高氧微环境刺激与细胞连续分裂在体外人髓核细胞老化中作用的初探

发布时间：2018-03-13 08:10

本文选题：微环境刺激　切入点：连续分裂　出处：《东南大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：探讨高氧微环境刺激与细胞连续分裂在体外人髓核细胞老化过程中的效应,通过比较两种因素作用后人髓核细胞分泌的基质金属蛋白酶(matrix metalloproteinases, MMP)-1,-3,-10及-12的改变幅度大小,明确两者的效应强度,为退变椎间盘的生物学治疗提供依据。方法：体外单层系统培养人椎间盘髓核细胞,制作阴性对照组(negative control, NC)、应激诱导的过早老化组(stress-induced premature senescence, SIPS)和复制性老化组(replicative senescence, RS)。通过SA-β-Gal染色计数,PI染色、流式细胞术,以及Annexin V-FITC/PI染色、流式细胞术评价各组细胞。通过Agilent人全基因4*44K芯片对产生差异的指标进行筛选,对于筛选出的MMP-1,-3,-10及-12四个指标,使用实时定量PCR技术测定在三组中的表达水平,并进行统计学分析。结果：1.细胞老化率测定：NC组为(4.67±1.83)%,SIPS组为(80.92±5.44)%,RS组为(77.08±3.32)%。SIPS组与RS组间无统计学差异(P0.05)：2.G1期细胞比例：NC组为(57.28±2.24)%,SIPS组为(84.48±1.08)%,RS组为(88.87±3.15)%。SIPS组与RS组间无统计学差异(P0.05)；3.细胞凋亡率：NC组为(2.51±0.45)%,SIPS组为(5.40±0.78)%,RS组为(8.69±0.92)%。4. Agilent人全基因4*44K芯片显示大量指标在三组样本中的表达发生变化,选取MMP-1,-3,-10及-12进行实时定量PCR检测：5.与NC组相比,两老化组中MMP-1,-3,-10及-12的表达水平均显著提高(P0.05)；两老化组间也存在差异性,上述MMPs在SIPS组中显著高于RS组(P0.05)。结论：1.高氧微环境刺激与细胞连续分裂均可在体外环境下,导致人髓核细胞的老化,使细胞老化率、G1期细胞比例以及细胞凋亡率增高；2.老化的髓核细胞除上述改变外,其生物学行为也发生变化,大量分泌表型发生显著改变;3.高氧微环境刺激与细胞连续分裂造成的老化人髓核细胞,其生物学改变具有差异性。通过比较部分MMPs表达的上调幅度发现,高氧微环境刺激在体外人髓核细胞老化中的作用更为显著。
[Abstract]:Objective: to investigate the effects of hyperoxia microenvironment stimulation and cell division on the aging process of human nucleus pulposus cells in vitro. In order to provide the basis for biological treatment of degenerative intervertebral disc methods: human disc nucleus pulposus cells were cultured in a monolayer system in vitro. Negative control, NCX, stress-induced premature senescence (SIPSs) and replicative senescence (RSV) were made in negative control group. Pi staining, flow cytometry, and Annexin V-FITC / Pi staining were performed by SA- 尾 -Gal staining. Flow cytometry (FCM) was used to evaluate the cells in each group. The differentially expressed indexes were screened by Agilent human complete gene 44K microarray, and the expression of MMP-1 + -3 + -10 and -12 were measured by real-time quantitative PCR technique in the three groups. Results 1. The cell aging rate was 4.67 卤1.83 in the control group and 80.92 卤5.44 in the SIPS group. There was no statistical difference between the two groups. There was no statistical difference between the two groups (P0.05: 2.G1 phase cell ratio: 57.28 卤2.24SPS group vs 84.48 卤1.08SPS group). There was no statistical difference between the SIPS group and the RS group. There was no statistical difference between the two groups (P 0.05). The apoptotic rate was 2.51 卤0.45 in the control group and 5.40 卤0.78 in the SIPS group. The percentage of apoptotic cells in the RS group was 8.69 卤0.92 卤0.92%. 4. The expression of a large number of markers in the three groups was detected by Agilent human gene 44-K microarray. Compared with NC group, the expression levels of MMP-1 + -3 + -10 and -12 in aging group were significantly higher than those in control group (P 0.05), and there were also differences between the two aging groups. The MMPs in SIPS group was significantly higher than that in RS group (P 0.05). Conclusion: 1. Hyperoxia microenvironment stimulation and continuous cell division can lead to the aging of human nucleus pulposus cells in vitro. In addition to the above changes, the biological behavior of aging nucleus pulposus cells also changed. The biological changes of aging human nucleus pulposus cells caused by hyperoxia microenvironment stimulation and continuous cell division were different. By comparing the up-regulation of partial MMPs expression, it was found that there were significant differences in the biological changes of the aging human nucleus pulposus cells caused by hyperoxia microenvironment stimulation and continuous cell division. Hyperoxia microenvironment stimulation plays a more important role in the aging of human nucleus pulposus cells in vitro.
【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

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