本文選題：PSMB5 + 蛋白酶體�。� 參考：《山西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：探討20S蛋白酶體β5亞單位（PSMB5）對(duì)體外擴(kuò)增培養(yǎng)人骨髓間充質(zhì)干細(xì)胞（humanbone marrow mesenchymal stem cells, hBMSCs）增殖能力的影響，明確調(diào)控hBMSCs細(xì)胞活力的有效靶點(diǎn)，從而為干細(xì)胞臨床移植奠定實(shí)驗(yàn)基礎(chǔ)。 方法：體外分離培養(yǎng)hBMSCs，依據(jù)體外傳代次數(shù)將其分為早期組（1-3代）和晚期組（12-14代）。1.檢測(cè)PSMB5在早期和晚期hBMSCs的表達(dá)變化。（1）通過(guò)溴脫氧尿嘧啶核苷（BrdU）摻入實(shí)驗(yàn)和流式細(xì)胞術(shù)檢測(cè)早期和晚期hBMSCs增殖能力。（2）應(yīng)用熒光酶標(biāo)儀檢測(cè)早期和晚期hBMSCs20S蛋白酶體活性。（3）應(yīng)用real-time PCR技術(shù)分析蛋白酶體β亞單位PSMB1、 PSMB5和PSMB6的表達(dá)變化；應(yīng)用Western blotting檢測(cè)早期和晚期20S蛋白酶體和PSMB5亞單位的表達(dá)變化。2.構(gòu)建攜帶綠色熒光蛋白（GFP）基因的PSMB5-shRNA慢病毒載體，并設(shè)錯(cuò)義序列對(duì)照，感染早期hBMSCs，觀(guān)察PSMB5基因沉默對(duì)hBMSCs增殖能力的影響。（1）慢病毒感染細(xì)胞后倒置熒光顯微鏡觀(guān)察綠色熒光蛋白表達(dá)情況，判斷感染效率。（2）應(yīng)用real-time PCR和Western blotting分別從基因和蛋白水平檢測(cè)PSMB5抑制效率。（3）熒光酶標(biāo)儀檢測(cè)20S蛋白酶體活性的變化。（4）應(yīng)用Ki67和BrdU免疫熒光染色檢測(cè)hBMSCs增殖率，Western blotting檢測(cè)細(xì)胞周期相關(guān)蛋白（Cyclin D1）的表達(dá)變化。 結(jié)果：1.（1）BrdU摻入實(shí)驗(yàn)結(jié)果證明早期hBMSCs BrdU陽(yáng)性率為68.92±7.08%，晚期hBMSCs陽(yáng)性率為27.67±2.76%，提示傳代晚期hBMSCs增殖能力較早期細(xì)胞顯著降低(P0.01)；流式細(xì)胞儀分析結(jié)果顯示，晚期hBMSCs S期細(xì)胞(4.42±0.98%)較早期hBMSCs(13.72±1.58%)顯著減少(P0.01)，細(xì)胞增殖指數(shù)較早期細(xì)胞降低11.02±1.88%(P0.05)，進(jìn)一步說(shuō)明擴(kuò)增培養(yǎng)晚期hBMSCs增殖活力降低。（2）晚期hBMSCs20S蛋白酶體活性較早期顯著降低52.99±13.08%(P0.01)，提示20S蛋白酶體活性可能與hBMSCs細(xì)胞活力有關(guān)。（3）real-time PCR結(jié)果表明，傳代晚期細(xì)胞蛋白酶體亞基PSMB1、PSMB5表達(dá)水平較早期分別降低13.0±1.0%和37.0±8.0%(P0.01)，且PSMB5下降最為明顯；同時(shí)，Western blotting實(shí)驗(yàn)進(jìn)一步表明晚期hBMSCs20S蛋白酶體和PSMB5表達(dá)水平分別比早期下降54.51±5.94%(P0.05)和52.99±13.08%(P0.05)，提示20S蛋白酶體核心亞單位PSMB5可能是調(diào)控hBMSCs細(xì)胞活力的關(guān)鍵因素。2.（1）PSMB5-shRNA慢病毒感染早期hBMSCs，24h后隱約可見(jiàn)部分細(xì)胞發(fā)出綠色熒光，但是信號(hào)較弱；72h后熒光信號(hào)增強(qiáng)，，約90%以上細(xì)胞可見(jiàn)綠色熒光。（2）real-time PCR和Western blotting結(jié)果證實(shí)，PSMB5-shRNA組PSMB5mRNA和蛋白的表達(dá)水平分別較GFP對(duì)照組下調(diào)93.3±0.34%(P0.01)和56.9±13.31%(P0.05)，提示慢病毒介導(dǎo)的shRNA能夠有效下調(diào)目的基因PSMB5的表達(dá)水平。（3）PSMB5-shRNA組20S蛋白酶體活性較GFP對(duì)照組降低69.87±1.32%(P0.05)，提示下調(diào)PSMB5表達(dá)水平能夠降低早期hBMSCs20S蛋白酶體活性。（4）Ki67免疫熒光染色結(jié)果顯示，PSMB5-shRNA組Ki67陽(yáng)性率較GFP對(duì)照組降低76.18±12.37%(P0.01)；同時(shí)BrdU摻入實(shí)驗(yàn)結(jié)果也證明，PSMB5-shRNA組BrdU陽(yáng)性率26.14±8.13%較GFP對(duì)照組49.53±11.18%顯著降低(P0.01)，表明PSMB5基因沉默能夠抑制早期hBMSCs增殖能力。此外，Western blotting實(shí)驗(yàn)結(jié)果證明PSMB5-shRNA組細(xì)胞周期相關(guān)蛋白Cyclin D1表達(dá)水平較GFP對(duì)照組降低54.55±5.49%(P0.05)，提示PSMB5基因下調(diào)可能通過(guò)抑制細(xì)胞周期進(jìn)程，從而影響hBMSCs增殖能力。 結(jié)論：（1）晚期hBMSCs增殖能力降低，并且20S蛋白酶體活性及其核心亞單位PSMB5表達(dá)水平下降。（2）應(yīng)用基因沉默技術(shù)手段下調(diào)PSMB5表達(dá)水平，能夠抑制早期hBMSCs增殖能力，提示PSMB5可能是影響hBMSCs增殖活力的關(guān)鍵靶點(diǎn)。
[Abstract]:Objective: To investigate the effect of 20S proteasome beta 5 subunit (PSMB5) on the proliferation of human bone marrow mesenchymal stem cells (humanbone marrow mesenchymal stem cells, hBMSCs) in vitro, and to clarify the effective targets for regulating the viability of hBMSCs cells, so as to lay an experimental basis for the clinical transplantation of stem cells.
Methods: hBMSCs was isolated and cultured in vitro. According to the number of passages in vitro, it was divided into early group (1-3 generation) and late group (12-14 generation) to detect the expression changes of PSMB5 in early and late hBMSCs. (1) detection of early and late hBMSCs proliferation by bromodeoxyuridine (BrdU) incorporation and flow cytometry. (2) the use of fluorescence enzyme labeling method. The activity of early and late hBMSCs20S proteasome was measured. (3) real-time PCR technique was used to analyze the expression of proteasome beta subunit PSMB1, PSMB5 and PSMB6, and Western blotting was used to detect the expression changes of early and late 20S proteasome and PSMB5 subunits and to construct the lentivirus carrying green color fluorescent protein (GFP) gene. Vector, and missense sequence control, infect early hBMSCs, observe the effect of PSMB5 gene silencing on hBMSCs