重症肌无力胸腺Foxp3表达及临床意义的研究
摘要
重症肌无力(MG)系累及神经肌肉接头乙酰胆碱受体(AchR)的自身免疫性疾病。尽管目前已经明确参与MG自身免疫反应的抗体与靶抗原,但是MG自身免疫反应的产生机制仍不清楚。多项研究表明,胸腺异常与MG关系密切,可能为自身免疫产生的主要原因。胸腺来源的CD4+CD25+调节性T淋巴细胞(Treg)对活化的效应T淋巴细胞具有显著的抑制功能,在维持自身免疫耐受中起着重要作用。Foxp3为抑制性转录调节基因,系Treg发育及功能形成所必须的重要条件。MG患者胸腺及外周血中存在CD4+CD25+Treg功能异常,但Foxp3作为Treg功能特征性标志,目前对其在MG胸腺内表达及变化的研究尚未见报道。
本研究结合HE染色与免疫组织化学,检测Foxp3在MG患者胸腺组织中的表达与定位分布;采用Western Blot与RT-PCR技术观察Foxp3基因及蛋白产物表达的变化;并利用流式细胞技术分析Foxp3阳性细胞(Foxp3+)在胸腺细胞亚群中的分布与变化,以及功能分子CTLA-4、TGF-β、IL-10的表达;通过RT-PCR与Western Blot分别检测MG胸腺TGF-β、IFN-γ、IL-10、IL-4 mRNA以及Smad3、Smad7的表达并探讨其与Foxp3表达的关系。
实验的主要结果:(1) MG增生胸腺、萎缩胸腺以及瘤旁胸腺的髓质区组织学改变较为相似,均表现为相对面积扩大,淋巴细胞数量增多;髓质区内淋巴滤泡形成,血管周围间隙增宽。增生胸腺及瘤旁胸腺CD25阳性细胞率显著高于对照。MG增生胸腺以及萎缩、瘤旁胸腺组织中Foxp3蛋白及mRNA水平无显著差异,但均显著低于正常胸腺而高于胸腺瘤。Foxp3蛋白与mRNA表达在不同的MGFA分型之间亦存在显著差异。(2)胸腺瘤组织以增生上皮细胞为主,间有数量不等淋巴细胞,其中CD25、Foxp3阳性细胞率,Foxp3蛋白及mRNA表达水平均显著低于对照胸腺、增生胸腺、萎缩胸腺及瘤旁胸腺。(3) Foxp3在CD4、CD8单阳性(SP)胸腺细胞以及CD4+CD8+双阳性(DP)胸腺细胞中均有表达,CD4+SP胸腺细胞中Foxp3+细胞比例非常显著高于CD8+SP、DP胸腺细胞(P＜0.01)。MG胸腺Foxp3+细胞在CD4+SP中的比例显著低于正常胸腺(P＜0.01),而在CD8+SP、DP中无显著改变。(4)正常胸腺CD25+细胞76%表达Foxp3,CD25-细胞0.32%表达Foxp3,差异非常显著(P＜0.01)。MG胸腺中Foxp3在CD25+细胞中的表达显著低于正常(P＜0.05),而CD25-细胞中Foxp3表达则非常显著高于正常(P＜0.01)。(5) MG胸腺组织Foxp3+细胞表达CTLA-4显著低于正常胸腺(P＜0.05),而TGF-β、IL-10的表达则无统计学差异。(6) MG胸腺组织中TGF-βmRNA表达水平、Smad3/Smad7比值均非常显著低于正常胸腺(P＜0.01)。其中,增生胸腺TGF-βmRNA水平非常显著高于胸腺瘤组织(P＜0.05)。(7) MG增生胸腺、萎缩胸腺、瘤旁胸腺及胸腺瘤组织中IFN-γmRNA表达存在显著性差异,增生胸腺IFN-γmRNA表达水平非常显著高于正常胸腺、萎缩胸腺、瘤旁胸腺以及胸腺瘤;萎缩胸腺、瘤旁胸腺中IFN-γmRNA水平亦显著高于胸腺瘤组织。(7) MG胸腺组织中Foxp3 mRNA表达水平与TGF-βmRNA呈正相关,与IFN-γmRNA表达呈负相关,与IL-10、IL-4 mRNA表达水平无显著相关性。。
研究结论:①MG萎缩胸腺、瘤旁胸腺与增生胸腺具有相似的髓质区组织学改变及与之相一致的CD25+、Foxp3+细胞分布特点;且三者Foxp3蛋白、mRNA表达水平无显著差异。提示增生胸腺、萎缩胸腺、瘤旁胸腺具有相同的免疫病理学异常,这可能是MG发病的共同基础。胸腺瘤的存在是否参与MG发病及其作用尚需深入研究。②MG胸腺组织中Foxp3阳性细胞率、蛋白及mRNA表达水平均显著低于正常胸腺,且与MGFA临床分型相关;CD4+SP胸腺细胞、CD25+胸腺细胞表达Foxp3显著降低,提示Foxp3表达异常可能在MG发病中起着重要作用。③MG胸腺Foxp3+性细胞表达CTLA-4水平显著低于正常胸腺,而TGF-β、IL-10表达则无显著改变,表明CTLA-4/B7介导的细胞接触抑制途径异常,可能系MG胸腺Treg功能降低的原因之一。④MG胸腺内TGF-βmRNA水平,以及Smad3/Smad7比值显著低于正常,并与Foxp3表达显著正相关,提示TGF水平的降低可能系MG胸腺Foxp3表达下降的原因之一。而IFN-γ表达水平上调,并与Foxp3、TGF-β呈显著负相关,可能参与MG发病。
Myasthenia Gravis(MG) is a prototypical CD4+ T cell-dependent autoimmune disease mediated by anti-acetylcholine receptor(AChR) autoantibodies affecting the neuromuscular transmission.Though characterized well,the mechanism of the autoimmune reaction is still unclear.The thymus is assumed to be the initiative site for the occurring of autoimmunity.Regulatory T cells(Treg),coming from thymus and characterized by expression of CD4+CD25+,is found to play a central role in dominant immune tolerance by inhibiting the immune response of CD4+ effective T cells.In a recent study,the CD4+CD25+Treg,which function is critically dependent on the expression of Foxp3,was found been dysfunction in myasthenic thymus.
