系统性红斑狼疮患者外周血单个核细胞中微管相关蛋白轻链3和溶酶体相关膜蛋白2基因表达及其临床意义
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摘要
目的检测系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)中巨自噬标志性分子微管相关蛋白轻链3 (LC3)和分子伴侣介导的自噬(CMA)标志性分子溶酶体相关膜蛋白2(LAMP-2)基因表达,探讨其与SLE发病的关系。方法纳入2017年11月至2018年3月在川北医学院附属医院风湿免疫科诊治的88例SLE患者(SLE),采用密度梯度离心法分离外周血单个核白细胞(PBMCs);采用实时荧光定量PCR法测定患者PBMCs中LC3和LAMP-2在mRNA水平的表达量;采用SLE疾病活动指数(SLEDAI)评分判断疾病活动度,分析LC3和LAMP-2表达与SLE患者临床特点及病情活动度的相关性。同期纳入年龄匹配的40例健康体检者作为正常对照组。结果正常对照组受检者PBMCs中LC3 mRNA和LAMP-2 mRNA相对表达量分别为1. 021±0. 551和1. 015±0. 667,SLE组患者分别为0. 783±0. 435和2. 402±2. 233,组间比较差异均有统计学意义(P=0. 020、0. 000)。SLE患者PBMCs中LAMP-2 mRNA表达水平与SLEDAI呈明显正相关(rs=0. 312,P=0. 003),LC3mRNA表达水平与SLEDAI无明显相关性(rs=-0. 175,P=0. 103)。LAMP-2 mRNA表达量升高患者肾脏损害发生率明显高于非升高患者(40. 7%vs. 16. 3%),LC3 mRNA表达量降低患者的血液系统受累率明显高于无LC3 mRNA表达降低患者(65. 2%vs. 32. 3%),差异均有统计学意义(P=0. 013、0. 006)。不同LC3 mRNA和LAMP-2 mRNA表达水平组实验室检查指标和治疗效果的患者分布差异均无统计学意义(均P>0. 05)。结论 SLE患者LC3 mRNA表达水平下调,LAMP-2 mRNA表达水平上调,提示SLE患者存在巨自噬功能不足。SLE患者CMA功能增强,并且与肾脏和血液系统受累有关。
Objective To detect the expression of macroautophagy microtubule-associated protein light chain 3( LC3) and chaperone-mediated autophagy( CMA) lysosomal-associated membrane protein tipe-2( LAMP-2) in peripheral blood mononuclear cells( PBMCs) from patients with systemic lupus erythematosus( SLE),and to explore the relationship between the expression and the pathogenesis of SLE. Methods From November 2017 to March 2018,88 SLE patients were recruited from the Department of Rheumatology and Immunology,Affiliated Hospital of North Sichuan Medical College. The PBMCs were separated by density gradient centrifugation,and the mRNA expression levels of LC3 and LAMP-2 in PBMCs were measured by real-time fluorescence quantitative PCR. Disease activity index( SLEDAI) score was used to determine disease activity,and then the correlation between clinical characteristics and disease activity of SLE patients was analyzed. In the same period,40 age-matched healthy subjects were enrolled as normal control group. Results The relative expressions of LC3 and LAMP-2 at mRNA levels in the normal control group were 1. 021 ± 0. 551 and 1. 015 ±0. 667,respectively,and in the SLE patients,they were 0. 783± 0. 435( P = 0. 02) and 2. 402± 2. 233( P =0. 000),respectively; The mRNA expression level of LAMP-2 in PBMCs of SLE patients was positively correlated with SLEDAI( rs= 0. 312,P = 0. 003) but the mRNA expression level of LC3 was not siginificantly correlated with SLEDAI( rs=-0. 175,P = 0. 103). The incidence of renal involvement in patients with increased LAMP-2 mRNA expression was significantly higher than that in the unelevated patients( 40. 7% vs. 16. 3%,P = 0. 013),and hematologic involvement in patients with decreased LC3 mRNA expression was significantly higher than that in unreduced patients( 65. 2% vs. 32. 3%,P = 0. 006). There were no significant differences in other laboratory indexes and therapeutic agents used among patients with different levels of LC3 mRNA and LAMP-2 mRNA expression( P>0. 05). Conclusions LC3 mRNA expression is down-regulated and LAMP-2 mRNA expression is up-regulated in SLE patients. It suggests that macrophagy may be deficient in SLE patients.CMA is enhanced in SLE patients,which is associated with the increase of renal and hematologic involvement.
