芹菜素在静水压下对椎间盘髓核蛋白多糖合成及相关基因表达的影响

【作者】 乔卫平；

【导师】 徐林；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 研究背景腰椎间盘退行性疾病是骨科的常见病和多发病,是导致腰腿痛、神经功能障碍的最根本原因。随着我国老龄化社会的到来,由椎间盘退变造成的腰椎疾病日益构成对人民健康的威胁并严重影响患者的生活质量。随着机械化及办公电脑的普及,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,是困扰现代人的主要问题之一。在我国,腰腿痛病人占外科门诊的30%左右。如何征服这一顽疾已成为国内乃至国际医学界的一项重大课题。腰椎间盘退变及突出症的保守治疗方法很多,其中药物治疗方面,无外乎使用非甾体抗炎药(NSAIDs)及活血镇痛中成药等用以缓解疼痛症状,随着椎间盘退变的进一步加重,症状严重的许多患者将面临骨科的脊柱外科手术,给患者在心理上和经济上带来严重的负担。因此,开发研制延缓腰椎间盘退变过程及促进再生的药物是新时代所需。研究目的1.以中药提取物芹菜素通过体外培养在静水压下对人腰椎间盘髓核蛋白多糖(Proteoglycan, PG)合成及核心蛋白、连接蛋白基因表达进行干预,对获得的数据进行分析,如果在退变的椎间盘髓核组织蛋白多糖的合成及核心蛋白、连接蛋白的基因表达中芹菜素的作用得到明了,提示可用芹菜素延缓椎间盘的退变过程,为临床上预防和延缓腰椎间盘退行性变开辟一条新的途径。2.深入了解蛋白多糖在腰椎间盘退变过程中的代谢(本课题主要研究合成)及其与各影响因素(本课题主要是静水压)之间的关系,以及蛋白多糖核心蛋白、连接蛋白的基因表达,在分子水平上揭示椎间盘退变的部分分子机制,对于腰椎间盘退行性疾病的预防、诊断和治疗有重要的意义。研究方法1.芹菜素对人腰椎间盘髓核蛋白多糖合成剂量依赖性研究12个椎间盘髓核样品,均系北京中医药大学东直门医院骨科因腰椎间盘突出症行髓核摘除术的志愿患者,年龄50-60岁,平均54.3岁。培养基中添加高中低不同剂量芹菜素(3.125-125μmol/L),在1atm下研究芹菜素对蛋白多糖合成影响的最佳剂量。2.芹菜素在静水压下对人腰椎间盘髓核蛋白多糖合成及相关基因表达的影响32个椎间盘髓核组织样品,年龄40-60岁,平均年龄51.6岁。其中40-50岁男、女性各8例,51-60岁男、女性各8例。每一例椎间盘组织分成9组(空白1atm组,空白3atm组,空白30atm组,L-NMMA3atm组,L-NMMA30atm组,芹菜素3atm组,芹菜素30atm组,SNAP3atm组,SNAP30atm组)。在静水压装置中培养2小时后提取上清液,测定上清液中蛋白多糖的含量,进行统计学分析。并对每组中的椎间盘髓核组织进行石蜡包埋、切片,进行HE、Masson染色、免疫组化和原位杂交,通过组织形态学分析来研究芹菜素对椎间盘髓核蛋白多糖的核心蛋白及连接蛋白的基因转录的调节作用。研究结果1.芹菜素可以抑制椎间盘髓核组织的一氧化氮释放量,促进蛋白多糖的合成,并且有剂量依赖性,其中培养基中添加62.5μmol/L芹菜素蛋白多糖合成较其他组高。2.9组中,芹菜素3atm组和L-NMMA3atm组蛋白多糖合成比率最高,两组间相比无显著性差异(P>0.05),与空白组相比有显著性差异(P<0.05),SNAP30atm组蛋白多糖合成比率最低,与各组间相比均有极显著性差异(P<0.01)。空白组、L-NMMA组、SNAP组和芹菜素组中3atm下蛋白多糖合成比率较30atm下蛋白多糖合成比率高,与之相比有显著性差异(P<0.05)；芹菜素3atm组和L-NMMA3atm组蛋白多糖核心蛋白免疫组化和原位杂交结果显示阳性髓核细胞数最多,平均光密度值(MOD)最大,蛋白多糖连接蛋白免疫组化和原位杂交结果显示阳性纤维连接蛋白面积百分比值最大。结论1.3atm的静水压可以降低椎间盘髓核组织外源性的一氧化氮(NO)产生量的比率,增加蛋白多糖(PG)的合成率,而30atm的静水压可以增加椎间盘髓核组织外源性的一氧化氮(NO)的产生量,降低蛋白多糖(PG)的合成率。2.培养基中添加芹菜素在3atm静水压条件下,NO产生量明显减少,PG合成率明显增加。另外,添加芹菜素使30atm的静水压下NO的产生量有所减少,也部分降低了30atm的静水压下对PG合成率的抑制,说明芹菜素确实能够增加蛋白多糖的合成率。3.本研究从基因水平上揭示了椎间盘退变的部分分子机制,数据及图像分析显示对于退变的椎间盘髓核组织,芹菜素有促进蛋白多糖核心蛋白及连接蛋白mRNA基因表达的作用,因此我们猜测芹菜素可能通过增加髓核细胞核的基因转录水平来提高细胞外间质蛋白质的合成,并能重组蛋白多糖核心蛋白和连接蛋白的生化成分。芹菜素对椎间盘髓核细胞的作用可能是通过稳定软骨细胞的基因表型来实现的,因此有必要对退变椎间盘细胞的基因转录片段进行进一步的研究,彻底明确芹菜素的作用机理。更多还原