Aim to explore the role of Foxp3 in the thymus of MG,the HE and immunohistochemistry staining(HIS) was used to detect the expression and location of Foxp3 in myasthenic thymus and thymoma tissues;Western Blot and RT-PCR were also carried out to examine the level of Foxp3 protein and mRNA.And we also checked the frequency of Foxp3+ cells in the CD4+ or CD8+ single positive(SP) and CD4+CD8+ double positive(DP) thymocytes,as well as the expression of CTLA-4,TGF-βand IL-10, which were thought to be mediators in the suppression of immune response.At last,we explored the expressions of some cytokines such as TGF-β,IFN-γ,IL-4 and IL-10,which function as inducers of Foxp3 in the myasthenic thymus.
The results from out study were described:1.Hyperplasic thymus(HT) from MG patients is characterized by the expansion of medullar area,lymphoid follicular and per vascular space(PVS),while some similar histological changes were found in atrophic thymus(AT) and the thymus near thymoma(Tnt).The ratio of CD25+ cell in HT and Tnt was significant higher than that in normal thymus,and Foxp3+ cell ratio was much lower in HT and AT compared with normal thymus.There was no significant difference of Foxp3 protein or mRNA expression level among HT,AT and Tnt tissues,while which was much lower than that in normal thymus but higher than that in thymoma.Moreover,significant differences of the expression was also found among different MGFA classification.2. Thymoma was characterized with hyperplastic epithelial cells with variant amount of lymphocytes.The ratio of CD25 positive cell and Foxp3 positive cell,as well as the expression of Foxp3 protein and mRNA,was much lower than that in HT and normal thymus.3.The expression of Foxp3 can be found both in CD4+,CD8+ SP and CD4+CD8+ DP thymocytes,CD4+ SP thymocytes had a higher percentage of Foxp3 expression than CD8+ SP and DP thymocyte.However,the ratio of Foxp3 in CD4+ SP thymocytes was much decreased compared with normal thymus,while no change of CD8+ SP and CD4+CD8+ DP thymocyte was found.Despite CD4 and CD8,more than half of CD25+ thymocyte was Foxp3 positive,with a sharp decreased ratio in the myasthenic thymus.4.The expression of CTLA-4 in the Foxp3 positive cell was significantly decreased in myasthenic thymus compared with normal thymus,while no change was found for the expression of TGF-β,IL-10.5.The level of TGF-βmRNA was significantly lower in myasthenic thymus than normal thymus,as well as the ratio Smad3/Smad7.Moreover, the expression of TGF-βwas significantly correlated with the expression of Foxp3 positively.We also found that the level of IFN-γmRNA,which was correlated with the expression of TGF-βand Foxp3 negatively,was much higher in HT than normal thymus or AT,Tnt and thymoma.Moreover,no significant changes was found of the expression of IL-10 and IL-4 mRNA.
