BclG_L在系统性红斑狼疮患者外周血CD4~+T细胞中的表达及其功能研究
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摘要
一、研究背景和目的
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种典型的、慢性的、可累及全身多系统的自身免疫性疾病,其临床表现复杂,严重影响患者,尤其是育龄期女性的身体健康[1]。免疫调控失衡是SLE发病的中心环节,其主要表现为自身反应性淋巴细胞异常活化,自身抗体大量产生,循环免疫复合物异常沉积于组织和器官,导致多器官系统的损害。然而,目前SLE的确切病因和发病机制仍未完全明确,可涉及遗传、环境、感染、药物等多种因素,其中细胞凋亡异常已成为SLE免疫发病机制关注的热点之一。
大量研究证实,凋亡紊乱是SLE免疫失衡的关键环节,也是疾病发生发展的始动因素[2]。在遗传及环境因子的作用下,机体细胞显著凋亡,核内隐蔽抗原异常暴露,而巨噬细胞及补体清除功能障碍,大量凋亡碎片不断聚集成为自身抗原的主要来源,导致自身反应性T、B淋巴细胞持续活化,自身抗体不断产生,在直接损伤细胞的同时又形成大量免疫复合物沉积于组织与器官,造成持久损害[3, 4]。
外周血淋巴细胞作为免疫系统的重要成份,在SLE免疫内环境复杂信号的异常调控下大量细胞异常凋亡,是导致SLE外周血淋巴细胞数量显著减少的主要原因,也是SLE自身免疫反应的重要特征[5, 6]。临床研究显示,SLE患者淋巴细胞本身即存在凋亡调控机制的显著失衡,表现为各淋巴细胞亚群凋亡异常紊乱,B细胞凋亡减少,而CD4+ T及CD8+ T细胞凋亡显著增加,特别是CD4+ T细胞更处于明显活跃的凋亡状态,导致外周血中CD4+ T/CD8+ T细胞的比率持续降低[7, 8]。值得关注的是,SLE患者体内CD4+CD25+ Foxp3+调节性T细胞及NK T细胞的凋亡也显著增加,是导致机体免疫抑制效应受损,自身耐受打破的重要基础[9- 11]。
CD4+ T细胞是机体调控免疫内环境平衡和自稳的“重要枢纽”[12],其凋亡失衡是导致SLE免疫耐受打破的关键环节[13- 15],而CD4+T细胞内凋亡信号分子的调控失衡是诱导其凋亡异常紊乱的重要原因以及分子基础。研究显示,SLE患者CD4+ T细胞内外源性及内源性凋亡信号通路均存在明显紊乱,外源性凋亡通路中Fas, TNFR, TRAIL等分子及内源性凋亡通路的Bcl-2分子表达均异常上调,Bim, PUMA等分子功能异常[16, 17],血清中游离可溶性Fas(soluble Fas,sFas)及TNF-α等分子显著增多,这种凋亡与抗凋亡调控机制的严重失衡导致了CD4+ T细胞凋亡的明显紊乱[18- 20]。然而,由于凋亡分子各成员间相互作用复杂,已有的研究未能完全阐明SLE淋巴细胞凋亡紊乱的分子机理,提示还可能存在其它未知因素参与了淋巴细胞的凋亡调控。因此,进一步研究SLE患者淋巴细胞凋亡紊乱的信号传递途径,深入探讨SLE患者淋巴细胞凋亡失衡的分子机制,是为最终明确SLE异常凋亡的发病机理,寻找纠正凋亡失衡状态、恢复免疫自稳有效治疗手段的必要前提。
在我们前期的研究中,通过长标签基因表达序列分析(long serial analysis of gene expression, LongSAGE)构建了1例新发SLE患者发病前及缓解后CD4+ T淋巴细胞差异表达基因文库,发现凋亡相关基因BclGL在SLE患者疾病活动期表达明显增高。BclGL基因是近年发现的Bcl-2家族中具有凋亡促进效应的一种特殊蛋白质,研究发现其除对部分肿瘤细胞有凋亡诱导效应之外,同时也可对部分细胞的活化起到抑制作用[21],提示其对不同类型细胞可能有不同的调控作用;然而,迄今其对淋巴细胞的凋亡、增殖等相关功能是否具有调控作用以及其可能的机制至今尚不明确,并且国内外也尚未见BclGL与SLE发病相关性的研究报道。我们推测BclGL在SLE患者外周血CD4+T细胞内高表达是否可能参与了SLE CD4+T细胞的异常凋亡,促进SLE疾病的发生与发展?因此,本研究我们拟首先观察BclGL在SLE患者外周血淋巴细胞亚群内的表达分布,分析其与SLE疾病指标间的相互关系,之后进一步探讨其在SLE CD4+T细胞凋亡异常中的可能机制,为深入研究SLE淋巴细胞凋亡紊乱的分子机理及今后SLE临床诊断与治疗分子靶标的筛选提供理论依据与实验线索。
二、方法与结果
1.基因芯片筛选SLE患者外周血CD4+T细胞凋亡相关差异表达基因免疫磁珠分离3名女性新发SLE患者外周血CD4+T细胞,流式细胞检测细胞纯度,并以相应年龄、地域、种族的健康女性CD4+T细胞为对照,表达谱基因芯片检测细胞基因差异表达情况。结果显示SLE患者CD4+T细胞中凋亡相关及I型干扰素诱导基因的表达较对照组显著增强;与前期构建的LongSAGE差异基因表达谱结合分析发现,BclGL分子表达在SLE患者外周血CD4+ T细胞中明显增高(p<0.01)。
2. SLE患者淋巴细胞亚群BclGL基因表达检测及其与细胞凋亡及疾病指标的相关性分析收集95例女性SLE患者,分析SLE疾病活动度及各项临床指标,同时以同年龄阶段、地域、种族、病程的43例类风湿关节炎(rheumatoid arthritis, RA)、30名皮肌炎(dermatomyositis,DM)患者及75名正常人为对照,分离外周血CD4+T, CD8+ T及B细胞,运用实时荧光定量PCR及Western blot方法检测BclGL的在各淋巴细胞亚群中的表达情况,Annexin V-FITC/PI双染法流式细胞仪(flow cytometry, FCM)检测各淋巴细胞亚群细胞凋亡情况。结果显示:BclGL在SLE患者外周血CD4+ T细胞中表达较各对照组均显著增高(p<0.01),而在CD8+ T及B细胞中表达无明显差异(p>0.05);SLE患者外周血CD4+ T,CD8+T细胞凋亡较各对照组显著上调(p<0.01, p<0.05),而B细胞凋亡无明显差异(p>0.05);SLE CD4+ T细胞中BclGL表达量与CD4+T细胞凋亡率及患者体内抗核抗体(anti-nuclear antibody, ANA)滴度、蛋白尿水平呈正相关(r= 0.82,0.70和0.77,p<0.05);抗双链DNA抗体(anti-dsDNA antibody)阳性患者及患有肾脏疾病、淋巴细胞减少症及浆膜炎的SLE患者其CD4+ T细胞BclGL表达量较阴性明显升高(p<0.05)。
