内质网应激参与SLE患者BM-MSCs衰老和凋亡过程
发布时间：2018-08-03 09:25
【摘要】：目的探讨内质网应激(endoplasmic reticulum stress, ERS)在系统性红斑狼疮(systemic lupus erythematosus patients, SLE)患者骨髓间充质干细胞(bone marrow-mesenchymal stem cells, BM-MSCs)衰老和凋亡过程中的作用及其机制。方法 (1)分离、培养SLE患者和止常对照组BM-MSCs,透射电子显微镜观察SLE患者BM-MSCs中内质网(endoplasmic reticulum,ER)是否处于应激状态：免螋印迹法(western blotting, WB)分析ERS标志蛋白GRP78、p-IRE-1 p-PERK、p-eIF 2α、和CHOP表达情况：(2)利用衰老相关p半乳糖苷酶(senescence associated β-galactosidase, SA-β-gal)试剂检测SA-β-gal活性,流式细胞仪检测细胞周期分布情况,用免疫荧光法(Immunofluorescence)观察细胞骨架的分布情况：(3)WB细胞周期相关蛋白p27、Skp2 在 BM-MSCs中的表达情况；用免疫荧光法(Immunofluorescence)观察GRP78、p27和Skp2在BM-MSCs中的定位变化以及细胞骨架的分布情况：转录多聚酶链反应(reverse transcription polymerase chain reaction, RT-PCR)检测p27和Skp2在BM-MSCs中的表达情况： (4)通过流式细胞仪、活化型caspase-3、 BAX含量测定、抗凋亡蛋白Bcl-2测定来检测SLE患者BM-MSCs是否存在细胞凋亡；用免疫荧光法(Immunofluorescence)观察CHOP和活化型caspase-3在BM-MSCs中的表达变化：(5)利用ERS抑制剂4苯基丁酸(4-phenylbutyric acid,4PBA)处理SLE患者BM-MSCs后,观察其SA-β-gal活性、细胞周期及细胞骨架变化情况：用通过流式细胞仪、活化型caspase-3含量测定、抗凋亡蛋白Bcl-2测定细胞凋亡情况：(5)利用ERS抑制剂4苯基丁酸(4-phenylbutyric acid,4PBA)处理SLE患者BM-MSCs, WB及免疫荧光法检测GRP78、p27、Skp2表达变化,并干扰p27表达后再分别检测上述细胞衰老相关特性及蛋白表达变化：利用WB及泛素化分析检测p27泛素化降解水平： (6)siRNA干扰p-PERK和CHOP表达后,用WB检测p-PERK、CHOP 和 Bcl-2表达变化；(7)用WB测定MAP K通路相关蛋白p-JNK1/2、p-ERK1/2和p-p38在SLE患者BM-MSCs表达情况,以及4PBA)处理后的表达差异；(8)siRNA干扰p-IRE-1和p-JNK1/2表达后,用WB检测p-IRE-1、p-JNK、/2和BAX表达变化。结果与正常对照组相比较,SLE患者BM-MSCs存在明显的ER肿胀、肥大等应激表现,ERS分子伴侣GRP78及标志蛋白p-IRE-1、 p-PERK和其下游分子p-eIF 2α、CHOP表达明显增加,而4PBA干预ERS后,SLE患者BM-MSCs细胞周期停滞、SA-β-gal阳性细胞数增多、细胞骨架分布紊乱及p27积聚等细胞衰老征象得以逆转,进一步研究发现,ERS通过抑制泛素化降解过程使p27大量积聚,最终导致SLE患者BM-MSCs衰老。同时,4PBA干预ERS后,SLE患者BM-MSCs凋亡细胞数明显减少,且活化型caspase-3、BAX含量降低,表明细胞凋亡征象得以逆转。并且ERS下游信号分子p-IRE-1/ p-JNK1/2/BAX和p-PERK/CHOP/Bcl-2参与调节SLE患者BM-MSCs凋亡过程。结论ERS参与了SLE患者BM-MSCs的衰老和凋亡过程。针对ERS可以逆转SLE患者MSCs衰老和凋亡增加,为SLE的治疗提供新的靶点。
[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 伪 and CHOP expression: (2) the expression of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 伪 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 伪 -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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.241
本文编号：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 伪 and CHOP expression: (2) the expression of ERS marker GRP78 p-IRE-1 p-PERKPERKp-eIF2 伪 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 伪 -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.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.241
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