修回日期: 2005-12-15
接受日期: 2005-12-31
在线出版日期: 2006-06-28
目的: 研究溃疡性结肠炎(UC)肠黏膜CD8 T细胞Fas/FasL、Bcl-2/Bax蛋白表达以及相互关系, 探讨细胞凋亡机制在UC发病中的作用.
方法: 采用免疫组化SP法检测60例UC肠黏膜组织以及60例正常肠黏膜组织CD8, Fas/FasL, Bcl-2/Bax蛋白表达.
结果: CD8阳性细胞在上皮间的浸润在UC组为52%, 正常组为78%, 两组相比差异显著(P<0.01); 急性期较缓解期也显著减少(20% vs 74%, P<0.01). CD8在UC患者急性期黏膜固有层的表达为80%, 高于缓解期的34%(P = 0.0006). Fas在UC上皮中表达62%, 正常组织30%, 两组相比差异显著(P<0.01); 急性期高于缓解期(84% vs 45%, P<0.01). FasL在固有膜炎性细胞中的表达UC组为62%, 正常组7%, 两组相比差异显著(P<0.01); 急性期(88%)高于缓解期(43%), 而且CD8与FasL在黏膜固有层炎性细胞的表达呈正相关(χ2 = 7.3, P<0.01). Bcl-2/Bax在UC肠黏膜上皮的表达率与正常组相近, 差异无显著性.
结论: UC肠黏膜组织Fas/FasL表达增强, Bcl-2/Bax表达无明显变化, CD8细胞与UC急性期Fas/FasL表达相关.
引文著录: 张姮, 夏冰, 杨桂芳, 李瑾. 溃疡性结肠炎肠黏膜组织CD8、Fas/FasL和Bcl-2/Bax的表达及关系. 世界华人消化杂志 2006; 14(18): 1795-1798
Revised: December 15, 2005
Accepted: December 31, 2005
Published online: June 28, 2006
AIM: To investigate the expression of Fas/FasL and Bcl-2/Bax and distribution of CD8 T cells as well as their correlations in ulcerative colitis (UC).
METHODS: Immunohistochemistry was used to detect the expression of CD8, Bcl-2/Bax and Fas/FasL in intestinal mucosal tissues from UC (n = 60) and normal controls (n = 60).
RESULTS: The positive rate of CD8 was significantly higher in the epithelia of UC tissues than that in the controls (52% vs 78%, P < 0.01), and rate in the active UC was also significantly lower than that in the remissive UC (20% vs 74%, P < 0.01). The positive rate of CD8 in the lamina propria tissues of UC at active stage was markedly higher than that at remissive stage (80% vs 34%, P = 0.0006). The expression of Fas was remarkably higher in the epithelia of UC tissues than that in the controls (62% vs 30%, P < 0.01), and its expression at active UC was also dramatically higher than that at remissive stage (84% vs 45%, P < 0.01). The expression of FasL was significantly increased in the inflammatory cells from the lamina propria of UC tissues as compared with that from normal mucosa (62% vs 7%, P < 0.01), and it was also a significant different between the active and remissive stage (88% vs 43%, P < 0.01). Furthermore, there was a correlation between the expression CD8 and FasL in the inflammatory cells from the lamina propria (χ2 = 7.3, P < 0.01). The expression of Bcl-2/Bax was not different between UC and normal mucosa (P > 0.05).
CONCLUSION: The expression of Fas/FasL is up-regulated in UC, but the expression of Bcl-2/Bax is not obviously changed. CD8 T cells play important roles in the development of UC and they are closely related with Fas/FasL system.
- Citation: Zhang H, Xia B, Yang GF, Li J. Distribution of CD8 T cells and expression of Fas/FasL and Bcl-2/Bax in ulcerative colitis. Shijie Huaren Xiaohua Zazhi 2006; 14(18): 1795-1798
- URL: https://www.wjgnet.com/1009-3079/full/v14/i18/1795.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v14.i18.1795
溃疡性结肠炎(ulcerative colitis, UC)是病因不明的结肠黏膜慢性炎症, 主要累及直肠和乙状结肠, 亦可累及全结肠. UC的病理表现有黏膜和黏膜下层的慢性炎性细胞浸润、隐窝脓肿、黏膜溃疡以及腺体和上皮的损伤. UC患者临床上有关节炎、硬化性胆管炎等免疫异常的肠外表现, 血清pANCA及抗结肠黏膜抗体阳性率增高, 肠黏膜T淋巴细胞激活以及Th1/Th2炎性细胞比例失衡, 以上观察和研究提示UC发病机制主要与T细胞免疫作用有关[1]. 研究表明细胞毒T细胞(CTL)在UC肠黏膜的损伤中起着重要作用[2-3]. CD8分子是CTL的重要表面标志[4], 其作用机制与Fas及穿孔素有关[5]. 近年发现UC上皮细胞损伤和黏膜溃疡与细胞凋亡有关[6], 而Bcl-2基因家族、Fas与其配体在细胞凋亡中起凋节作用. 有研究发现UC中Fas系统凋控的肠上皮细胞凋亡增加, 并参与上皮细胞损伤和炎症[7], 而Bcl-2/Bax系统可能不参与UC上皮细胞凋亡的凋控[8]. 至今尚未见CD8细胞与UC细胞凋亡相关性研究的报道. 我们探讨UC患者黏膜组织内CD8阳性细胞与细胞凋亡分子表达的关系, 研究Bcl-2/Bax及Fas/FasL二个细胞凋亡系统在UC发病机制中的作用.
