静水压下芹菜素对人椎间盘髓核细胞凋亡及基质代谢的影响

【作者】 陈江；

【导师】 徐林；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 研究背景由于腰椎间盘退变导致的腰椎间盘突出、腰椎管狭窄等疾病是骨科门诊的常见病。随着我国老龄化社会的到来,腰椎退行性疾患对人民健康已构成威胁并严重影响患者的生活质量。随着机械化及电脑办公的普及,以坐姿进行工作的人越来越多,由此导致的腰椎间盘突出症患者呈上升趋势,且发病趋于年轻化,因此腰椎间盘退行性病变已成为当今研究的热点问题。椎间盘退变已成为困扰骨科医生的问题之一,椎间盘一旦退变难以逆转。目前的治疗方法有保守治疗和手术治疗,手术在一定程度上缓解了临床症状,但仍未阻断椎间盘的退变进程。而且脊柱内固定手术会导致脊柱生物力学结构的改变,加速邻近节段椎间盘的退变,同时给患者在心理上和经济上带来严重的负担。因此,如何阻止或延缓腰椎间盘退变成为研究的焦点。研究目的探讨人髓核细胞在体外构建的静水压环境下,芹菜素(Apigenin, API)对其功能状态、凋亡率及基质金属蛋白酶MMP-3及其抑制因子TIMP-1的影响。研究芹菜素作为一种中药类一氧化氮合酶(NOS)抑制剂在延缓腰椎间盘退变方面的可能性并揭示其部分作用机理,为中医药防治椎间盘退行性疾病寻求新的思路。研究方法1.选择由美国ScienCell Research Laboratories提供的人椎间盘髓核细胞Human Nucleus Pulposus Cells (HNPC)进行实验室培养扩增、传代,并对各代次(P1-P7)髓核细胞进行形态学观察,生长增殖活性检测以及主要细胞外基质表达(蛋白多糖、Ⅰ型胶原、Ⅱ型胶原)进行分析,选择合适代次的髓核细胞进行药物及静水压干预研究。2.研究芹菜素对体外单层培养的人髓核细胞NO产生量的影响及药物的剂量依赖性,使用经典一氧化氮供体药物(Sodium Nitroprusside,Dihydrate, SNAP)及一氧化氮合酶抑制剂(NG-Monomethyl-L-arginine, Monoacetate Salt, L-NMMA)与芹菜素进行对照分析研究,探讨芹菜素作为一种类一氧化氮合酶(NOS)抑制剂的可行性。3.对原英国牛津大学生理学研究所和日本宇宙开发医学研究中心共同研制的静水压装置进行合理改进,构建一种加压方便,操作安全的体外单层细胞静水压加载系统。分析不同静水压环境下髓核细胞在形态、存活率以及功能状态等方面的差异,论证该细胞静水压加载系统的可行性,确定下一步实验所采用的静水压力值及加载时间。4.使用已构建的单层细胞静水压加载系统及具有NOS抑制作用药物芹菜素对人髓核细胞进行综合干预。观察不同静水压环境下芹菜素对人髓核细胞的影响,对髓核细胞凋亡率、MMP-3及TIMP-1产生量以及Ⅱ型胶原表达情况进行检测,对实验结果进行统计学处理,分析芹菜素在不同静水压环境下对髓核细胞凋亡率、MMP-3与TIMP.1的平衡表达及细胞外基质的影响。探讨中药芹菜素用于延缓腰椎间盘退变的可行性并揭示部分作用机理。研究结果1.人正常椎间盘髓核细胞可以在体外单层培养条件下获得良好的生长状态。前4代细胞形态良好,增殖能力强,并且表型表达稳定,属于正常状态的人髓核细胞。随着传代次数的增多会出现“去分化”现象：传5代时不仅蛋白多糖和Ⅱ型胶原表达减少,同时细胞增殖能力降低,而且出现Ⅰ型胶原的表达,说明细胞出现老化现象,不宜作为椎间盘髓核细胞研究的对象。2.芹菜素和L-NMMA在抑制髓核细胞NO产生方面具有类似的作用,当芹菜素浓度为12.5μmol/L时能明显降低髓核细胞NO产生量,L-NMMA药物浓度为200μmol/L时也能明显降低髓核细胞NO产生量,两者在抑制作用方面没有明显差异,但芹菜素药用浓度明显低于L-NMMA。SNAP药物浓度在500μmol/L时能明显增加髓核细胞NO产生量。3.构建的细胞静水压加载系统能提供较宽范围的应力水平,符合多种实验研究的需要,且具有良好的生物相容性。单层培养的髓核细胞在0.3Mpa静水压下,持续受压120min时,髓核细胞的形态较好,存活率相对较高,蛋白多糖等胞外基质表达旺盛,而在3Mpa静水压下持续受压120min时,髓核细胞的存活率下降,胞外基质表达明显较少,处于功能抑制状态。4.芹菜素作为一种NOS抑制剂能够对髓核细胞的功能状态、凋亡率以及基质金属蛋白酶的代谢产生影响,而且这种影响在不同的静水压环境下有所不同：在0.3Mpa静水压下,芹菜素通过调节基质金属蛋白酶MMP-3与TIMP-1之间的平衡能减少髓核细胞外基质PG及Ⅱ型胶原的分解,改善由于高浓度NO引起的髓核细胞功能抑制状态；而在3Mpa静水压下,芹菜素在降低髓核细胞的凋亡率,减少胞外基质的过度分解方面有一定作用。结论芹菜素是一种安全有效的中药类NOS合酶抑制剂,能够减少髓核细胞的NO产生量。芹菜素通过调节基质金属蛋白酶MMP-3与TIMP-1之间的平衡减少髓核细胞外基质PG及Ⅱ型胶原的分解,改善由于NO刺激引起的髓核细胞功能抑制状态,并能降低高静水压下髓核细胞的凋亡率,从而对椎间盘髓核细胞及胞外基质起到一定的保护作用。更多还原

【Abstract】 BackgroundAs a result of lumbar disc degeneration, lumbar disc herniation and spinal stenosis become common diseases in orthopedics department. With the advent of an aging society in China, lumbar degenerative diseases pose a threat to the health of the people and has serious impact on the life quality of patients. With the popularity of mechanical and computer office, more and more people work in a sitting position, which led to the lumbar disc herniation, and the disease tends to younger. So lumbar intervertebral disc degeneration has become the hot research issue.Disc degeneration has become one of the problems plagued orthopedic surgeon, and its unlikely to reverse in case of degeneration. Current