proliferation ability. (1) the expression of green fluorescent protein was observed by inverted fluorescence microscope after lentivirus infection, and infection efficiency was judged. (2) real-time PCR and Western blotting were used to detect PSMB5 inhibition from gene and protein levels, respectively. Efficiency. (3) the changes in the activity of 20S proteasome were detected by the fluorescent enzyme labeling apparatus. (4) the proliferation rate of hBMSCs was detected by Ki67 and BrdU immunofluorescence staining, and the expression of cell cycle related protein (Cyclin D1) was detected by Western blotting.
Results: the results of 1. (1) BrdU incorporation showed that the positive rate of early hBMSCs BrdU was 68.92 + 7.08% and the positive rate of late hBMSCs was 27.67 + 2.76%, suggesting that the proliferation ability of late hBMSCs was significantly lower than that of early cells (P0.01). The results of flow cytometry showed that the late hBMSCs S phase cells (4.42 + 0.98%) were significantly higher than those of early hBMSCs (13.72 + 1.58%). Decrease (P0.01), the cell proliferation index decreased by 11.02 + 1.88% (P0.05), further indicating that the proliferation activity of advanced hBMSCs was reduced. (2) late hBMSCs20S proteasome activity was significantly reduced by 52.99 + 13.08% (P0.01), suggesting that the viability of 20S proteasome may be related to the viability of hBMSCs cells. (3) real-time PCR results showed that The advanced cell proteasome subunit PSMB1, PSMB5 expression level decreased by 13 + 1% and 37 + 8% (P0.01), respectively, and the PSMB5 decreased most obviously. Meanwhile, Western blotting experiment further indicated that the expression level of late hBMSCs20S proteasome and PSMB5 decreased by 54.51 + 5.94% (P0.05) and 52.99 + 13.08% (P0.05), respectively. The core subunit PSMB5 of 20S proteasome may be a key factor in the regulation of the activity of hBMSCs cells,.2. (1) PSMB5-shRNA lentivirus infection early hBMSCs. After 24h, some cells emit green fluorescence, but the signal is weak; after 72h, the fluorescence signal is enhanced, and more than 90% cells can be seen green fluorescence. (2) real-time PCR and Western blotting The results showed that the expression level of PSMB5mRNA and protein in PSMB5-shRNA group was down 93.3 + 0.34% (P0.01) and 56.9 + 13.31% (P0.05), respectively, suggesting that lentivirus mediated shRNA could effectively reduce the expression level of PSMB5 in the target gene. (3) the activity of 20S proteasome in PSMB5-shRNA group decreased by 69.87 + 1.32% (P0.05) in the GFP control group, suggesting down regulation. The expression level of PSMB5 could reduce the activity of early hBMSCs20S proteasome. (4) the results of Ki67 immunofluorescence staining showed that the positive rate of Ki67 in the PSMB5-shRNA group was 76.18 + 12.37% lower than that of the GFP control group (P0.01), and the BrdU incorporation test results also showed that the positive rate of BrdU in the PSMB5-shRNA group was 26.14 + 8.13% compared with the GFP control group (49.53 + 11.18%) (P0.01). It was indicated that PSMB5 gene silencing could inhibit the early hBMSCs proliferation ability. In addition, the Western blotting test results showed that the expression level of cell cycle related protein Cyclin D1 in PSMB5-shRNA group was 54.55 + 5.49% (P0.05) lower than that of the GFP control group, suggesting that the PSMB5 gene downregulation may affect the hBMSCs proliferation by inhibiting the cell cycle process.
Conclusion: (1) the proliferation ability of late hBMSCs decreased, and the activity of 20S proteasome and the expression level of PSMB5 in the core subunit decreased. (2) the application of gene silencing technique to reduce the expression of PSMB5 could inhibit the early hBMSCs proliferation ability, suggesting that PSMB5 may be the key target for the proliferation of hBMSCs.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R329.2