3.血清刺激后淋巴细胞BclGL表达及细胞凋亡检测以SLE,RA,DM患者及正常人血清(n=12)刺激正常人外周血淋巴细胞(n=6) 4h,16h,24h后,分别检测CD4+T,CD8+T及B细胞内BclGL基因表达量及细胞凋亡率。结果显示:SLE血清刺激的正常CD4+T细胞BclGL基因表达量及细胞凋亡率较各对照组均显著上调(p<0.01),上调的BclGL基因表达与细胞凋亡呈正比(r=0.73, p<0.05),并且具有时间依赖性;SLE血清虽可刺激CD8+T凋亡,但BclGL在CD8+T及B细胞中表达较对照组相比无明显差异(p>0.05)。
4.构建BclGL慢病毒表达载体,观察其对淋巴细胞凋亡的影响为进一步探讨BclGL基因对淋巴细胞凋亡效应的影响,体外构建BclGL慢病毒表达载体Lenti-BclGL-GFP,并包装浓缩慢病毒,测定病毒滴度,体外转染Jurkat细胞及正常人CD4+T细胞,免疫荧光观察GFP+表达率,流式细胞仪检测转染效率,实时荧光定量PCR及Western blot检测BclGL基因表达量,Annexin V-APC/PI双染检测细胞凋亡变化。结果显示:经酶切测序鉴定,慢病毒表达载体Lenti-BclGL-GFP构建成功,包装浓缩后重组慢病毒滴度可达2.0×106/ml,转染细胞后两种细胞内BclGL表达量均显著上调且细胞凋亡率也均明显增高(p<0.05),表明BclGL过表达对Jurkat细胞及CD4+T淋巴细胞具有显著的促凋亡效应。
5.构建针对BclGL基因的shRNA逆转录病毒,检测其干扰效率及对T淋巴细胞凋亡、增殖的影响利用shRNA预测软件设计合成3条针对BclGL基因的shRNA编码序列,插入pSilencer5.1-H1 Retro构建重组逆转录病毒siRNA表达载体,脂质体法转染PT67细胞,嘌呤霉素筛选阳性细胞克隆,包装并浓缩重组逆转录病毒pSilencer5.1-H1-BclGL(P1,P2,P3),以空载体包装病毒(Pneg)为对照,体外感染Jurkat细胞56h,实时荧光定量PCR及Western blot检测BclGL基因干扰效率, CCK-8法检测细胞增殖力。将干扰效率最高病毒转染过表达BclGL的正常人CD4+T细胞56 h后检测干扰效率;并将病毒转染正常人CD4+T细胞72h后用SLE患者血清继续刺激细胞24h检测细胞凋亡情况。结果显示:3组BclGL shRNA逆转录病毒干扰载体的酶切及测序结果均正确,证实病毒载体构建成功;3组病毒稳定包装细胞筛选成功,滴度可达105CFU/mL以上,对BclGL的表达均具有明显的沉默效应,其中P2组的干扰效率最高可达62%。BclGL表达下调后可促进Jurkat细胞的增殖力;并且可部分抑制SLE血清对CD4+T细胞的凋亡诱导效应。
三、结论
1.与RA、DM及正常对照相比,BclGL在SLE患者CD4+ T细胞中显著高表达,而在CD8+ T及B细胞内表达无明显差异;上调的BclGL表达量与SLE CD4+ T细胞凋亡率及患者ANA滴度,蛋白尿水平呈正相关;抗ds-DNA抗体阳性患者及具有肾脏疾病、淋巴细胞减少症、浆膜炎的患者BclGL的表达量相对较高。
2.与RA、DM患者及正常血清相比,SLE患者血清可同时诱导正常人CD4+ T细胞内BclGL基因表达及促进CD4+ T细胞凋亡,而BclGL表达在CD8+ T及B细胞内无明显差异;上调的BclGL表达量与CD4+ T细胞凋亡率呈正相关,并具有时间依赖性。
3.在体外,BclGL基因过表达可明显诱导人T淋巴细胞系Jurkat及正常人原代CD4+ T细胞凋亡;并且干扰BclGL表达后可促进Jurkat细胞的增殖,并且可部分抑制SLE血清对正常CD4+T细胞的凋亡促进效应。提示BclGL表达对T淋巴细胞的凋亡及增殖具有重要的调控作用;其表达异常可能参与了SLE血清诱导的内源性凋亡信号通路,并在SLE CD4+T细胞凋亡紊乱中发挥重要作用。
4.BclGL在SLE患者CD4+T细胞中高表达,可能通过促进SLE CD4+ T细胞的异常凋亡而导致了SLE免疫紊乱与内环境失衡,加速疾病的发生与发展。BclGL可能是SLE患者CD4+T细胞凋亡调节中的一个重要因素,但并非决定性因素,深入研究BclGL的分子调控机制将为进一步阐明SLE淋巴凋亡紊乱的分子机理以及临床寻找SLE诊断与治疗的新靶位和新策略提供必要的理论基础与实验依据。
Background and objectives
Systemic lupus erythematosus (SLE), which develops predominantly in women of childbearing age, is a chronic autoimmune disease with a wide spectrum of clinical and immunological abnormalities. It is characterized by overactivation of autoreactive T and B lymphocytes, the presence of various autoantibodies, especially those directed to dsDNA,and circulating immune complexes deposit in tissues and organs. Both the accurate etiology and the pathogenesis of SLE are still unexplained, which might involve genetics, environment, infection and drugs and so on. In recent years, insight into the pathogenesis of SLE has deepened. In particular studies on apoptosis disorders in lupus have shed a new and intriguing light on the development and course of the disease.
