Received 2002-04-22 Accepted 2002-06-26
This work was supported by the National Natural Science Foundation of China (No.30140023).
*Corresponding author. Tel: +86-754-8900405; Fax: +86-754-8900192:
E-mail: shaohenghe@hotmail.com
**Now an attending physician at the General Hospital of Shengyang Military Region.
生理学报, Dec. 2002, 54 (6): 531-534
Acta Physiologica Sinica
研究论文
蛋白酶激活受体2(PAR-2)激动剂对肥大细胞释放
类胰蛋白酶的影响
何韶衡1,*, 谢 华1,**, 何永松2
1汕头大学医学院变态反应学与炎症学研究所, 汕头 515031;
2四川大学华西医学院检验系, 成都 610044
摘 要: 研究反肉桂酰-亮-异亮-甘-精-亮-鸟-[酰胺] (tc-LIGRLO), 一种PAR-2激动剂, 对肥大细胞类胰蛋白酶释放的影响。结果显示, 经过15 min的培养, tc-LIGRLO可引起比基础分泌量增加1倍以上的类胰蛋白酶释放, 作用强度超过抗IgE抗体和钙离子导入剂 (calcium ionophore A23187, CI)。而反PAR-2激动剂反肉桂酰-鸟-亮-精-甘-异亮-亮-[酰胺] (tc-OLRGIL)无此作用。培养时间延长到30 min时对tc-LIGRLO的作用无明显影响。其时间关系曲线表明, tc-LIGRLO的作用从1 min开始, 3 min后达高峰。结果表明, PAR-2激动剂tc-LIGRLO是一种高效类胰蛋白酶释放刺激剂, 在肥大细胞上可能有PAR-2存在。
关键词: 蛋白酶激活受体2; 类胰蛋白酶; 肥大细胞
中图分类号: R33
Effect of a
proteinase-activated receptor-2 (PAR-2) agonist on tryptase release from human mast cells
HE Shao-Heng1, , XIE Hua1, **, HE Yong-Song2
1Allergy and Inflammation Research Institute, the Medical College of Shantou University, Shantou 515031;2Laboratory Medicine Department of Western China Medical College, Sichuan University, Chengdou 610044
Abstract: Proteinase-activated receptor-2 (PAR-2) expression has been observed on numerous cell types. However, little is known about the functional expression of PAR-2 in human mast cells. In the current study, the actions of a PAR-2 agonist trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (tc-LIGRLO) on tryptase release from dispersed human colonic mast cells were examined. The results showed that tc-LIGRLO was able to induce a fold increase in tryptase release over the basal level following a 15 min incubation of colonic mast cells, whereas tc-OLRGIL did not have any effect on tryptase release. The potency of tc-LIGRLO appeared greater than that of anti-IgE and calcium ionophore A23187 (CI) in induction of tryptase release. Extending the incubation time to 30 min had no significant effect on the actions of tc-LIGRLO or anti-IgE. In the time course study, it was observed that the tryptase release from mast cells induced by tc-LIGRLO started at 1 min and peaked at 3 min following incubation. The above-mentioned results indicate that tc-LIGRLO is a potent stimulus of tryptase release from human mast cells, which strongly suggests that PAR-2s are expressed in human mast cells.
Key words: proteinase activated receptor-2; tryptase; mast cell
在人类, 组胺、类胰蛋白酶、类糜蛋白酶、肝素等是肥大细胞产生的主要分泌性介质, 其中以类胰蛋白酶的含量为最大, 约占肥大细胞分泌颗粒中蛋白质总量的一半[1]。 近年来, 人们先后发现类胰蛋白酶具有增加血管通透性[2]、诱导炎性细胞浸润[3]、刺激上皮细胞增生并释放IL-8[4]等功能, 提示在某些细胞上可能有它的受体存在。最近, 鉴定出一组蛋白酶激活受体 (proteinase activated receptors; PARs)[5], 其中PAR-2被认为是类胰蛋白酶的受体[6, 7], 在大鼠结肠细胞[8]、人类角化细胞[9]、人类冠状动脉的血管内皮细胞[10]和人类支气管平滑肌细胞[11]上均有表达。现已证实, 以多肽丝-亮-异亮-甘-赖-丙 (SLIGKV)为代表的PAR-2激动剂具有激活人类中性粒细胞[12]和大鼠腹腔肥大细胞[13]的能力, 因此这类多肽应当具有激活人类肥大细胞的能力。本文利用人类大肠组织的肥大细胞和肥大细胞激活的体外研究系统来评价PAR-2激动剂tc-LIGRLO对肥大细胞的激活作用。
1 材料和方法
1.1 材料