The conclusions of our study can be drawn as follows:ⅰ.There were similar pathologic changes at the medullar area of HT,AT and Tnt thymus,and the same location can also be found for Foxp3,CD25 positive cells.Moreover,there were no significant changes for the level of Foxp3 protein and mRNA among the three type of myasthenic thymus,while the expressions were much higher than that of thymoma.These kinds of changes indicated that there were some common basis for HT,AT and Tnt myasthenic thymus in the mechanisms of MG autoimmunity,which might be different from thymoma, the thymus tissue itself of MG patients combined with thymoma maybe play a similar role as HT in the autoimmune disease,ⅱ.The percentage of Foxp3 positive cells in CD4+ SP thymocytes or CD25+ thymocytes,as well as the expression of Foxp3 protein and mRNA of myasthenic thymus were significant decreased than that of normal thymus,and were significantly correlated with the MGFA clinical classification,which meant an important role of Foxp3 for the autoimmunity,ⅲ.Compared with normal thymus,the expression of CTLA-4 of Foxp3 positive cells in myasthenic hyperplasia thymus was significant decreased,which suggested that abnormal intercellular contact mediated by CTLA-4/B7 might account for the autoimmunity,ⅳ.The significant lower expression of TGF-βmRNA,as well as the ratio of Smad3/Smad7,was relevant to the expression of Foxp3.It suggested that the down-regulation of TGF might be account for the decreasing of Foxp3 expression in myasthenic thymus.In our study,an up-regulated IFN-γmRNA in myasthenic thymus suggested a important role for the occurrence of the disease.
本研究结合HE染色与免疫组织化学,检测Foxp3在MG患者胸腺组织中的表达与定位分布;采用Western Blot与RT-PCR技术观察Foxp3基因及蛋白产物表达的变化;并利用流式细胞技术分析Foxp3阳性细胞(Foxp3+)在胸腺细胞亚群中的分布与变化,以及功能分子CTLA-4、TGF-β、IL-10的表达;通过RT-PCR与Western Blot分别检测MG胸腺TGF-β、IFN-γ、IL-10、IL-4 mRNA以及Smad3、Smad7的表达并探讨其与Foxp3表达的关系。