Increasing knowledge has considered that the defective regulation of apoptosis is the central defects of immune function of SLE. Under the circumstances of the genetic and environmental factors, excessive nuclear antigens released from apoptotic cells without being removed by impaired clearance result in overloaded self-antigens, abnormal activation of autoreactive lymphocytes, end-organ damage by immunocomplexes deposition on organs in SLE.
Many studies have confirmed that an increased rate of peripheral blood lymphocytes apoptosis could be in part responsible for the lymphopenia of SLE patients and the skewed apoptotic signal pathways result in abnormal lymphocytes subsets apoptosis. The apoptosis of CD4+T and CD8+T cells increase but not B cells. CD4+ T cells are at a state of active apoptosis and the ratio of CD4+ / CD8+ T cells keep on decreasing. Especially natural CD4+ CD25+ regulatory T cell depletion and high apoptotic sensitivity of NK T cells result in autoreactive lymphocytes overactivation in SLE.
CD4+ T cells are considered to play a pivotal role in the pathogenesis of SLE. A dysfunction in their regulatory action can account for the altered immunological homeostasis and the generation of losing self-tolerance. Although CD4+ T cells regulate multiple layers of autoimmunity, apoptotic signaling is an essential component of this balance. Increasing knowledge have confirmed that there is an altered expression of apoptosis-related molecules in CD4+ T cells of SLE. Extrinsic apoptotic pathways mediated by death receptor are evidently aberrant. Increased Fas, TRAIL, soluble Fas(sFas) and TNF-αexpression results in a higher apoptotic susceptibility of CD4+ T cells. Intrinsic apoptotic pathways mediated by the Bcl-2 families are also remarkably disordered in SLE with Bim, PUMA,Bcl-XL upregulated in SLE and so on. These unbalanced apoptotic signal result in the aberrant apoptosis of SLE CD4+ T cells. But, the complicated interaction mechanisms of apoptotic molecules and unclear signal regulation of SLE lymphocytes indicates that there must exist other unknown factors taking part in the disordered cell apoptosis. It is evidently important to investigate the aberrant apoptotic signal pathways in order to further elucidate the pathogenesis of SLE and find the effective methods to correct the imbalance of apoptosis.
We have previously obtained genomewide gene expression profiles of CD4+ T cells from a female SLE patient at severe activity and nonactivity state of disease by using long serial analysis of gene expression (LongSAGE) and found a novel apoptosis-related gene, BclGL expression was significantly higher in peripheral blood CD4+ T cells. BclGL is a newly identified special pro-apoptotic members of Bcl-2 families. Besides its apoptotsis inducing effects on several tumor cell lines, it has been demonstrated to have the inhibiting effects on cell activation, which indicates that it may have different effects on different kinds of cells. But, up to now, its regulatory function on lymphocytes apoptosis and proliferation and its precise mechanisms are still unknown, and there is no the relative reports of BclGL in the pathogenesis of SLE. So, we conjecture that the increased BclGL expression in SLE CD4+ T cells may participate in the abnormal apoptosis of CD4+ T cells. In order to verify this presumption, in the present study, we first want to detect the expression distribution of BclGL in peripheral blood lymphocytes of SLE patients and analyze its relationships with cell apoptosis and clinical parameters, and further investigate its possible regulatory mechanisms in aberrant SLE CD4+T apoptosis. Our study will provide experimental clues and theoretical evidences for discovering molecular mechanisms of disordered lymphocytes apoptosis and screening diagnostic and therapeutic targets of SLE in the future.
Methods and results
1. Screening apoptosis-related differential genes in CD4+T cells of SLE patients by microarray analyses Peripheral CD4+T cells of 3 recent-onset SLE female patients and age-, area-,ethnically matched normal controls were isolated with immunomagnetic beads. The purity of isolated cells were identified by flow cytometry(FCM). The results showed that the expression of apoptosis-related genes and type I interferon signaling genes are evidently increased in SLE CD4+T cells compared to normal controls. Combining with LongSAGE profiles, we found the expression of a novel apoptosis-related gene BclGL was greatly increased in SLE CD4+T cells(p<0.01).
