內(nèi)質(zhì)網(wǎng)應(yīng)激參與SLE患者BM-MSCs衰老和凋亡過程
發(fā)布時(shí)間：2018-08-03 09:25
【摘要】：目的探討內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress, ERS)在系統(tǒng)性紅斑狼瘡(systemic lupus erythematosus patients, SLE)患者骨髓間充質(zhì)干細(xì)胞(bone marrow-mesenchymal stem cells, BM-MSCs)衰老和凋亡過程中的作用及其機(jī)制。方法 (1)分離、培養(yǎng)SLE患者和止常對照組BM-MSCs,透射電子顯微鏡觀察SLE患者BM-MSCs中內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum,ER)是否處于應(yīng)激狀態(tài)：免螋印跡法(western blotting, WB)分析ERS標(biāo)志蛋白GRP78、p-IRE-1 p-PERK、p-eIF 2α、和CHOP表達(dá)情況：(2)利用衰老相關(guān)p半乳糖苷酶(senescence associated β-galactosidase, SA-β-gal)試劑檢測SA-β-gal活性,流式細(xì)胞儀檢測細(xì)胞周期分布情況,用免疫熒光法(Immunofluorescence)觀察細(xì)胞骨架的分布情況：(3)WB細(xì)胞周期相關(guān)蛋白p27、Skp2 在 BM-MSCs中的表達(dá)情況；用免疫熒光法(Immunofluorescence)觀察GRP78、p27和Skp2在BM-MSCs中的定位變化以及細(xì)胞骨架的分布情況：轉(zhuǎn)錄多聚酶鏈反應(yīng)(reverse transcription polymerase chain reaction, RT-PCR)檢測p27和Skp2在BM-MSCs中的表達(dá)情況： (4)通過流式細(xì)胞儀、活化型caspase-3、 BAX含量測定、抗凋亡蛋白Bcl-2測定來檢測SLE患者BM-MSCs是否存在細(xì)胞凋亡；用免疫熒光法(Immunofluorescence)觀察CHOP和活化型caspase-3在BM-MSCs中的表達(dá)變化：(5)利用ERS抑制劑4苯基丁酸(4-phenylbutyric acid,4PBA)處理SLE患者BM-MSCs后,觀察其SA-β-gal活性、細(xì)胞周期及細(xì)胞骨架變化情況：用通過流式細(xì)胞儀、活化型caspase-3含量測定、抗凋亡蛋白Bcl-2測定細(xì)胞凋亡情況：(5)利用ERS抑制劑4苯基丁酸(4-phenylbutyric acid,4PBA)處理SLE患者BM-MSCs, WB及免疫熒光法檢測GRP78、p27、Skp2表達(dá)變化,并干擾p27表達(dá)后再分別檢測上述細(xì)胞衰老相關(guān)特性及蛋白表達(dá)變化：利用WB及泛素化分析檢測p27泛素化降解水平： (6)siRNA干擾p-PERK和CHOP表達(dá)后,用WB檢測p-PERK、CHOP 和 Bcl-2表達(dá)變化；(7)用WB測定MAP K通路相關(guān)蛋白p-JNK1/2、p-ERK1/2和p-p38在SLE患者BM-MSCs表達(dá)情況,以及4PBA)處理后的表達(dá)差異；(8)siRNA干擾p-IRE-1和p-JNK1/2表達(dá)后,用WB檢測p-IRE-1、p-JNK、/2和BAX表達(dá)變化。結(jié)果與正常對照組相比較,SLE患者BM-MSCs存在明顯的ER腫脹、肥大等應(yīng)激表現(xiàn),ERS分子伴侶GRP78及標(biāo)志蛋白p-IRE-1、 p-PERK和其下游分子p-eIF 2α、CHOP表達(dá)明顯增加,而4PBA干預(yù)ERS后,SLE患者BM-MSCs細(xì)胞周期停滯、SA-β-gal陽性細(xì)胞數(shù)增多、細(xì)胞骨架分布紊亂及p27積聚等細(xì)胞衰老征象得以逆轉(zhuǎn),進(jìn)一步研究發(fā)現(xiàn),ERS通過抑制泛素化降解過程使p27大量積聚,最終導(dǎo)致SLE患者BM-MSCs衰老。同時(shí),4PBA干預(yù)ERS后,SLE患者BM-MSCs凋亡細(xì)胞數(shù)明顯減少,且活化型caspase-3、BAX含量降低,表明細(xì)胞凋亡征象得以逆轉(zhuǎn)。并且ERS下游信號(hào)分子p-IRE-1/ p-JNK1/2/BAX和p-PERK/CHOP/Bcl-2參與調(diào)節(jié)SLE患者BM-MSCs凋亡過程。結(jié)論ERS參與了SLE患者BM-MSCs的衰老和凋亡過程。針對ERS可以逆轉(zhuǎn)SLE患者M(jìn)SCs衰老和凋亡增加,為SLE的治療提供新的靶點(diǎn)。
[Abstract]:Objective to investigate the role and mechanism of endoplasmic reticulum stress (endoplasmic reticulum stress, ERS) in the senescence and apoptosis of bone marrow-mesenchymal stem cells (BM-MSCs) in patients with systemic lupus erythematosus (systemic lupus erythematosus patients, SLE). Method (1) Separation, BM-MSCs were cultured in SLE patients and normal controls. Transmission electron microscopy was used to observe whether the endoplasmic reticulum ER in BM-MSCs of SLE patients was in stress state. (western blotting, WB) analysis of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 偽 and CHOP expression: (2) the expression of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 偽 was analyzed by (western blotting, WB). The activity of SA- 尾 -gal was detected by (senescence associated 尾 -galactosidase (SA- 尾 -gal) reagent. The cell cycle distribution was detected by flow cytometry and the cytoskeleton distribution was observed by immunofluorescence (Immunofluorescence). (3) the expression of cell cycle associated protein p27 Skp2 in BM-MSCs; The localization of GRP78 p27 and Skp2 in BM-MSCs and the distribution of cytoskeleton were observed by