-3、MMP-13呈正相關(guān)。(2)r Htr A1刺激HNPCs后,MMP-1、MMP-3、MMP-13的m RNA以及蛋白的表達均上調(diào),并且r Htr A1可引起ERK1/2和ROCK的磷酸化。(3)ERK1/2和ROCK的抑制劑干預(yù)之后,MMP-1、MMP-3、MMP-13的m RNA以及蛋白的表達均下調(diào),同時,細胞上清中的MMP-1、MMP-3、MMP-13亦下調(diào)。
[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揪姘村鉤涓嶮MP-1,MMP-3,MMP-7,MMP-13涔嬮棿鐨勭浉鍏蟲，

本文編號：1713585