2. BclGL gene expression in SLE lymphocytes and its relationships with cell apoptosis and clinical parameters 95 Chinese female SLE patients were consecutively recruited and the clinical parameters and disease activity determined with SLEDAI score were analyzed at time of blood acquisition. Seventy-five age-, gender-, course-matched and ethnically matched healthy female subjects, 43 active rheumatoid arthritis (RA) and 30 dermatomyositis (DM) patients were included from the same geographical area as controls. BclGL gene expression was detected by real-time PCR and wester blot in the peripheral blood CD4+T, CD8+ T and B cells. The percentage of apoptotic lymphocyte subsets were detected by FCM with Annexin V-FITC/PI double staining. The results showed that BclGL expression was significantly increased in SLE CD4+T cells (p<0.05) but not in CD8+T and B cells(p>0.05) compared to controls. The apoptotic rates of CD4+T and CD8+T cells in SLE patients were higher than other controls(p<0.01,p<0.05).The apoptosis of B cells could be seen but not reach significant levels(p>0.05). The increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis, antinuclear antibody (ANA) titer and proteinnuria(r=0.82,0.70 and 0.77,p<0.05).The SLE patients who have positive anti-dsDNA antibody, renal diseases, lymphopenia and serositis had relatively higher BclGL expression than those without these(p<0.05).
3. Detection of BclGL expression and cell apoptosis after different sera stimulation BclGL expression and cell apoptotic rates of normal human CD4+T, CD8+ T and B cells (n=6) were detected after stimulation with different sera of SLE, RA, DM patients and normal controls(NHS) for 4h,16h and 24h. The results showed that BclGL expression and cell apoptosis in SLE sera stimulated CD4+T cells were significantly increased as compared with other sera(p<0.05). Increased BclGL expression was positively correlated with CD4+T cells apoptotic rates(r=0.73,p<0.05) with time-dependence. The apoptosis rates of CD8+T cells were also increased but the expression of BclGL were not increased in CD8+T and B cells by SLE sera stimulation(p>0.05).
4. Construction of lentivirus FUGW-BclGL and detection of its apoptotic inducing effects A recombinant lentivirus vector Lenti-BclGL-GFP was constructed in vitro and transfected into 293FT cells to package lentivirus.The virus titer was detected after concentration.The virus was tranducted into Jurkat cell line and normal human CD4+T cells. Green fluorescent protein(GFP) expression was detected by immunofluorensence microscopy and FCM. BclGL expression was detected by real-time PCR and Western blot. Cell apoptosis was detected by FCM with Annexin V-APC/PI double staining. The results showed that Lenti-BclGL-GFP vector was successfully constructed by enzyme cutting and sequencing. The virus titer could reach 2.0×106/ml after packaging and concentration. The BclGL expression and cell apoptosis were both simultanously increased in transfected Jurkat and normal CD4+T cells(p<0.05),which indicated that BclGL had apoptosis-inducing effects on CD4+T cell apoptosis
5. Construction of retrovirus pSilencer5.1-H1-BclGL and detection of BclGL silence efficiency and its effects on apoptosis and proliferation of T lymphocytes Three shRNA encoding sequences were synthesized by shRNA analysis software and retrovirus vectors pSilencer5.1-H1-BclGL (P1,P2, P3) were constructed and transfected into PT67 packaging cells. Stable cell clones were set up by puromycin selection. Three recombinant retrovirus and empty control retrovirus were packaged,concentrated and tranducted into Jurkat cells for 56h. Silence efficiency of BclGL was detected by real-time PCR and Western blot. The proliferation of cells was detected by CCK-8. The silence efficiency of normal CD4+T cells previously was detected by infection retrovirus into BclGL overexpressed CD4+T cells. After normal CD4+T cells were infected by shRNA retrovirus for 72h, the cell apoptosis by SLE sera stimulation was detected. The results showed that 3 retovirus vectors were all successfully constructed and the titers were all above 105CFU/mL. BclGL expression could be silenced by 3 siRNA retrovirus and P2 was most efficient. The proliferation of Jurkat cells was increased after BclGL silence. SLE serum-induced CD4+T cell apoptosis could be partially inhibited by BclGL silence.
Conclusions
1. BclGL expression was significantly increased in peripheral blood CD4+T cells of SLE patients compared with RA,DM patients and normal controls, but there was no significant differences of BclGL expression in CD8+T and B cells. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis. The patients with positive anti-dsDNA, renal diseases, lymphopenia and serisitis had relatively higher BclGL expression compared those without these.
2. SLE sera could induce BclGL expression in normal CD4+T cells but not in CD8+T and B cells, and CD4+T cell apoptosis was also simultaneously enhanced by SLE sera stimulation. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis with time-dependence.
3. BclGL gene overexpression could induce Jurkat and normal CD4+T cells apoptosis in vitro. The proliferation of Jurkat cells could be increased and the apoptosis-inducing effects of SLE serum could be inhibited after BclGL gene silencing, which indicated that BclGL might play important roles in regulating T lymphocyte apoptosis and proliferation, and its abnormal expression could participate in the intracellular apoptotic signal pathways induced by SLE serum stimulation and play important roles in the pathogenesis of SLE CD4+T cells apoptosis.
4. Abnormal BclGL expression in SLE CD4+T cells could contribute to the aberrant SLE CD4+T cell apoptosis which results in immune disorder and imbalanced homeostasis in SLE. BclGL might be an important factor in regulating SLE CD4+T cell apoptosis but not the decisive factor. Deeply investigation of the BclGL signal pathways is significant and will provide worthy experimental clue and theoretical evidences for elucidating the pathogenesis and uncover useful and potential diagnostic and therapeutic targets of SLE in the future.
