人老化髓核细胞的炎性因子表达变化分析

发布时间：2018-03-12 06:16

  本文选题：老化髓核细胞　切入点：细胞表型　出处：《东南大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：研究体外培养人类正常和退变腰椎间盘髓核细胞的基因表达谱,分析人老化髓核细胞的炎性因子表达变化。方法：1、体外传代培养人正常髓核细胞系(Scien Cell4800)。连续性1：3传代、培养(理论上可传至13代),诱导髓核细胞复制性老化。2、取P8代髓核细胞按1×106个/孔接种于六孔板,按双氧水浓度100μmol/L、作用时间2h/天、连续四天处理,氧化应激诱导髓核细胞过早老化。3、分别对步骤1中每一代细胞及步骤2中的细胞老化前及应激诱导老化后行显微镜下观察髓核细胞的形态、SA-β-Gal染色并计数细胞老化率,制备两组髓核细胞老化模型。(注：因目前尚无公认的髓核细胞老化模型标准,本研究中拟定老化率≥80%、G1期细胞≥80%造模组为合格的髓核细胞老化模型。)4、取P9代髓核细胞(老化阴性对照组)、复制性老化模型、应激诱导的P9代过早老化模型3组细胞,采用实时定量RT-PCR检测3组细胞TNF-a及IL-1β、 IL-6、IL-8等的相对表达含量。结果：1、在诱导髓核细胞复制性老化的过程中,随着细胞传代次数的增加,髓核细胞形态逐渐由类圆形、星形、短梭形向长梭形、不规则形等形态转变。细胞体积逐渐增大、胞质变脆、胞浆空泡化明显。SA-β-Gal染色计数髓核细胞老化率、流式细胞仪检测G1期细胞比例均逐渐增加,传至P13代时三者比例分别为81.1%、84.5%,差异与正常P9代髓核细胞相比有统计学意义(P0.05)。2、在氧化应激诱导髓核细胞过早老化的过程中,随着双氧水作用天数的延长,细胞形态逐渐由类圆形向长梭形转变,细胞体积增加、胞浆空泡化明显。SA-β-Gal染色计数髓核细胞的老化率、流式细胞仪检测G1期细胞比例均逐渐增大,且100μmol/L双氧水作用P9代髓核细胞2h/天、连续四天后三者比例分别为80.3%、80.6%,差异与正常P9代髓核细胞相比有统计学意义(P0.05)。3、RT-PCR实验结果显示,在复制性老化模型中,TNF-α、IL-1β、IL-6、IL-8的表达量较对照组均有所增加,差异较对照组有统计学意义(P0.05)。在应激诱导的过早老化模型中,TNF-α、IL-1β、IL-6、IL-8的表达量较对照组均有所增加,差异较对照组有统计学意义(P0.05)。复制老化模型组增幅与应激老化模型组中TNF-α、IL-1β、IL-6、IL-8的表达量相近,差异无统计学意义(P0.05)。结论：1、通过连续性传代培养人正常髓核细胞系(Scien Cell4800),成功建立髓核细胞复制性老化模型(P13代髓核细胞)；2、通过100μmol/L双氧水作用于P9代髓核细胞2h/天,连续作用四天,成功建立应激诱导的髓核细胞过早老化模型；3、在髓核细胞复制性老化与双氧水诱导应激老化模型中,TNF-α、IL-1β、IL-6、 IL-8表达量均较正常P9代对照组明显增高；4、TNF-α、IL-1β、IL-6、IL-8在应激诱导的髓核细胞过早老化模型中表达量与其在复制老化模型组中表达量无明显差异。
[Abstract]:Objective: to study the gene expression profiles of human normal and degenerated lumbar disc nucleus pulposus cells cultured in vitro, and to analyze the expression of inflammatory factors in human aging nucleus pulposus cells. Methods: the normal human nucleus pulposus cell line Scien Cell4800 was subcultured in vitro. Culture (in theory can be transmitted to 13 passages, induce nucleus pulposus cells to replicate aging. 2. P8 passage of nucleus pulposus cells were inoculated at 1 脳 106 cells per well on the six-hole plate, at the concentration of 100 渭 mol / L of hydrogen peroxide for 2 h / day for 4 days, and the cells were treated for 4 days in a continuous period of 4 days, 100 渭 mol 路L ~ (-1) of H _ 2O _ 2). Oxidative stress induced premature aging of nucleus pulposus cells. The morphology of nucleus pulposus cells was observed under microscope before and after aging of each generation of cells in step 1 and step 2, and the rate of cell aging was counted. Preparation of two groups of nucleus pulposus aging model. (note: there is no accepted model of nucleus pulposus aging model standards, In this study, the aging rate 鈮，

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