20S蛋白酶体参与人骨髓间充质干细胞复制性衰老进程

发布时间：2018-05-26 06:02

  本文选题：蛋白酶体 + 人骨髓间充质干细胞　； 参考：《山西医科大学》2010年硕士论文

【摘要】： 目的：通过观察人骨髓间充质干细胞(human bone marrow stromal cells, hBMSCs)的复制性衰老进程以及衰老进程中hBMSCs的生物学特征变化,探讨20S蛋白酶体在hBMSCs复制性衰老进程中的调控作用,从而揭示hBMSCs复制性衰老的机制,以期为hBMSCs的临床应用提供指导依据。 方法：体外培养hBMSCs,依据传代次数将细胞分为早期组(1-3代)、中期组(7-9代)和晚期组(12-14代)：(1)倒置相差显微镜下观察不同时期细胞形态特征,β-半乳糖苷酶染色鉴定细胞衰老进程,噻唑蓝(MTT)比色分析法和溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入实验检测细胞增殖能力。(2)免疫荧光染色观察20S蛋白酶体在hBMSCs复制性衰老进程中的表达变化。(3)将10μM蛋白酶体特异性抑制剂MG132作用于早期hBMSCs,每日作用2h,反复刺激4d,随后通过β-半乳糖苷酶染色,观察衰老标志物β-半乳糖苷酶的表达变化,并应用MTT比色法和BrdU掺入实验检测细胞增殖活性,检测蛋白酶体抑制剂对早期hBMSCs增殖能力的影响。 结果：(1)早期hBMSCs胞体较小、核仁清晰,细胞呈梭形,似成纤维细胞样外形。伴随体外传代次数增多hBMSCs胞体变大、突起增多,细胞折光度降低,胞浆内出现许多细小颗粒,细胞粘附能力增加,生长速度减慢。衰老标志物β-半乳糖苷酶染色结果显示,早期、中期和晚期组阳性细胞率分别为9%±7%,54%±8%,90%±8%,三组之间比较p0.05,提示体外培养的hBMSCs存在复制性衰老。MTT比色法检测不同培养阶段hBMSCs细胞活力早期组OD值为0.61±0.04,中期组为0.53±0.05,晚期组为0.37±0.03,三组之间比较p0.05,表明在复制性衰老进程中细胞活力显著降低,同时BrdU掺入实验结果也表明晚期组细胞BrdU阳性率(8%±2%)较早期组(73%±11%)显著降低,且p0.05,进一步表明在复制性衰老进程中hBMSCs增殖活力降低。(2)免疫荧光染色结果显示hBMSCs表达20S蛋白酶体,早期组与中期组20S蛋白酶体阳性率为100%,然而晚期细胞20S蛋白酶体表达水平降低,其阳性率降至31%,提示蛋白酶体活性降低可能与hBMSCs复制性衰老有关。(3)早期hBMSCs经MG132作用后β-半乳糖苷酶染色增强,β-半乳糖苷酶阳性率达87%±13.2%,较DMSO组(对照组)(19%±6.8%)明显升高,且p0.05,MTT检测结果证实MG132组OD值(0.36±0.06)较DMSO组(0.47±0.08)显著降低,且p0.05,并且Brdu掺入实验也显示MG132组Brdu阳性率(7%±2%)较对照组(54%±4%)明显降低,且p0.05,表明蛋白酶体抑制剂可诱导早期hBMSCs出现衰老细胞的表型,进一步说明蛋白酶体活性降低与hBMSCs复制性衰老有关。 结论：(1)体外培养的hBMSCs存在复制性衰老；(2)20S蛋白酶体在hBMSCs复制性衰老进程中表达水平降低。(3)应用蛋白酶体特异性抑制剂可诱导hBMSCs呈现衰老细胞的表型,提示蛋白酶体活性降低可能是导致hBMSCs衰老的重要原因。
[Abstract]:Objective: to investigate the role of 20s proteasome in the process of replicative senescence of human bone marrow mesenchymal stem cells (hBMSCs) by observing the process of replicative senescence and the changes of biological characteristics of hBMSCs in the process of senescence. So as to reveal the mechanism of hBMSCs replicative senescence, in order to provide guidance for clinical application of hBMSCs. Methods: hBMSCs were cultured in vitro. The cells were divided into three groups according to the times of passage. The cells were divided into early group (1-3 generations), middle group (7-9 passages) and late group (12-14 generations). The morphological characteristics of cells were observed under phase contrast microscope. 尾 -galactosidase staining was used to identify the process of cell senescence. MTT colorimetric assay and bromodeoxyuridine (BrdU) incorporation assay to detect cell proliferation ability. (2) immunofluorescence staining to observe the expression changes of 20s proteasome in the process of hBMSCs replicative senescence. The inhibitor MG132 was treated with hBMSCs for 2 hours a day, stimulated repeatedly for 4 days, and then stained with 尾 -galactosidase (尾 -galactosidase). The expression of 尾 -galactosidase was observed, and the proliferative activity of cells was detected by MTT colorimetry and BrdU incorporation assay, and the effect of proteasome inhibitor on the proliferation of early hBMSCs was detected. Results in the early stage, hBMSCs cells had smaller body, clear nucleoli, fusiform cells and fibroblast-like shape. With the increase of passage times in vitro, the hBMSCs cell body became larger, the processes increased, the cell diopter decreased, many small granules appeared in the cytoplasm, the cell adhesion ability increased and the growth rate slowed down. The results of 尾 -galactosidase staining showed that, The positive rate of hBMSCs cells in middle and late stage groups was 9% 卤7% 卤8% and 90% 卤8%, respectively. The comparison between the three groups was p0.05, which suggested that the OD value of hBMSCs cultured in vitro was 0.61 卤0.04 in early stage, 0.53 卤0.05 in metaphase group, and 0.53 卤0.05 in late stage. 0.37 卤0.03, compared with the three groups (p0.05), which indicated that the cell viability decreased significantly in the process of replicative senescence. The results of BrdU incorporation also showed that the positive rate of BrdU in the late group was 8% 卤2) significantly lower than that in the early group (73% 卤11), and p0.05, which further indicated that the proliferative activity of hBMSCs decreased in the process of replicative senescence. The immunofluorescence staining showed that hBMSCs expressed 20s proteasome. The positive rate of 20s proteasome was 100 in early and middle stage, but the expression level of 20s proteasome was decreased in late stage cells. The positive rate of 尾 -galactosidase was decreased to 31%, suggesting that the decrease of proteasome activity might be related to hBMSCs replicative senescence. The positive rate of 尾 -galactosidase staining in early hBMSCs treated with MG132 was increased, and the positive rate of 尾 -galactosidase was 87% 卤13.2%, which was significantly higher than that in DMSO group (control group, 19% 卤6.8 points). The OD value of MG132 group (0.36 卤0.06) was significantly lower than that of DMSO group (0.47 卤0.08), and the Brdu incorporation test also showed that the positive rate of Brdu in MG132 group was 7% 卤2%) significantly lower than that in control group (54% 卤4). P0.05, which indicated that proteasome inhibitor could induce the appearance of senescent cell phenotype in hBMSCs at early stage, which further indicated that the decrease of proteasome activity was related to hBMSCs replicative senescence. Conclusion: hBMSCs cultured in vitro has the ability to induce hBMSCs to present the phenotype of senescent cells by using proteasome specific inhibitor. It is suggested that the decrease of proteasome activity may be an important cause of hBMSCs senescence.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【引证文献】

相关硕士学位论文 前1条

1 朱茜;20S蛋白酶体参与调控神经干细胞衰老进程[D];山西医科大学;2012年

，

本文编号：1936204