系统性红斑狼疮(systemic lupus erythematosus, SLE)是一种典型的、慢性的、可累及全身多系统的自身免疫性疾病,其临床表现复杂,严重影响患者,尤其是育龄期女性的身体健康[1]。免疫调控失衡是SLE发病的中心环节,其主要表现为自身反应性淋巴细胞异常活化,自身抗体大量产生,循环免疫复合物异常沉积于组织和器官,导致多器官系统的损害。然而,目前SLE的确切病因和发病机制仍未完全明确,可涉及遗传、环境、感染、药物等多种因素,其中细胞凋亡异常已成为SLE免疫发病机制关注的热点之一。
大量研究证实,凋亡紊乱是SLE免疫失衡的关键环节,也是疾病发生发展的始动因素[2]。在遗传及环境因子的作用下,机体细胞显著凋亡,核内隐蔽抗原异常暴露,而巨噬细胞及补体清除功能障碍,大量凋亡碎片不断聚集成为自身抗原的主要来源,导致自身反应性T、B淋巴细胞持续活化,自身抗体不断产生,在直接损伤细胞的同时又形成大量免疫复合物沉积于组织与器官,造成持久损害[3, 4]。
外周血淋巴细胞作为免疫系统的重要成份,在SLE免疫内环境复杂信号的异常调控下大量细胞异常凋亡,是导致SLE外周血淋巴细胞数量显著减少的主要原因,也是SLE自身免疫反应的重要特征[5, 6]。临床研究显示,SLE患者淋巴细胞本身即存在凋亡调控机制的显著失衡,表现为各淋巴细胞亚群凋亡异常紊乱,B细胞凋亡减少,而CD4+ T及CD8+ T细胞凋亡显著增加,特别是CD4+ T细胞更处于明显活跃的凋亡状态,导致外周血中CD4+ T/CD8+ T细胞的比率持续降低[7, 8]。值得关注的是,SLE患者体内CD4+CD25+ Foxp3+调节性T细胞及NK T细胞的凋亡也显著增加,是导致机体免疫抑制效应受损,自身耐受打破的重要基础[9- 11]。
CD4+ T细胞是机体调控免疫内环境平衡和自稳的“重要枢纽”[12],其凋亡失衡是导致SLE免疫耐受打破的关键环节[13- 15],而CD4+T细胞内凋亡信号分子的调控失衡是诱导其凋亡异常紊乱的重要原因以及分子基础。研究显示,SLE患者CD4+ T细胞内外源性及内源性凋亡信号通路均存在明显紊乱,外源性凋亡通路中Fas, TNFR, TRAIL等分子及内源性凋亡通路的Bcl-2分子表达均异常上调,Bim, PUMA等分子功能异常[16, 17],血清中游离可溶性Fas(soluble Fas,sFas)及TNF-α等分子显著增多,这种凋亡与抗凋亡调控机制的严重失衡导致了CD4+ T细胞凋亡的明显紊乱[18- 20]。然而,由于凋亡分子各成员间相互作用复杂,已有的研究未能完全阐明SLE淋巴细胞凋亡紊乱的分子机理,提示还可能存在其它未知因素参与了淋巴细胞的凋亡调控。因此,进一步研究SLE患者淋巴细胞凋亡紊乱的信号传递途径,深入探讨SLE患者淋巴细胞凋亡失衡的分子机制,是为最终明确SLE异常凋亡的发病机理,寻找纠正凋亡失衡状态、恢复免疫自稳有效治疗手段的必要前提。
在我们前期的研究中,通过长标签基因表达序列分析(long serial analysis of gene expression, LongSAGE)构建了1例新发SLE患者发病前及缓解后CD4+ T淋巴细胞差异表达基因文库,发现凋亡相关基因BclGL在SLE患者疾病活动期表达明显增高。BclGL基因是近年发现的Bcl-2家族中具有凋亡促进效应的一种特殊蛋白质,研究发现其除对部分肿瘤细胞有凋亡诱导效应之外,同时也可对部分细胞的活化起到抑制作用[21],提示其对不同类型细胞可能有不同的调控作用;然而,迄今其对淋巴细胞的凋亡、增殖等相关功能是否具有调控作用以及其可能的机制至今尚不明确,并且国内外也尚未见BclGL与SLE发病相关性的研究报道。我们推测BclGL在SLE患者外周血CD4+T细胞内高表达是否可能参与了SLE CD4+T细胞的异常凋亡,促进SLE疾病的发生与发展?因此,本研究我们拟首先观察BclGL在SLE患者外周血淋巴细胞亚群内的表达分布,分析其与SLE疾病指标间的相互关系,之后进一步探讨其在SLE CD4+T细胞凋亡异常中的可能机制,为深入研究SLE淋巴细胞凋亡紊乱的分子机理及今后SLE临床诊断与治疗分子靶标的筛选提供理论依据与实验线索。
二、方法与结果
1.基因芯片筛选SLE患者外周血CD4+T细胞凋亡相关差异表达基因免疫磁珠分离3名女性新发SLE患者外周血CD4+T细胞,流式细胞检测细胞纯度,并以相应年龄、地域、种族的健康女性CD4+T细胞为对照,表达谱基因芯片检测细胞基因差异表达情况。结果显示SLE患者CD4+T细胞中凋亡相关及I型干扰素诱导基因的表达较对照组显著增强;与前期构建的LongSAGE差异基因表达谱结合分析发现,BclGL分子表达在SLE患者外周血CD4+ T细胞中明显增高(p<0.01)。
2. SLE患者淋巴细胞亚群BclGL基因表达检测及其与细胞凋亡及疾病指标的相关性分析收集95例女性SLE患者,分析SLE疾病活动度及各项临床指标,同时以同年龄阶段、地域、种族、病程的43例类风湿关节炎(rheumatoid arthritis, RA)、30名皮肌炎(dermatomyositis,DM)患者及75名正常人为对照,分离外周血CD4+T, CD8+ T及B细胞,运用实时荧光定量PCR及Western blot方法检测BclGL的在各淋巴细胞亚群中的表达情况,Annexin V-FITC/PI双染法流式细胞仪(flow cytometry, FCM)检测各淋巴细胞亚群细胞凋亡情况。结果显示:BclGL在SLE患者外周血CD4+ T细胞中表达较各对照组均显著增高(p<0.01),而在CD8+ T及B细胞中表达无明显差异(p>0.05);SLE患者外周血CD4+ T,CD8+T细胞凋亡较各对照组显著上调(p<0.01, p<0.05),而B细胞凋亡无明显差异(p>0.05);SLE CD4+ T细胞中BclGL表达量与CD4+T细胞凋亡率及患者体内抗核抗体(anti-nuclear antibody, ANA)滴度、蛋白尿水平呈正相关(r= 0.82,0.70和0.77,p<0.05);抗双链DNA抗体(anti-dsDNA antibody)阳性患者及患有肾脏疾病、淋巴细胞减少症及浆膜炎的SLE患者其CD4+ T细胞BclGL表达量较阴性明显升高(p<0.05)。