immunofluorescence (Immunofluorescence). The expression of p27 and Skp2 in BM-MSCs was detected by transcriptional polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR): (4) flow cytometry was used to detect the expression of p27 and Skp2 in BM-MSCs. The contents of activated caspase-3, BAX and anti-apoptotic protein Bcl-2 were determined to detect whether there was apoptosis in BM-MSCs of SLE patients. The expression of CHOP and activated caspase-3 in BM-MSCs was observed by immunofluorescence (Immunofluorescence). (5) the SA- 尾 -gal activity, cell cycle and cytoskeleton of SLE patients treated with ERS inhibitor 4-phenylbutyric acidine 4PBA were observed by flow cytometry. The content of activated caspase-3 and anti-apoptotic protein Bcl-2 were measured. (5) BM-MSCs was treated with ERS inhibitor 4-phenylbutyric acid (4-phenylbutyric acidine 4PBA), and the expression of GRP78p27p27 Skp2 was detected by WB and immunofluorescence. After interfering with the expression of p27, the senescence related characteristics and protein expression of the above cells were detected. The degradation level of p27 ubiquitin was detected by WB and ubiquitin analysis. (6) after siRNA interfered with the expression of p-PERK and CHOP, the expression of p-PERKHop and Bcl-2 were detected by WB. (7) the expression of p-JNK1 / 2p-ERK1 / 2 and p-p38 in SLE patients and the difference of BM-MSCs expression after 4PBA treatment were detected by WB. (8) after siRNA interfered with the expression of p-IRE-1 and p-JNK1/2, the expression of p-IRE-1pJNK2 / 2 and BAX were detected by WB. Results compared with the control group, the expression of ER swelling, hypertrophy, GRP78, p-IRE-1 and p-IRE-1, p-PERK and the downstream molecule p-eIF 2 偽 -chop were significantly increased in patients with BM-MSCs. After 4PBA intervention, the number of SA- 尾 -gal positive cells in BM-MSCs patients with BM-MSCs cell cycle arrest increased, the cellular cytoskeleton distribution disorder and p27 accumulation were reversed. Eventually lead to SLE patients with BM-MSCs aging. At the same time, the number of BM-MSCs apoptotic cells and the content of activated caspase-3 and Bax in BM-MSCs patients were significantly decreased after PBA intervention, which indicated that the apoptotic signs could be reversed. ERS downstream signaling molecules p-IRE-1/ p-JNK1/2/BAX and p-PERK/CHOP/Bcl-2 are involved in the regulation of BM-MSCs apoptosis in SLE patients. Conclusion ERS is involved in the senescence and apoptosis of BM-MSCs in SLE patients. ERS can reverse the aging and apoptosis of MSCs in SLE patients and provide a new target for the treatment of SLE.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.241
本文編號(hào)：2161309
[Abstract]:Objective to investigate the role and mechanism of endoplasmic reticulum stress (endoplasmic reticulum stress, ERS) in the senescence and apoptosis of bone marrow-mesenchymal stem cells (BM-MSCs) in patients with systemic lupus erythematosus (systemic lupus erythematosus patients, SLE). Method (1) Separation, BM-MSCs were cultured in SLE patients and normal controls. Transmission electron microscopy was used to observe whether the endoplasmic reticulum ER in BM-MSCs of SLE patients was in stress state. (western blotting, WB) analysis of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 偽 and CHOP