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  本文選題：蛋白酶體 + 人骨髓間充質(zhì)干細胞�。� 參考：《山西醫(yī)科大學》2010年碩士論文

【摘要】： 目的：通過觀察人骨髓間充質(zhì)干細胞(human bone marrow stromal cells, hBMSCs)的復制性衰老進程以及衰老進程中hBMSCs的生物學特征變化,探討20S蛋白酶體在hBMSCs復制性衰老進程中的調(diào)控作用,從而揭示hBMSCs復制性衰老的機制,以期為hBMSCs的臨床應用提供指導依據(jù)。 方法：體外培養(yǎng)hBMSCs,依據(jù)傳代次數(shù)將細胞分為早期組(1-3代)、中期組(7-9代)和晚期組(12-14代)：(1)倒置相差顯微鏡下觀察不同時期細胞形態(tài)特征,β-半乳糖苷酶染色鑒定細胞衰老進程,噻唑藍(MTT)比色分析法和溴脫氧尿嘧啶核苷(bromodeoxyuridine,BrdU)摻入實驗檢測細胞增殖能力。(2)免疫熒光染色觀察20S蛋白酶體在hBMSCs復制性衰老進程中的表達變化。(3)將10μM蛋白酶體特異性抑制劑MG132作用于早期hBMSCs,每日作用2h,反復刺激4d,隨后通過β-半乳糖苷酶染色,觀察衰老標志物β-半乳糖苷酶的表達變化,并應用MTT比色法和BrdU摻入實驗檢測細胞增殖活性,檢測蛋白酶體抑制劑對早期hBMSCs增殖能力的影響。 結果：(1)早期hBMSCs胞體較小、核仁清晰,細胞呈梭形,似成纖維細胞樣外形。伴隨體外傳代次數(shù)增多hBMSCs胞體變大、突起增多,細胞折光度降低,胞漿內(nèi)出現(xiàn)許多細小顆粒,細胞粘附能力增加,生長速度減慢。衰老標志物β-半乳糖苷酶染色結果顯示,早期、中期和晚期組陽性細胞率分別為9%±7%,54%±8%,90%±8%,三組之間比較p0.05,提示體外培養(yǎng)的hBMSCs存在復制性衰老。MTT比色法檢測不同培養(yǎng)階段hBMSCs細胞活力早期組OD值為0.61±0.04,中期組為0.53±0.05,晚期組為0.37±0.03,三組之間比較p0.05,表明在復制性衰老進程中細胞活力顯著降低,同時BrdU摻入實驗結果也表明晚期組細胞BrdU陽性率(8%±2%)較早期組(73%±11%)顯著降低,且p0.05,進一步表明在復制性衰老進程中hBMSCs增殖活力降低。(2)免疫熒光染色結果顯示hBMSCs表達20S蛋白酶體,早期組與中期組20S蛋白酶體陽性率為100%,然而晚期細胞20S蛋白酶體表達水平降低,其陽性率降至31%,提示蛋白酶體活性降低可能與hBMSCs復制性衰老有關。(3)早期hBMSCs經(jīng)MG132作用后β-半乳糖苷酶染色增強,β-半乳糖苷酶陽性率達87%±13.2%,較DMSO組(對照組)(19%±6.8%)明顯升高,且p0.05,MTT檢測結果證實MG132組OD值(0.36±0.06)較DMSO組(0.47±0.08)顯著降低,且p0.05,并且Brdu摻入實驗也顯示MG132組Brdu陽性率(7%±2%)較對照組(54%±4%)明顯降低,且p0.05,表明蛋白酶體抑制劑可誘導早期hBMSCs出現(xiàn)衰老細胞的表型,進一步說明蛋白酶體活性降低與hBMSCs復制性衰老有關。 結論：(1)體外培養(yǎng)的hBMSCs存在復制性衰老；(2)20S蛋白酶體在hBMSCs復制性衰老進程中表達水平降低。(3)應用蛋白酶體特異性抑制劑可誘導hBMSCs呈現(xiàn)衰老細胞的表型,提示蛋白酶體活性降低可能是導致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% 鹵7% 鹵8% and 90% 鹵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 鹵0.04 in early stage, 0.53 鹵0.05 in metaphase group, and 0.53 鹵0.05 in late stage. 0.37 鹵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% 鹵2) significantly lower than that in the early group (73% 鹵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% 鹵13.2%, which was significantly higher than that in DMSO group (control group, 19% 鹵6.8 points). The OD value of MG132 group (0.36 鹵0.06) was significantly lower than that of DMSO group (0.47 鹵0.08), and the Brdu incorporation test also showed that the positive rate of Brdu in MG132 group was 7% 鹵2%) significantly lower than that in control group (54% 鹵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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R329

【引證文獻】

相關碩士學位論文 前1條

1 朱茜;20S蛋白酶體參與調(diào)控神經(jīng)干細胞衰老進程[D];山西醫(yī)科大學;2012年

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本文編號：1936204