3.血清刺激后淋巴细胞BclGL表达及细胞凋亡检测以SLE,RA,DM患者及正常人血清(n=12)刺激正常人外周血淋巴细胞(n=6) 4h,16h,24h后,分别检测CD4+T,CD8+T及B细胞内BclGL基因表达量及细胞凋亡率。结果显示:SLE血清刺激的正常CD4+T细胞BclGL基因表达量及细胞凋亡率较各对照组均显著上调(p<0.01),上调的BclGL基因表达与细胞凋亡呈正比(r=0.73, p<0.05),并且具有时间依赖性;SLE血清虽可刺激CD8+T凋亡,但BclGL在CD8+T及B细胞中表达较对照组相比无明显差异(p>0.05)。
4.构建BclGL慢病毒表达载体,观察其对淋巴细胞凋亡的影响为进一步探讨BclGL基因对淋巴细胞凋亡效应的影响,体外构建BclGL慢病毒表达载体Lenti-BclGL-GFP,并包装浓缩慢病毒,测定病毒滴度,体外转染Jurkat细胞及正常人CD4+T细胞,免疫荧光观察GFP+表达率,流式细胞仪检测转染效率,实时荧光定量PCR及Western blot检测BclGL基因表达量,Annexin V-APC/PI双染检测细胞凋亡变化。结果显示:经酶切测序鉴定,慢病毒表达载体Lenti-BclGL-GFP构建成功,包装浓缩后重组慢病毒滴度可达2.0×106/ml,转染细胞后两种细胞内BclGL表达量均显著上调且细胞凋亡率也均明显增高(p<0.05),表明BclGL过表达对Jurkat细胞及CD4+T淋巴细胞具有显著的促凋亡效应。
5.构建针对BclGL基因的shRNA逆转录病毒,检测其干扰效率及对T淋巴细胞凋亡、增殖的影响利用shRNA预测软件设计合成3条针对BclGL基因的shRNA编码序列,插入pSilencer5.1-H1 Retro构建重组逆转录病毒siRNA表达载体,脂质体法转染PT67细胞,嘌呤霉素筛选阳性细胞克隆,包装并浓缩重组逆转录病毒pSilencer5.1-H1-BclGL(P1,P2,P3),以空载体包装病毒(Pneg)为对照,体外感染Jurkat细胞56h,实时荧光定量PCR及Western blot检测BclGL基因干扰效率, CCK-8法检测细胞增殖力。将干扰效率最高病毒转染过表达BclGL的正常人CD4+T细胞56 h后检测干扰效率;并将病毒转染正常人CD4+T细胞72h后用SLE患者血清继续刺激细胞24h检测细胞凋亡情况。结果显示:3组BclGL shRNA逆转录病毒干扰载体的酶切及测序结果均正确,证实病毒载体构建成功;3组病毒稳定包装细胞筛选成功,滴度可达105CFU/mL以上,对BclGL的表达均具有明显的沉默效应,其中P2组的干扰效率最高可达62%。BclGL表达下调后可促进Jurkat细胞的增殖力;并且可部分抑制SLE血清对CD4+T细胞的凋亡诱导效应。
三、结论
1.与RA、DM及正常对照相比,BclGL在SLE患者CD4+ T细胞中显著高表达,而在CD8+ T及B细胞内表达无明显差异;上调的BclGL表达量与SLE CD4+ T细胞凋亡率及患者ANA滴度,蛋白尿水平呈正相关;抗ds-DNA抗体阳性患者及具有肾脏疾病、淋巴细胞减少症、浆膜炎的患者BclGL的表达量相对较高。
2.与RA、DM患者及正常血清相比,SLE患者血清可同时诱导正常人CD4+ T细胞内BclGL基因表达及促进CD4+ T细胞凋亡,而BclGL表达在CD8+ T及B细胞内无明显差异;上调的BclGL表达量与CD4+ T细胞凋亡率呈正相关,并具有时间依赖性。
3.在体外,BclGL基因过表达可明显诱导人T淋巴细胞系Jurkat及正常人原代CD4+ T细胞凋亡;并且干扰BclGL表达后可促进Jurkat细胞的增殖,并且可部分抑制SLE血清对正常CD4+T细胞的凋亡促进效应。提示BclGL表达对T淋巴细胞的凋亡及增殖具有重要的调控作用;其表达异常可能参与了SLE血清诱导的内源性凋亡信号通路,并在SLE CD4+T细胞凋亡紊乱中发挥重要作用。
4.BclGL在SLE患者CD4+T细胞中高表达,可能通过促进SLE CD4+ T细胞的异常凋亡而导致了SLE免疫紊乱与内环境失衡,加速疾病的发生与发展。BclGL可能是SLE患者CD4+T细胞凋亡调节中的一个重要因素,但并非决定性因素,深入研究BclGL的分子调控机制将为进一步阐明SLE淋巴凋亡紊乱的分子机理以及临床寻找SLE诊断与治疗的新靶位和新策略提供必要的理论基础与实验依据。
Background and objectives
Systemic lupus erythematosus (SLE), which develops predominantly in women of childbearing age, is a chronic autoimmune disease with a wide spectrum of clinical and immunological abnormalities. It is characterized by overactivation of autoreactive T and B lymphocytes, the presence of various autoantibodies, especially those directed to dsDNA,and circulating immune complexes deposit in tissues and organs. Both the accurate etiology and the pathogenesis of SLE are still unexplained, which might involve genetics, environment, infection and drugs and so on. In recent years, insight into the pathogenesis of SLE has deepened. In particular studies on apoptosis disorders in lupus have shed a new and intriguing light on the development and course of the disease.