Objective To detect the expression of macroautophagy microtubule-associated protein light chain 3( LC3) and chaperone-mediated autophagy( CMA) lysosomal-associated membrane protein tipe-2( LAMP-2) in peripheral blood mononuclear cells( PBMCs) from patients with systemic lupus erythematosus( SLE),and to explore the relationship between the expression and the pathogenesis of SLE. Methods From November 2017 to March 2018,88 SLE patients were recruited from the Department of Rheumatology and Immunology,Affiliated Hospital of North Sichuan Medical College. The PBMCs were separated by density gradient centrifugation,and the mRNA expression levels of LC3 and LAMP-2 in PBMCs were measured by real-time fluorescence quantitative PCR. Disease activity index( SLEDAI) score was used to determine disease activity,and then the correlation between clinical characteristics and disease activity of SLE patients was analyzed. In the same period,40 age-matched healthy subjects were enrolled as normal control group. Results The relative expressions of LC3 and LAMP-2 at mRNA levels in the normal control group were 1. 021 ± 0. 551 and 1. 015 ±0. 667,respectively,and in the SLE patients,they were 0. 783± 0. 435( P = 0. 02) and 2. 402± 2. 233( P =0. 000),respectively; The mRNA expression level of LAMP-2 in PBMCs of SLE patients was positively correlated with SLEDAI( rs= 0. 312,P = 0. 003) but the mRNA expression level of LC3 was not siginificantly correlated with SLEDAI( rs=-0. 175,P = 0. 103). The incidence of renal involvement in patients with increased LAMP-2 mRNA expression was significantly higher than that in the unelevated patients( 40. 7% vs. 16. 3%,P = 0. 013),and hematologic involvement in patients with decreased LC3 mRNA expression was significantly higher than that in unreduced patients( 65. 2% vs. 32. 3%,P = 0. 006). There were no significant differences in other laboratory indexes and therapeutic agents used among patients with different levels of LC3 mRNA and LAMP-2 mRNA expression( P>0. 05). Conclusions LC3 mRNA expression is down-regulated and LAMP-2 mRNA expression is up-regulated in SLE patients. It suggests that macrophagy may be deficient in SLE patients.CMA is enhanced in SLE patients,which is associated with the increase of renal and hematologic involvement.
引文
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[2] Klionsky DJ,Abdelmohsen K,Abe A,et al. Guidelines for the use and interpretation of assays for monitoring autophagy(3rd edition)[J]. Autophagy,2016,12:1-222.
[3] Petri M. Review of classification criteria for systemic lupus erythematosus[J]. Rheum Dis Clin North Am, 2005, 31:245-254.
[4] Yang Z,Goronzy JJ,WeyandCM. Autophagy in autoimmune disease[J]. J Mol Med(Berl),2015,93:707-717.
[5] Deng Q,Wang Z,Wang L,et al. Lower mRNA and protein expression levels of LC3 and Beclin1,markers of autophagy,were correlated with progression of renal clear cell carcinoma[J]. Jpn J Clin Oncol,2013,43:1261-1268.
[6] Clarke AJ, Ellinghaus U, Cortini A, et al. Autophagy is activated in systemic lupus erythematosus and required for plasmablast development[J]. Ann Rheum Dis, 2014, 74:912-920.
[7] Alessandri C,Barbati C,Vacirca D,et al. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy[J]. FASEB J,2012,26:4722-4732.
[8]罗雄燕,杨明辉,夏艳辉,等.系统性红斑狼疮患者外周血单个核细胞微管相关蛋白1轻链3 mRNA表达水平及其意义[J].中华内科杂志,2015,54:134-138.
[9] Harr MW,Mc Coll KS,Zhong F,et al. Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes[J]. Autophagy, 2010, 6:912-921.
[10] Ramos-Barr-n A,Pi1era-Haces C,Gómez-Alamillo C,et al.Prevention of murine lupus disease in(NZBxNZW)F1 mice by sirolimus treatment[J]. Lupus,2007,16:775-781.
[11] Lui SL,Tsang R,Chan KW,et al. Rapamycin attenuates the severity of established nephritis in lupus-prone NZB/W F1 mice[J]. Nephrol Dial Transplant,2008,23:2768-2776.
[12] Page N,Gros F,Schall N,et al. HSC70 blockade by the therapeutic peptide P140 affects autophagic processes and endogenous MHCII presentation in murine lupus[J]. Ann Rheum Dis,2011,70:837-843.
[13] Morell C, Bort A, Vara-Ciruelos D, et al. Up-regulated expression of LAMP2 and autophagy activity during neuroendocrine differentiation of prostate cancer LNCa P cells[J/OL]. PLoS One,2016,11:e0162977[2018-03-10]. http://journals. plos. org/plosone/article? id=10. 1371/journal. pone. 0162977.