本文選題：老化髓核細(xì)胞　切入點(diǎn)：細(xì)胞表型　出處：《東南大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究體外培養(yǎng)人類正常和退變腰椎間盤髓核細(xì)胞的基因表達(dá)譜,分析人老化髓核細(xì)胞的炎性因子表達(dá)變化。方法：1、體外傳代培養(yǎng)人正常髓核細(xì)胞系(Scien Cell4800)。連續(xù)性1：3傳代、培養(yǎng)(理論上可傳至13代),誘導(dǎo)髓核細(xì)胞復(fù)制性老化。2、取P8代髓核細(xì)胞按1×106個(gè)/孔接種于六孔板,按雙氧水濃度100μmol/L、作用時(shí)間2h/天、連續(xù)四天處理,氧化應(yīng)激誘導(dǎo)髓核細(xì)胞過早老化。3、分別對步驟1中每一代細(xì)胞及步驟2中的細(xì)胞老化前及應(yīng)激誘導(dǎo)老化后行顯微鏡下觀察髓核細(xì)胞的形態(tài)、SA-β-Gal染色并計(jì)數(shù)細(xì)胞老化率,制備兩組髓核細(xì)胞老化模型。(注：因目前尚無公認(rèn)的髓核細(xì)胞老化模型標(biāo)準(zhǔn),本研究中擬定老化率≥80%、G1期細(xì)胞≥80%造模組為合格的髓核細(xì)胞老化模型。)4、取P9代髓核細(xì)胞(老化陰性對照組)、復(fù)制性老化模型、應(yīng)激誘導(dǎo)的P9代過早老化模型3組細(xì)胞,采用實(shí)時(shí)定量RT-PCR檢測3組細(xì)胞TNF-a及IL-1β、 IL-6、IL-8等的相對表達(dá)含量。結(jié)果：1、在誘導(dǎo)髓核細(xì)胞復(fù)制性老化的過程中,隨著細(xì)胞傳代次數(shù)的增加,髓核細(xì)胞形態(tài)逐漸由類圓形、星形、短梭形向長梭形、不規(guī)則形等形態(tài)轉(zhuǎn)變。細(xì)胞體積逐漸增大、胞質(zhì)變脆、胞漿空泡化明顯。SA-β-Gal染色計(jì)數(shù)髓核細(xì)胞老化率、流式細(xì)胞儀檢測G1期細(xì)胞比例均逐漸增加,傳至P13代時(shí)三者比例分別為81.1%、84.5%,差異與正常P9代髓核細(xì)胞相比有統(tǒng)計(jì)學(xué)意義(P0.05)。2、在氧化應(yīng)激誘導(dǎo)髓核細(xì)胞過早老化的過程中,隨著雙氧水作用天數(shù)的延長,細(xì)胞形態(tài)逐漸由類圓形向長梭形轉(zhuǎn)變,細(xì)胞體積增加、胞漿空泡化明顯。SA-β-Gal染色計(jì)數(shù)髓核細(xì)胞的老化率、流式細(xì)胞儀檢測G1期細(xì)胞比例均逐漸增大,且100μmol/L雙氧水作用P9代髓核細(xì)胞2h/天、連續(xù)四天后三者比例分別為80.3%、80.6%,差異與正常P9代髓核細(xì)胞相比有統(tǒng)計(jì)學(xué)意義(P0.05)。3、RT-PCR實(shí)驗(yàn)結(jié)果顯示,在復(fù)制性老化模型中,TNF-α、IL-1β、IL-6、IL-8的表達(dá)量較對照組均有所增加,差異較對照組有統(tǒng)計(jì)學(xué)意義(P0.05)。在應(yīng)激誘導(dǎo)的過早老化模型中,TNF-α、IL-1β、IL-6、IL-8的表達(dá)量較對照組均有所增加,差異較對照組有統(tǒng)計(jì)學(xué)意義(P0.05)。復(fù)制老化模型組增幅與應(yīng)激老化模型組中TNF-α、IL-1β、IL-6、IL-8的表達(dá)量相近,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論：1、通過連續(xù)性傳代培養(yǎng)人正常髓核細(xì)胞系(Scien Cell4800),成功建立髓核細(xì)胞復(fù)制性老化模型(P13代髓核細(xì)胞)；2、通過100μmol/L雙氧水作用于P9代髓核細(xì)胞2h/天,連續(xù)作用四天,成功建立應(yīng)激誘導(dǎo)的髓核細(xì)胞過早老化模型；3、在髓核細(xì)胞復(fù)制性老化與雙氧水誘導(dǎo)應(yīng)激老化模型中,TNF-α、IL-1β、IL-6、 IL-8表達(dá)量均較正常P9代對照組明顯增高；4、TNF-α、IL-1β、IL-6、IL-8在應(yīng)激誘導(dǎo)的髓核細(xì)胞過早老化模型中表達(dá)量與其在復(fù)制老化模型組中表達(dá)量無明顯差異。
[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，

本文編號(hào)：1600280