实验的主要结果:(1) MG增生胸腺、萎缩胸腺以及瘤旁胸腺的髓质区组织学改变较为相似,均表现为相对面积扩大,淋巴细胞数量增多;髓质区内淋巴滤泡形成,血管周围间隙增宽。增生胸腺及瘤旁胸腺CD25阳性细胞率显著高于对照。MG增生胸腺以及萎缩、瘤旁胸腺组织中Foxp3蛋白及mRNA水平无显著差异,但均显著低于正常胸腺而高于胸腺瘤。Foxp3蛋白与mRNA表达在不同的MGFA分型之间亦存在显著差异。(2)胸腺瘤组织以增生上皮细胞为主,间有数量不等淋巴细胞,其中CD25、Foxp3阳性细胞率,Foxp3蛋白及mRNA表达水平均显著低于对照胸腺、增生胸腺、萎缩胸腺及瘤旁胸腺。(3) Foxp3在CD4、CD8单阳性(SP)胸腺细胞以及CD4+CD8+双阳性(DP)胸腺细胞中均有表达,CD4+SP胸腺细胞中Foxp3+细胞比例非常显著高于CD8+SP、DP胸腺细胞(P＜0.01)。MG胸腺Foxp3+细胞在CD4+SP中的比例显著低于正常胸腺(P＜0.01),而在CD8+SP、DP中无显著改变。(4)正常胸腺CD25+细胞76%表达Foxp3,CD25-细胞0.32%表达Foxp3,差异非常显著(P＜0.01)。MG胸腺中Foxp3在CD25+细胞中的表达显著低于正常(P＜0.05),而CD25-细胞中Foxp3表达则非常显著高于正常(P＜0.01)。(5) MG胸腺组织Foxp3+细胞表达CTLA-4显著低于正常胸腺(P＜0.05),而TGF-β、IL-10的表达则无统计学差异。(6) MG胸腺组织中TGF-βmRNA表达水平、Smad3/Smad7比值均非常显著低于正常胸腺(P＜0.01)。其中,增生胸腺TGF-βmRNA水平非常显著高于胸腺瘤组织(P＜0.05)。(7) MG增生胸腺、萎缩胸腺、瘤旁胸腺及胸腺瘤组织中IFN-γmRNA表达存在显著性差异,增生胸腺IFN-γmRNA表达水平非常显著高于正常胸腺、萎缩胸腺、瘤旁胸腺以及胸腺瘤;萎缩胸腺、瘤旁胸腺中IFN-γmRNA水平亦显著高于胸腺瘤组织。(7) MG胸腺组织中Foxp3 mRNA表达水平与TGF-βmRNA呈正相关,与IFN-γmRNA表达呈负相关,与IL-10、IL-4 mRNA表达水平无显著相关性。。
研究结论:①MG萎缩胸腺、瘤旁胸腺与增生胸腺具有相似的髓质区组织学改变及与之相一致的CD25+、Foxp3+细胞分布特点;且三者Foxp3蛋白、mRNA表达水平无显著差异。提示增生胸腺、萎缩胸腺、瘤旁胸腺具有相同的免疫病理学异常,这可能是MG发病的共同基础。胸腺瘤的存在是否参与MG发病及其作用尚需深入研究。②MG胸腺组织中Foxp3阳性细胞率、蛋白及mRNA表达水平均显著低于正常胸腺,且与MGFA临床分型相关;CD4+SP胸腺细胞、CD25+胸腺细胞表达Foxp3显著降低,提示Foxp3表达异常可能在MG发病中起着重要作用。③MG胸腺Foxp3+性细胞表达CTLA-4水平显著低于正常胸腺,而TGF-β、IL-10表达则无显著改变,表明CTLA-4/B7介导的细胞接触抑制途径异常,可能系MG胸腺Treg功能降低的原因之一。④MG胸腺内TGF-βmRNA水平,以及Smad3/Smad7比值显著低于正常,并与Foxp3表达显著正相关,提示TGF水平的降低可能系MG胸腺Foxp3表达下降的原因之一。而IFN-γ表达水平上调,并与Foxp3、TGF-β呈显著负相关,可能参与MG发病。
Myasthenia Gravis(MG) is a prototypical CD4+ T cell-dependent autoimmune disease mediated by anti-acetylcholine receptor(AChR) autoantibodies affecting the neuromuscular transmission.Though characterized well,the mechanism of the autoimmune reaction is still unclear.The thymus is assumed to be the initiative site for the occurring of autoimmunity.Regulatory T cells(Treg),coming from thymus and characterized by expression of CD4+CD25+,is found to play a central role in dominant immune tolerance by inhibiting the immune response of CD4+ effective T cells.In a recent study,the CD4+CD25+Treg,which function is critically dependent on the expression of Foxp3,was found been dysfunction in myasthenic thymus.