Increasing knowledge has considered that the defective regulation of apoptosis is the central defects of immune function of SLE. Under the circumstances of the genetic and environmental factors, excessive nuclear antigens released from apoptotic cells without being removed by impaired clearance result in overloaded self-antigens, abnormal activation of autoreactive lymphocytes, end-organ damage by immunocomplexes deposition on organs in SLE.
Many studies have confirmed that an increased rate of peripheral blood lymphocytes apoptosis could be in part responsible for the lymphopenia of SLE patients and the skewed apoptotic signal pathways result in abnormal lymphocytes subsets apoptosis. The apoptosis of CD4+T and CD8+T cells increase but not B cells. CD4+ T cells are at a state of active apoptosis and the ratio of CD4+ / CD8+ T cells keep on decreasing. Especially natural CD4+ CD25+ regulatory T cell depletion and high apoptotic sensitivity of NK T cells result in autoreactive lymphocytes overactivation in SLE.
CD4+ T cells are considered to play a pivotal role in the pathogenesis of SLE. A dysfunction in their regulatory action can account for the altered immunological homeostasis and the generation of losing self-tolerance. Although CD4+ T cells regulate multiple layers of autoimmunity, apoptotic signaling is an essential component of this balance. Increasing knowledge have confirmed that there is an altered expression of apoptosis-related molecules in CD4+ T cells of SLE. Extrinsic apoptotic pathways mediated by death receptor are evidently aberrant. Increased Fas, TRAIL, soluble Fas(sFas) and TNF-αexpression results in a higher apoptotic susceptibility of CD4+ T cells. Intrinsic apoptotic pathways mediated by the Bcl-2 families are also remarkably disordered in SLE with Bim, PUMA,Bcl-XL upregulated in SLE and so on. These unbalanced apoptotic signal result in the aberrant apoptosis of SLE CD4+ T cells. But, the complicated interaction mechanisms of apoptotic molecules and unclear signal regulation of SLE lymphocytes indicates that there must exist other unknown factors taking part in the disordered cell apoptosis. It is evidently important to investigate the aberrant apoptotic signal pathways in order to further elucidate the pathogenesis of SLE and find the effective methods to correct the imbalance of apoptosis.
We have previously obtained genomewide gene expression profiles of CD4+ T cells from a female SLE patient at severe activity and nonactivity state of disease by using long serial analysis of gene expression (LongSAGE) and found a novel apoptosis-related gene, BclGL expression was significantly higher in peripheral blood CD4+ T cells. BclGL is a newly identified special pro-apoptotic members of Bcl-2 families. Besides its apoptotsis inducing effects on several tumor cell lines, it has been demonstrated to have the inhibiting effects on cell activation, which indicates that it may have different effects on different kinds of cells. But, up to now, its regulatory function on lymphocytes apoptosis and proliferation and its precise mechanisms are still unknown, and there is no the relative reports of BclGL in the pathogenesis of SLE. So, we conjecture that the increased BclGL expression in SLE CD4+ T cells may participate in the abnormal apoptosis of CD4+ T cells. In order to verify this presumption, in the present study, we first want to detect the expression distribution of BclGL in peripheral blood lymphocytes of SLE patients and analyze its relationships with cell apoptosis and clinical parameters, and further investigate its possible regulatory mechanisms in aberrant SLE CD4+T apoptosis. Our study will provide experimental clues and theoretical evidences for discovering molecular mechanisms of disordered lymphocytes apoptosis and screening diagnostic and therapeutic targets of SLE in the future.
Methods and results
1. Screening apoptosis-related differential genes in CD4+T cells of SLE patients by microarray analyses Peripheral CD4+T cells of 3 recent-onset SLE female patients and age-, area-,ethnically matched normal controls were isolated with immunomagnetic beads. The purity of isolated cells were identified by flow cytometry(FCM). The results showed that the expression of apoptosis-related genes and type I interferon signaling genes are evidently increased in SLE CD4+T cells compared to normal controls. Combining with LongSAGE profiles, we found the expression of a novel apoptosis-related gene BclGL was greatly increased in SLE CD4+T cells(p<0.01).