【Abstract】 ObjectivesTo Study Apigenin with Hydrostatic Pressure Mediates the Change of Proteoglycan Synthesis and core protein and link protein related gene expression in the Human Lumbar Intervertebral. Disc Nucleus Pulposus, and analyze the data obtained, if Apigenin can improve the synthesis of proteoglycan and increase core protein with link protein gene expression,suggesting that apigenin can slow down the process of disc degeneration, which open up a new way to prevent and delay the clinical lumbar intervertebral disc degeneration.In-depth understanding the relationship between proteoglycans synthesis metabolism in lumbar intervertebral disc degeneration and the various Hydrostatic Pressure,clearing the proteoglycan core protein,link protein gene expression,and revealing lumbar intervertebral disc degeneration’s molecular mechanisms, which have an important significance to lumbar intervertebral disc degenerative disease’s prevention,diagnosis and treatment.Methods1.Apigenin’s dose-dependent study on the intervertebral disc nucleus pulposus proteoglycan synthesis.12 samples of intervertebral disc nucleus pulposus,which are obtained from the volunteer patients due to lumbar disc herniation discectomy operation in the Department of Beijing University of Chinese Medicine Dongzhimen Hospital orthopedics,aged 50-60 years,mean 54.3 years of age. Add a medium low or high doses of apigenin (3.125-125μmol/L),to study the best dose apigenin with latm mediate the proteoglycan synthesis.2.Apigenin with Hydrostatic Pressure Mediates the Change of Proteoglycan Synthesisand core protein and link protein related gene expression in the Human Lumbar Interverte-bral Disc 32 samples of intervertebral disc nucleus pulposus,aged 40-60 years old with a mean age of 51.6 years.In which 40-50 years old male and female of all 8 cases,51-60-years old male and female of all 8 cases.Each is divided into nine cases of nucleus pulposus group(blank latm group,blank 3atm group,blank 30atm group,L-NMMA3atm group,L-NMMA 30atm group, apigenin 3atm group,apigenin 30atm group,SNAP3atm group,SNAP30atm group).cultured for 2 hours in the hydrostatic pressure devices,then measured the proteoglycan content in the supernatant and statistically analyzed the resul-ts.Eachgroup in the nucleus pulposus embedded in paraffin,sliced,forHE,Masson staining,immunohistochemistry and in situ hybridization,through the organizational and morphological analysis to study the apigenin on the intervertebral disc nucleus pulposus proteoglycan core protein and link protein gene transcription in vitro.Results1.Apigenin can inhibit nitric oxide release and promote synthesis of proteoglycan of the intervertebral disc nucleus pulposus,and there are dose-dependent manner,proteoglycan synthesis higher than other groups when adding 62.5μmol/L apigenin in the medium.2.To 9 group,apigenin 3atm group and L-NMMA 3atm group have the highest rate of proteoglycan synthesis,there was no significant difference between the two groups (P>0.05), there was significant difference compared with other groups(P<0.05),SNAP 30atm group have the lowest rate of proteoglycan synthesis,there were significantly different compared with the various groups (P＜0.01). In Control group, L-NMA group, SNAP group and apigenin group under the 3atm proteoglycan synthesis rates are higher than under the 30atm,compared with a significant difference (P<0.05); apigenin 3atm group and the L-NMMA 3 atm group proteoglycan core protein by immunohistochemistry and in situ hybridization results showed the largest number of positive nucleuspulposus cells,with a largest average optical density (MOD).and proteoglycan link protein’s imm-unohistochemistry results showed the largest link protein’s area percentage.Conclusions1.3atm’s hydrostatic pressure can reduce exogenous nitric oxide(NO) the ratio of output and increase proteoglycan (PG) synthesis ratio of the intervertebral disc nucleus pulpo-sus,30atm’s hydrostatic pressure can can increase exogenous nitric oxide(NO) the ratio of output and reduce proteoglycan(PG)synthesis ratio of the intervertebral disc nucleus pulposus.2.Apigenin can increase proteoglycan synthesis ratio of the intervertebral disc nucleus pulposus under 3atm’s hydrostatic pressure,and reduce the 30atm’s hydrostatic pressure’s inhibition on proteoglycan synthesis ratio.3.This study reveals the part of molecular mechanisms of disc degeneration from the genetic level.Data and imagation analysis showed that for the degeneration intervertebral disc nucleus pulposus,Apigenin plays an important role in the promotion of proteogl-ycan core proteins and link protein mRNA gene expression,so we guess Apigenin may increase the extracellular matrix protein synthesis by increasing gene transcription levels of the nucleus, and can restruct proteoglycan core protein and link protein in the biochemical composition.Apigenin may take an action by stabilizing chondrocyte phenotype,it is necessary to study gene transcription fragment of the degeneration intervertebral disc nucleus pulposus cells,and thoroughly clear the mechanism of apigenin.更多还原