【相似文獻(xiàn)】

相關(guān)會(huì)議論文 前2條

1 呂書(shū)晴;王健民;楊建民;胡曉霞;黃崇媚;許曉倩;周虹;程輝;;PSMB5基因過(guò)表達(dá)導(dǎo)致多發(fā)性骨髓瘤患者對(duì)硼替佐米為基礎(chǔ)的化療耐藥[A];第13屆全國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文摘要[C];2011年

2 呂書(shū)晴;楊建民;龔勝藍(lán);周虹;李津嬰;韓鳳來(lái);郭列平;何毅;王健民;;蛋白酶體β5亞基基因(PSMB5)的擴(kuò)增和G322A點(diǎn)突變可導(dǎo)致T淋巴母細(xì)胞白血病細(xì)胞系對(duì)蛋白酶體抑制劑硼替唑米耐藥[A];第11次中國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文匯編[C];2007年

相關(guān)博士學(xué)位論文 前1條

1 梁俊波;與睪丸高表達(dá)蛋白質(zhì)RNF138相互作用的蛋白激酶NLK的功能研究和睪丸特異表達(dá)蛋白質(zhì)RSA-14-44的功能研究[D];北京協(xié)和醫(yī)學(xué)院;2010年

相關(guān)碩士學(xué)位論文 前2條

1 魏姣龍;PSMB5對(duì)人骨髓間充質(zhì)干細(xì)胞增殖能力的影響[D];山西醫(yī)科大學(xué);2014年

2 陳孟璋;乙型肝炎病毒聚合酶潛在的宿主作用因子的篩選與鑒定[D];暨南大學(xué);2011年

本文編號(hào)：1964543