2. BclGL gene expression in SLE lymphocytes and its relationships with cell apoptosis and clinical parameters 95 Chinese female SLE patients were consecutively recruited and the clinical parameters and disease activity determined with SLEDAI score were analyzed at time of blood acquisition. Seventy-five age-, gender-, course-matched and ethnically matched healthy female subjects, 43 active rheumatoid arthritis (RA) and 30 dermatomyositis (DM) patients were included from the same geographical area as controls. BclGL gene expression was detected by real-time PCR and wester blot in the peripheral blood CD4+T, CD8+ T and B cells. The percentage of apoptotic lymphocyte subsets were detected by FCM with Annexin V-FITC/PI double staining. The results showed that BclGL expression was significantly increased in SLE CD4+T cells (p<0.05) but not in CD8+T and B cells(p>0.05) compared to controls. The apoptotic rates of CD4+T and CD8+T cells in SLE patients were higher than other controls(p<0.01,p<0.05).The apoptosis of B cells could be seen but not reach significant levels(p>0.05). The increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis, antinuclear antibody (ANA) titer and proteinnuria(r=0.82,0.70 and 0.77,p<0.05).The SLE patients who have positive anti-dsDNA antibody, renal diseases, lymphopenia and serositis had relatively higher BclGL expression than those without these(p<0.05).
3. Detection of BclGL expression and cell apoptosis after different sera stimulation BclGL expression and cell apoptotic rates of normal human CD4+T, CD8+ T and B cells (n=6) were detected after stimulation with different sera of SLE, RA, DM patients and normal controls(NHS) for 4h,16h and 24h. The results showed that BclGL expression and cell apoptosis in SLE sera stimulated CD4+T cells were significantly increased as compared with other sera(p<0.05). Increased BclGL expression was positively correlated with CD4+T cells apoptotic rates(r=0.73,p<0.05) with time-dependence. The apoptosis rates of CD8+T cells were also increased but the expression of BclGL were not increased in CD8+T and B cells by SLE sera stimulation(p>0.05).
4. Construction of lentivirus FUGW-BclGL and detection of its apoptotic inducing effects A recombinant lentivirus vector Lenti-BclGL-GFP was constructed in vitro and transfected into 293FT cells to package lentivirus.The virus titer was detected after concentration.The virus was tranducted into Jurkat cell line and normal human CD4+T cells. Green fluorescent protein(GFP) expression was detected by immunofluorensence microscopy and FCM. BclGL expression was detected by real-time PCR and Western blot. Cell apoptosis was detected by FCM with Annexin V-APC/PI double staining. The results showed that Lenti-BclGL-GFP vector was successfully constructed by enzyme cutting and sequencing. The virus titer could reach 2.0×106/ml after packaging and concentration. The BclGL expression and cell apoptosis were both simultanously increased in transfected Jurkat and normal CD4+T cells(p<0.05),which indicated that BclGL had apoptosis-inducing effects on CD4+T cell apoptosis
5. Construction of retrovirus pSilencer5.1-H1-BclGL and detection of BclGL silence efficiency and its effects on apoptosis and proliferation of T lymphocytes Three shRNA encoding sequences were synthesized by shRNA analysis software and retrovirus vectors pSilencer5.1-H1-BclGL (P1,P2, P3) were constructed and transfected into PT67 packaging cells. Stable cell clones were set up by puromycin selection. Three recombinant retrovirus and empty control retrovirus were packaged,concentrated and tranducted into Jurkat cells for 56h. Silence efficiency of BclGL was detected by real-time PCR and Western blot. The proliferation of cells was detected by CCK-8. The silence efficiency of normal CD4+T cells previously was detected by infection retrovirus into BclGL overexpressed CD4+T cells. After normal CD4+T cells were infected by shRNA retrovirus for 72h, the cell apoptosis by SLE sera stimulation was detected. The results showed that 3 retovirus vectors were all successfully constructed and the titers were all above 105CFU/mL. BclGL expression could be silenced by 3 siRNA retrovirus and P2 was most efficient. The proliferation of Jurkat cells was increased after BclGL silence. SLE serum-induced CD4+T cell apoptosis could be partially inhibited by BclGL silence.
Conclusions
1. BclGL expression was significantly increased in peripheral blood CD4+T cells of SLE patients compared with RA,DM patients and normal controls, but there was no significant differences of BclGL expression in CD8+T and B cells. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis. The patients with positive anti-dsDNA, renal diseases, lymphopenia and serisitis had relatively higher BclGL expression compared those without these.
2. SLE sera could induce BclGL expression in normal CD4+T cells but not in CD8+T and B cells, and CD4+T cell apoptosis was also simultaneously enhanced by SLE sera stimulation. Increased BclGL expression was positively correlated with enhanced CD4+T cell apoptosis with time-dependence.
3. BclGL gene overexpression could induce Jurkat and normal CD4+T cells apoptosis in vitro. The proliferation of Jurkat cells could be increased and the apoptosis-inducing effects of SLE serum could be inhibited after BclGL gene silencing, which indicated that BclGL might play important roles in regulating T lymphocyte apoptosis and proliferation, and its abnormal expression could participate in the intracellular apoptotic signal pathways induced by SLE serum stimulation and play important roles in the pathogenesis of SLE CD4+T cells apoptosis.
4. Abnormal BclGL expression in SLE CD4+T cells could contribute to the aberrant SLE CD4+T cell apoptosis which results in immune disorder and imbalanced homeostasis in SLE. BclGL might be an important factor in regulating SLE CD4+T cell apoptosis but not the decisive factor. Deeply investigation of the BclGL signal pathways is significant and will provide worthy experimental clue and theoretical evidences for elucidating the pathogenesis and uncover useful and potential diagnostic and therapeutic targets of SLE in the future.
引文
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