1996/2000武汉大学中南医院门诊及住院部明确诊断的UC患者60例, 诊断标准参照中华医学会消化学分会2001年"对炎症性肠病诊断治疗规范的建议"[9]. 年龄13-62(平均39)岁, 病程1/12-25(平均10.5) a. 男34例, 女26例. 复习和阅读临床资料及病理切片, 根据组织学标准[10], 将UC分为活动期及缓解期. 活动期镜下表现为肠上皮及隐窝上皮变性、中性白细胞浸润、隐窝脓肿以及溃疡, 固有膜内弥漫性淋巴细胞、浆细胞、单核细胞浸润. 缓解期表现为黏膜面呈绒毛样外观, 隐窝变短、不规则、潘氏细胞化生, 隐窝基底部下方小淋巴细胞及浆细胞增多. 同时选取存档组织蜡块用于免疫组化测定. 对照组为正常肠黏膜60例, 取自结肠肿瘤10 cm以外的正常组织, 年龄为20-64(平均42)岁, 男37例, 女23例, 与UC组差别无统计学意义. CD8, Bcl-2, Bax鼠抗人mAb, Fas, FasL兔抗人多克隆抗体, 超敏SP(鼠/兔)试剂盒均购自福州迈新生物技术公司.
采用SP法进行免疫组化染色. 切片常规脱蜡水化, PBS冲洗, 热微波法修复10 min, 过氧化酶阻断溶液, 室温10 min, PBS冲洗; 非免疫性动物血清50 μL, 室温10 min, 去血清; 滴加一抗50 μL 4℃过夜; 生物素标记第二抗, 室温10 min; 链亲和素-过氧化物酶溶液, 室温10 min, PBS冲洗; DAB显色; 苏木素复染; 中性树胶封片, 阴性对照以PBS代替一抗作阴性对照. 购买福州迈新生物技术公司提供的阳性对照片作阳性对照, 每次实验均设对照组. CD8, Fas, Bcl-2定位表达于胞膜和胞质; FasL, Bax定位表达于细胞质. 着色强度评分标准: 无着色, (-), 为0分; 浅黄色, (+), 为1分; 棕黄色, (++), 为2分; 深棕色, (+++), 为3分. 阳性细胞数评分标准: 无细胞着色为0分; 阳性细胞数≤10%为1分; 11%-50%为2分; 51%-75%为3分; >75%为4分. 2次得分相乘>3者, 定为阳性病例. CD8阳性细胞数占炎性细胞的比例, 作为CD8阳性细胞数评分标准.
统计学处理 采用χ2和Fisher's exact检验, P<0.05为有显著性统计学意义.
CD8阳性染色定位于细胞膜和胞质. UC的肠上皮细胞间CD8阳性细胞总表达率低于正常对照组(52% vs 78%, P = 0.0038, OR = 3.382, 95%CI = 1.525-7.498), 且活动期UC低于缓解期UC (20% vs 74%, P<0.0001, OR = 0.0865, 95%CI = 0.025-0.299). 肠固有膜UC活动期的CD8阳性细胞表达率高于缓解期(80% vs 34%, P = 0.0006, OR = 7.667, 95%CI = 2.301-25.541). 肠上皮细胞间与肠固有膜UC的CD8阳性细胞表达率无显著性差别(52% vs 53%, P = 0.233, OR = 1.565, 95%CI = 0.800-3.061).
Fas阳性细胞染色定位于胞质和胞膜, FasL表达定位于胞质. UC肠上皮Fas表达(62%)明显高于正常肠上皮(30%), 且活动期(84%)高于缓解期(45%). UC及正常肠黏膜上皮几乎不表达FasL, 然而, Fas及FasL在UC及正常肠黏膜固有膜内炎性细胞均有表达. Fas阳性在UC及正常肠黏膜固有膜炎性细胞中表达无显著性差别(70% vs 83%, P = 0.13). 然而, UC肠黏膜固有层炎性细胞FasL的表达(62%)显著高于正常肠黏膜(7%), 且活动期(88%)高于缓解期(43%).