treatment methods are conservative treatment and surgical treatment. To some extent, operation can alleviate the clinical symptoms, but can’t block the process of disc degeneration. Spinal surgery with Internal fixation will lead to changes in the structure, speed up the adjacent segment disc degeneration, while giving patients a serious psychological and economic burden. Therefore, how to prevent or delay the process of disc degeneration become the focus of research.ObjectiveDiscuss the effect Apigenin on human nucleus pulposus cells in vitro hydrostatic pressure environment, including functional status, apoptosis and matrix metalloproteinase MMP-3 and its inhibitor TIMP-1.And to demonstrate the possibilities that Apigenin as a nitric oxide synthase(NOS) inhibitors in delaying the process of disc degeneration and reveal part mechanism of action. To seek some new ideas that Chinese medicine for the prevention and treatment of degenerative disc disease.Methods1. Human Nucleus Pulposus Cells (HNPC) are provided by U.S ScienCell Research Laboratories. In this study, we use Laboratory culture, proliferation and passage. The passages 1-7 were observed, proliferation activity was detected and the main extracellular matrix expression (proteoglycans, collagen-Ⅰ, collagen-Ⅱ) were analysis. Select the appropriate passage for hydrostatic pressure and drug intervention.2. Research the effect of Apigenin on the NO output and drug dose-dependent of human nucleus pulposus cells which were monolayer cultured in vitro. Classic nitric oxide donor drugs SNAP and nitric oxide synthase inhibitor L-NMMA were used to compare with Apigenin for the Comparative Analysis. Discussion the feasibility of using Apigenin as a nitric oxide synthase (NOS) inhibitor.3. Build a convenient and safe hydrostatic pressure system which was designed by Oxford University Physiology Institute and Space Development Medical Research Center in Japan. We improved the system in order to realize hydrostatic pressure on cells that were Monolayer cultured in vitro. Analysis the differences of Nucleus Pulposus Cells in morphology, survival rate and functional status under different hydrostatic pressure. From the result, we demonstrate the feasibility of the hydrostatic pressure system and identify the value and loading-time of hydrostatic pressure in the next step.4. Integrated intervention on human nucleus pulposus cells with hydrostatic pressure system and Apigenin. Were detected nucleus pulposus cells apoptosis rate, MMP-3 and TIMP-1 production volume and collagen-Ⅱexpression. The experimental results were analyzed statistically. Analysis the effect of Apigenin on nucleus pulposus cells including apoptosis, balance expression of MMP-3 and TIMP-1 and extracellular matrix in different hydrostatic pressure environment. Discuss the feasibility of using traditional Chinese medicine Apigenin for