expression: (2) the expression of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 偽 was analyzed by (western blotting, WB). The activity of SA- 尾 -gal was detected by (senescence associated 尾 -galactosidase (SA- 尾 -gal) reagent. The cell cycle distribution was detected by flow cytometry and the cytoskeleton distribution was observed by immunofluorescence (Immunofluorescence). (3) the expression of cell cycle associated protein p27 Skp2 in BM-MSCs; The localization of GRP78 p27 and Skp2 in BM-MSCs and the distribution of cytoskeleton were observed by immunofluorescence (Immunofluorescence). The expression of p27 and Skp2 in BM-MSCs was detected by transcriptional polymerase chain reaction (reverse transcription polymerase chain reaction, RT-PCR): (4) flow cytometry was used to detect the expression of p27 and Skp2 in BM-MSCs. The contents of activated caspase-3, BAX and anti-apoptotic protein Bcl-2 were determined to detect whether there was apoptosis in BM-MSCs of SLE patients. The expression of CHOP and activated caspase-3 in BM-MSCs was observed by immunofluorescence (Immunofluorescence). (5) the SA- 尾 -gal activity, cell cycle and cytoskeleton of SLE patients treated with ERS inhibitor 4-phenylbutyric acidine 4PBA were observed by flow cytometry. The content of activated caspase-3 and anti-apoptotic protein Bcl-2 were measured. (5) BM-MSCs was treated with ERS inhibitor 4-phenylbutyric acid (4-phenylbutyric acidine 4PBA), and the expression of GRP78p27p27 Skp2 was detected by WB and immunofluorescence. After interfering with the expression of p27, the senescence related characteristics and protein expression of the above cells were detected. The degradation level of p27 ubiquitin was detected by WB and ubiquitin analysis. (6) after siRNA interfered with the expression of p-PERK and CHOP, the expression of p-PERKHop and Bcl-2 were detected by WB. (7) the expression of p-JNK1 / 2p-ERK1 / 2 and p-p38 in SLE patients and the difference of BM-MSCs expression after 4PBA treatment were detected by WB. (8) after siRNA interfered with the expression of p-IRE-1 and p-JNK1/2, the expression of p-IRE-1pJNK2 / 2 and BAX were detected by WB. Results compared with the control group, the expression of ER swelling, hypertrophy, GRP78, p-IRE-1 and p-IRE-1, p-PERK and the downstream molecule p-eIF 2 偽 -chop were significantly increased in patients with BM-MSCs. After 4PBA intervention, the number of SA- 尾 -gal positive cells in BM-MSCs patients with BM-MSCs cell cycle arrest increased, the cellular cytoskeleton distribution disorder and p27 accumulation were reversed. Eventually lead to SLE patients with BM-MSCs aging. At the same time, the number of BM-MSCs apoptotic cells and the content of activated caspase-3 and Bax in BM-MSCs patients were significantly decreased after PBA intervention, which indicated that the apoptotic signs could be reversed. ERS downstream signaling molecules p-IRE-1/ p-JNK1/2/BAX and p-PERK/CHOP/Bcl-2 are involved in the regulation of BM-MSCs apoptosis in SLE patients. Conclusion ERS is involved in the senescence and apoptosis of BM-MSCs in SLE patients. ERS can reverse the aging and apoptosis of MSCs in SLE patients and provide a new target for the treatment of SLE.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.241
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