Aim to explore the role of Foxp3 in the thymus of MG,the HE and immunohistochemistry staining(HIS) was used to detect the expression and location of Foxp3 in myasthenic thymus and thymoma tissues;Western Blot and RT-PCR were also carried out to examine the level of Foxp3 protein and mRNA.And we also checked the frequency of Foxp3+ cells in the CD4+ or CD8+ single positive(SP) and CD4+CD8+ double positive(DP) thymocytes,as well as the expression of CTLA-4,TGF-βand IL-10, which were thought to be mediators in the suppression of immune response.At last,we explored the expressions of some cytokines such as TGF-β,IFN-γ,IL-4 and IL-10,which function as inducers of Foxp3 in the myasthenic thymus.
The results from out study were described:1.Hyperplasic thymus(HT) from MG patients is characterized by the expansion of medullar area,lymphoid follicular and per vascular space(PVS),while some similar histological changes were found in atrophic thymus(AT) and the thymus near thymoma(Tnt).The ratio of CD25+ cell in HT and Tnt was significant higher than that in normal thymus,and Foxp3+ cell ratio was much lower in HT and AT compared with normal thymus.There was no significant difference of Foxp3 protein or mRNA expression level among HT,AT and Tnt tissues,while which was much lower than that in normal thymus but higher than that in thymoma.Moreover,significant differences of the expression was also found among different MGFA classification.2. Thymoma was characterized with hyperplastic epithelial cells with variant amount of lymphocytes.The ratio of CD25 positive cell and Foxp3 positive cell,as well as the expression of Foxp3 protein and mRNA,was much lower than that in HT and normal thymus.3.The expression of Foxp3 can be found both in CD4+,CD8+ SP and CD4+CD8+ DP thymocytes,CD4+ SP thymocytes had a higher percentage of Foxp3 expression than CD8+ SP and DP thymocyte.However,the ratio of Foxp3 in CD4+ SP thymocytes was much decreased compared with normal thymus,while no change of CD8+ SP and CD4+CD8+ DP thymocyte was found.Despite CD4 and CD8,more than half of CD25+ thymocyte was Foxp3 positive,with a sharp decreased ratio in the myasthenic thymus.4.The expression of CTLA-4 in the Foxp3 positive cell was significantly decreased in myasthenic thymus compared with normal thymus,while no change was found for the expression of TGF-β,IL-10.5.The level of TGF-βmRNA was significantly lower in myasthenic thymus than normal thymus,as well as the ratio Smad3/Smad7.Moreover, the expression of TGF-βwas significantly correlated with the expression of Foxp3 positively.We also found that the level of IFN-γmRNA,which was correlated with the expression of TGF-βand Foxp3 negatively,was much higher in HT than normal thymus or AT,Tnt and thymoma.Moreover,no significant changes was found of the expression of IL-10 and IL-4 mRNA.
The conclusions of our study can be drawn as follows:ⅰ.There were similar pathologic changes at the medullar area of HT,AT and Tnt thymus,and the same location can also be found for Foxp3,CD25 positive cells.Moreover,there were no significant changes for the level of Foxp3 protein and mRNA among the three type of myasthenic thymus,while the expressions were much higher than that of thymoma.These kinds of changes indicated that there were some common basis for HT,AT and Tnt myasthenic thymus in the mechanisms of MG autoimmunity,which might be different from thymoma, the thymus tissue itself of MG patients combined with thymoma maybe play a similar role as HT in the autoimmune disease,ⅱ.The percentage of Foxp3 positive cells in CD4+ SP thymocytes or CD25+ thymocytes,as well as the expression of Foxp3 protein and mRNA of myasthenic thymus were significant decreased than that of normal thymus,and were significantly correlated with the MGFA clinical classification,which meant an important role of Foxp3 for the autoimmunity,ⅲ.Compared with normal thymus,the expression of CTLA-4 of Foxp3 positive cells in myasthenic hyperplasia thymus was significant decreased,which suggested that abnormal intercellular contact mediated by CTLA-4/B7 might account for the autoimmunity,ⅳ.The significant lower expression of TGF-βmRNA,as well as the ratio of Smad3/Smad7,was relevant to the expression of Foxp3.It suggested that the down-regulation of TGF might be account for the decreasing of Foxp3 expression in myasthenic thymus.In our study,an up-regulated IFN-γmRNA in myasthenic thymus suggested a important role for the occurrence of the disease.
引文
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