delaying the process of disc degeneration and reveal part mechanism of action.Results1. Normal human nucleus pulposus cells can be monolayer cultured for good growth state in vitro. The nucleus pulposus cells before passage 4th are in normal state:having good morphology and proliferative ability, as well as stabile phenotypic expression. With the increase of passage will appear "dedifferentiation" phenomenon:the passage 5th decreased cell proliferation, and the expression of collagen-Ⅰcan be detected which indicates cell aging, so is not suitable for seed cells;2. Apigenin and L-NMMA have similar effect in inhibiting NO production of nucleus pulposus cells. Apigenin can significantly decreased NO production of nucleus pulposus cells when concentration is 12.5μmol/L.When L-NMMA concentration is 200μmol/L can also decrease NO output which is similar to Apigenin. However, the drug concentration of Apigenin was significantly lower than L-NMMA. SNAP drug can significantly increase NO production of nucleus pulposus cells when concentration is 500μmol/L.3. The constructed hydrostatic pressure system can provide a wide range of stress levels to meet the needs of a variety of experimental studies and has good biocompatibility. When nucleus pulposus cells were under 0.3Mpa hydrostatic pressure sustained for 120min, they have better morphology, survival rate is relatively high, strong expression of extracellular matrix. When under 3Mpa hydrostatic pressure for 120min, the nucleus pulposus cells survival rate was reduced, the expression of extracellular matrix was significantly less in the function inhibition state.4. Apigenin as a NOS inhibitors can impact nucleus pulposus cells’function status, apoptosis rate and the metabolism of matrix metalloproteinase.And the effect is different in different hydrostatic pressure environment:under 0.3Mpa hydrostatic pressure, Apigenin can reduce the degradation of extracellular matrix PG and collagen-Ⅱby regulating the balance between matrix metalloproteinases MMP-3 and TIMP-1.Improve nucleus pulposus cells inhibition status due to the high concentration of NO. In 3Mpa hydrostatic pressure, Apigenin have a role in reducing the apoptosis rate of nucleus pulposus cells and reduce the excessive breakdown of extracellular matrix.ConclusionsApigenin is a safe and effective Chinese medicine like NOS synthase inhibitor, can reduce the amount of NO production by nucleus pulposus cells. Apigenin can reduce the degradation of extracellular matrix PG and collagen-Ⅱby regulating the balance between matrix metalloproteinases MMP-3 and TIMP-1 and improve nucleus pulposus cells function inhibition status due to the high concentration of NO, reducing the apoptosis rate of nucleus pulposus in high hydrostatic pressure. Overall, Apigenin play a protective role on the intervertebral disc nucleus pulposus cells and extracellular matrix.更多还原