老化髓核细胞的致退变效应及其机制的探索

发布时间：2018-07-09 15:09

本文选题：椎间盘退变 + 髓核细胞　；参考：《东南大学》2015年硕士论文

【摘要】：目的：体外建立人髓核细胞复制性老化和血清剥夺诱导老化的模型,探索两种老化髓核细胞对正常髓核细胞的致退变效应并探索其机制。方法：1、体外连续传代、培养人正常髓核细胞,模拟髓核细胞复制性老化过程；2、体外无血清培养基培养第9代髓核细胞,诱导髓核细胞老化；3、分别对步骤1中的各代细胞及步骤2中的细胞行显微镜观察髓核细胞形态、SA-β-Gal染色计数细胞老化率、流式细胞仪检测G1期细胞比例,制备两组髓核细胞老化模型(本研究中拟定老化率≥80%、G1期细胞≥80%的造模组为合格的髓核细胞老化模型)；4、免疫荧光法观察P9代髓核细胞中TGF-β1的表达,同时对第9代髓核细胞、复制性老化模型、血清剥夺诱导老化模型3组细胞,采用QRT-PCR检测3组细胞TGF-β1的表达含量；5、建立老化髓核细胞和正常髓核细胞共培养体系,观察共培养体系中正常髓核细胞的活力指标,Western-blot检测共培养体系中正常髓核细胞中ERK. Akt两条信号传导通路的表达情况。结果：1、在建立髓核细胞复制性老化模型过程中,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,传至P13代时两者比例分别为84.13±4.7%、87.59±3.0%；2、在无血清培养基培养髓核细胞的模型中,第7d观察髓核细胞形态,细胞形态呈不规则形、长梭形转变,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期细胞比例较含10%胎牛血清组明显增高,其中SA-β-Gal染色计数髓核细胞老化率为86.70±6.0%,流式细胞仪检测G1期细胞比例为88.34±4.4%；3、免疫荧光法检测提示P9代髓核细胞表达TGF-β1, QRT-PCR实验结果显示,在复制性老化模型和血清剥夺诱导老化模型中,TGF-β1的表达量较正常髓核细胞均有所减少,差异较对照组有统计学意义(P0.05),两个老化模型组中TGF-β1的表达量相近,差异无统计学意义(P0.05)；4、在老化髓核细胞与正常髓核细胞共培养体系中,第7d观察共培养体系中正常髓核细胞的活力指标,发现实验组A(复制老化髓核细胞(P13代)与正常髓核细胞共培养)、实验组B(血清剥夺诱导老化的髓核细胞与正常髓核细胞共培养)中老化率及G1期细胞比例较对照组(单纯正常髓核细胞培养)中均有所上升,差异无统计学意义(P0.05)；Western-blot检测提示对照组、实验组A、实验组B中正常髓核细胞中ERK、Akt表达无明显差异,实验组A、实验组B中正常髓核细胞的pERK.pAkt较对照组表达减少。结论：1、通过体外连续性传代培养人正常髓核细胞(至P13代髓核细胞),其老化率及G1期细胞比例逐渐增加,成功建立髓核细胞复制性老化模型；2、通过体外无血清培养基培养人正常髓核细胞,其老化率及G1期细胞比例较含10%胎牛血清组明显增高,成功建立血清剥夺诱导髓核细胞老化模型；3、人髓核细胞中可表达TGF-β1,两个老化模型组髓核细胞中TGF-β1的表达较正常髓核细胞减少；4、老化髓核细胞与正常髓核细胞共培养可加速正常髓核细胞老化,与老化髓核细胞共培养组中的正常髓核细胞中ERK、Akt两条信号传导通路的活化相对减少。
[Abstract]:Objective : To establish a model of human nucleus pulposus cell replicative aging and serum deprivation induced aging in vitro , explore the effect of two aging nucleus pulposus cells on normal nucleus pulposus cells and explore its mechanism .
2 . No serum - free medium was cultured in vitro for the ninth generation of nucleus pulposus cells , which induced the aging of nucleus pulposus cells .
3 , observing the morphology of the nucleus pulposus cells , the SA - beta - Gal staining counting cell aging rate and the flow cytometry to detect the cell aging rate of the nucleus pulposus cells in each of the cells in the step 1 and the cell line microscope in the step 2 , and preparing two groups of nucleus pulposus cell aging models ( the aging rate is not more than or equal to 80 percent in the study , and the G1 phase cells are more than or equal to 80 percent of the cell modules are qualified nucleus pulposus cell aging models ) ;
4 . The expression of TGF - 尾1 in the nucleus pulposus cells was observed by immunofluorescence . At the same time , the expression of TGF - 尾1 was detected by QRT - PCR in the 3rd generation nucleus pulposus cells , the replicative aging model , the serum deprivation induced aging model 3 cells .
5 . Establish the co - culture system of aged nucleus pulposus cells and normal nucleus pulposus cells , observe the activity index of normal nucleus pulposus cells in the co - culture system , and detect ERK in normal nucleus pulposus cells in the co - culture system by Western - blot . Results : 1 . In the process of establishing the replicative aging model of nucleus pulposus cells , the morphology of nucleus pulposus cells gradually increased with the increase of the number of cells passaged , the shape of nucleus pulposus cells gradually changed from round shape , star shape , short spindle shape to long shuttle shape , irregular shape and so on .
2 . In the model of culturing the nucleus pulposus cells without serum culture medium , the morphology of the nucleus pulposus cells was observed on the 7th day , the morphology of the cells was irregular , the cytoplasmic vacuolation was obvious . The aging rate of the nucleus pulposus cells was significantly increased by the SA - 尾 - Gal staining . The aging rate of the SA - 尾 - Gal staining was 86.70 卤 6.0 % , and the percentage of the flow cytometry was 88.34 卤 4.4 % .
3 . The expression levels of TGF - 尾1 and QRT - PCR showed that TGF - 尾1 was decreased in the model of replication and the model of serum deprivation induced aging ( P0.05 ) . The expression of TGF - 尾1 was similar between the two aging models ( P0.05 ) .
4 . In the co - culture system of aged nucleus pulposus cells and normal nucleus pulposus cells , the activity index of normal nucleus pulposus cells in the co - culture system was observed on the 7th day . In the experimental group , the aging rate and the percentage of G1 phase in the experimental group B ( the cultured nucleus pulposus cells were co - cultured with normal nucleus pulposus cells ) were higher than those in the control group ( normal nucleus pulposus cells ) , and the difference was not statistically significant ( P0.05 ) .
Western - blot analysis showed that there was no significant difference in the expression of ERK in normal nucleus pulposus cells in experimental group A and experimental group B . Conclusion : 1 . The aging rate and the percentage of G1 phase of normal nucleus pulposus cells in experimental group A and experimental group B were gradually increased , and the replication - aging model of nucleus pulposus cells was established successfully .
2 . Normal nucleus pulposus cells were cultured in vitro without serum culture medium . The aging rate and the percentage of G1 phase cells were significantly higher than those in 10 % fetal bovine serum group , and the aging model of serum deprivation induced nucleus pulposus cells was established successfully .
3 . TGF - 尾1 can be expressed in human nucleus pulposus cells , and the expression of TGF - 尾1 in the nucleus pulposus cells of two aging models was lower than that of normal nucleus pulposus cells .
4 . The co - culture of aged nucleus pulposus cells with normal nucleus pulposus cells can accelerate the aging of normal nucleus pulposus cells , and the activation of two signal transduction pathways in normal nucleus pulposus cells in the co - culture group with aged nucleus pulposus cells is relatively reduced .
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5

【相似文献】