CD8与FasL在UC固有膜的表达如表1所示, 经χ2检验, 二者具有相关性.
| CD8 | FasL | |
| 阳性 | 阴性 | |
| 阳性 | 28 | 4 |
| 阴性 | 10 | 18 |
Bcl-2阳性细胞染色定位于胞质和胞膜, Bax表达定位于胞质. Bcl-2在UC病变肠黏膜表达率为60%, 与正常肠黏膜(67%)相比差异无显著性(P = 0.500), 且活动期与缓解期相近(64% vs 57%), Bax在UC肠黏膜与正常肠黏膜中的表达也无显著性差别(57% vs 58%, P = 0.50), 且活动期与缓解期相比差异无显著性(P = 0.63).
溃疡性结肠炎发病机制尚不明确, 目前认为细胞免疫起主要作用, T细胞是主要的免疫细胞, T细胞介导的免疫效应有迟发型超敏反应以及细胞毒淋巴细胞(CTL)介导的特异性细胞毒作用. CD8 T细胞亚群为职业性细胞毒细胞, 其作用机制与Fas及穿孔素有关, 以穿孔素-颗粒酶为基础的CTL杀伤作用, 必须直接接触靶细胞才能发挥细胞毒作用. 我们观察到UC组肠上皮CD8细胞数量较正常组明显减少且活动期减少较缓解期明显. 据此推测, UC患者急性期肠黏膜的破坏较少涉及到CTL的穿孔素一颗粒酶为基础的参与机制.
细胞凋亡是由基因控制的细胞自主性死亡方式, 是机体自身稳定的重要凋节机制. Bcl-2属原癌基因, 是研究最早的与细胞凋亡有关的分子. Bcl-2及Bcl-xL抑制caspase激活的细胞凋亡, 延长细胞存活时间. 相反, 凋亡前分子Bax则通过作用于线粒体, 凋节caspase活性, 以二聚体或多聚体形式与Bcl-2结合, 拮抗Bcl-2的作用. 一些研究发现Bcl-2与Bax, 这两种蛋白表达比例对控制细胞凋亡的强弱起到关键作用[11]. Bax增高, 促进细胞凋亡; Bcl-2增高, 抑制细胞凋亡. 日本Iimura et al[8]观察到UC肠黏膜中Bax蛋白及mRNA表达均下凋, 而Bcl-2与正常对照无明显差异, 提示Bax/Bcl-2系统不参与UC肠上皮细胞凋亡的凋控. 我们观察到Bcl-2、Bax表达在UC组与正常组肠黏膜上皮细胞的表达无差异, 支持上述结论. Fas、FasL是近年来认识较深入的与细胞凋亡信号传递有关的分子系统. Fas与其相应配体的相互作用是诱导细胞凋亡的重要途径之一[12]. Apo-1/Fas是一种跨膜糖蛋白分子, 是肿瘤坏死因子受体和神经生长因子受体家族的细胞表面分子. Fas配体是肿瘤坏死因子家族的细胞表面分子, FasL与其受体结合导致携带Fas的细胞凋亡[13]. 我们观察到UC肠上皮Fas表达明显高于正常肠上皮, 且活动期高于缓解期, UC组肠黏膜固有膜中Fas表达与正常组相比无明显差异, FasL在UC组及正常组织上皮细胞仅有少量表达, 而FasL在UC组固有膜中炎性细胞的表达明显高于正常组, 且与CD8的表达呈正相关, 并且FasL表达活动期高于缓解期, 推测FasL在UC的发病中作为病理因子而导致UC患者肠上皮细胞的损害, 与CD8有一定的关联. 基于本实验的结果, 我们认为Fas及FasL介导的细胞凋亡在UC的发病中起重要作用, 并且CD8细胞与Fas, FasL有关.
目前认为T细胞免疫在溃疡性结肠炎发病机制中起重要作用, UC肠黏膜的损伤与细胞毒T细胞(CTL)密切相关. CD8分子是CTL的重要表面标志, 其作用机制与Fas及穿孔素有关. 近年发现UC上皮细胞损伤和黏膜溃疡还与细胞凋亡有关. 有研究发现UC中Fas系统调控的肠上皮细胞凋亡增加, 并参与上皮细胞损伤和炎症, 而Bax/Bcl-2系统可能不参与UC上皮细胞凋亡的调控. 至今尚未见CD8细胞与UC细胞凋亡相关性研究的报道.
电编:张敏 编辑:潘伯荣
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