Ⅰ型胶原酶、Ⅰ型透明质酸酶、含25 mmol/L HEPES的MEM、绵羊抗鼠免疫球蛋白(calcium ionophore A23187, CI)、extr-抗生物素蛋白过氧化物酶和邻苯二胺均购于Sigma 公司。山羊抗人IgE抗体购于Serotec (Kidlington, Oxford, UK)。 类胰蛋白酶特异性抗体由Mark G. Buckley 博士 (University of Southampton, UK)馈赠。 tc-LIGRLO和 tc-OLRGIL购于美联 (西安)生物科技有限公司。
1.2 肥大细胞悬浮
详细方法见参考文献[14]。 手术切除的大肠组织被切成小块后放入含Ⅰ型胶原酶和Ⅰ型透明质酸酶的消化液中, 在37℃摇动水浴箱中孵育70 min。悬浮的细胞通过滤过器 (孔径100 μm)与未消化的组织碎片分离, 然后放入含10%小牛血清的MEM培养液中于室温下培养16 h。肥大细胞经甲苯胺蓝染色后在血细胞计数器上计数。大肠细胞悬液中肥大细胞的纯度为4%-5%。
1.3 肥大细胞激发试验
详细方法参阅参考文献[14]。 悬浮细胞经不含钙、镁离子的HBSS (HEPES缓冲盐溶液) (不全HBSS)冲洗2次后, 再重新悬浮于含钙、镁离子的HBSS (全HBSS)中。激发过程在LP4试管中完成, 即在预先置有50 μl tc-LIGRLO、tc-OLRGIL或全HBSS的试管中加入100 μl细胞悬液, 并在37℃水浴箱中孵育15 min。激发反应由向置于冰上的试管中立即加入150 μl冰冷的不全HBSS, 并立即离心而终止。上清液与细胞分离后于－20℃冻存。
1.4 类胰蛋白酶水平测定
采用ELISA法[15], 即以抗类胰蛋白酶抗血清作为I抗铺盘, 4℃过夜。用3% BSA阻断非特异性蛋白质结合位点后, 依次加入类胰蛋白酶标准品或待测样品 (每孔50 μl)、抗类胰蛋白酶鼠单克隆抗体、生物素化的羊抗鼠免疫球蛋白 (为Ⅱ抗)及extr-抗生物素蛋白过氧化物酶。最后用邻苯二胺显色以酶标仪于波长490 nm测定光吸收 (A值)。
1.5 类胰蛋白酶释放量占最大释放量的百分率
分别测量不同时间点的类胰蛋白酶释放量, 并设最大类胰蛋白酶释放量为100% (本试验在10 min时)。类胰蛋白酶释放量占最大释放量的百分率为实际量/最大释放量×100%。
1.6 统计学
全部统计分析均采用SPSS (Version 10.0)。 数据以mean±SEM表示。配对t检验用于组间成对数据的比较, P<0.05为统计学上有显著差异。
2 结果
经过15 min的培养, tc-LIGRLO可引起比基础分泌量增加1倍以上的类胰蛋白酶释放, 作用强度超过抗IgE抗体和CI。但在试验浓度为1-300 μmol/L时, 未形成明显的浓度关系曲线。
图 1. tc-LIGRLO、tc-OLRGIL、抗IgE抗体和钙离子导入剂(CI)对肥大细胞类胰蛋白酶释放的影响
Fig. 1. Effects of tc-LIGRLO, tc-OLRGIL, anti-IgE and CI on the release of tryptase from mast cells. The values shown are mean±SEM of four separate experiments performed in duplicate. Stimulus or control was incubated with cells for 15 min before termination of the reactions. *P<0.05 compared with complete HBSS alone group (paired student's t test).
试验浓度降低到0.01和0.1 μmol/L时, tc-LIGRLO不能诱导类胰蛋白酶释放增加。反PAR-2激动剂tc-OLRGIL在试验浓度为1-300 μmol/L时对类胰蛋白酶释放无影响 (图1)。本培养时间延长到 30 min 时, 除 CI引起的类胰蛋白酶释放量减少外, 对其它3种刺激物的作用无明显影响 (表1)。时间关系曲线显示, tc-LIGRLO的作用从加样后1 min开始, 3 min后达高峰并至少持续10 min (图2)。
图 2. tc-LIGRLO引起类胰蛋白酶释放的时间过程
Fig. 2. Time course that tc-LIGRLO induced tryptase release. Data shown are mean±SEM of four separate experiments performed in duplicate.
表 1. 培养30 min后, tc-LIGRLO、tc-OLRGIL、抗IgE抗体和CI对肥大细胞类胰蛋白酶释放的影响
Table 1. Effects of tc-LIGRLO, tc-OLRGIL, anti-IgE and CI on tryptase release from colonic mast cells following 30 min incubation
|
Concentration of stimulus |
Tryptase release (ng/ml) |
|
Complete HBSS |
8.2±0.6 |
|
tc-LIGRLO 300 μmol/L |
18±2.7* |
|
tc-OLRGIL 300 μmol/L |
8.4±2.8 |
|
Anti-IgE 1% |
17±5.3* |
|
CI 1 μmol/L |
13±0.6* |
The values shown are mean±SEM of four separate experiments. *P<0.05 compared with complete HBSS alone group (paired Student's t test). HBSS, HEPES buffered salt solution.
3 讨论
应用肥大细胞悬浮、激活和类胰蛋白酶测量的实验系统, 本文发现, tc-LIGRLO在浓度为1-300 μmol/L的范围内具有诱导大肠肥大细胞释放类胰蛋白酶的作用, 而且其作用强度超过抗IgE抗体和CI。由于tc-OLRGIL (一种用作tc-LIGRLO阴性对照的分子量和氨基酸组成与tc-LIGRLO完全相同的, 但氨基酸顺序不同的多肽)在同一试验条件下无诱导大肠肥大细胞释放类胰蛋白酶的作用, 所以进一步证明了tc-LIGRLO作用的特异性和可靠性。
同抗IgE一样, tc-LIGRLO的作用可持续至少30 min, 而CI刺激类胰蛋白酶的作用则在15-30 min之间消减了大约35%。CI的这种作用消减现象在以组胺为肥大细胞激活标记物的试验系统中未曾发现[14]。tc-LIGRLO的作用从加样后1 min开始, 3 min后达高峰并至少持续10 min的发现很有趣, 因为它作用达高峰的时间正巧介于神经介质P物质[16]和免疫激活剂抗IgE抗体[14]之间, 提示这种小肽类物质可能会具有某些神经介质的特性。
PAR-2激动剂不仅能够激活肥大细胞, 而且还能刺激皮肤角化细胞分泌IL-8[17]、气道上皮细胞释放基质金属蛋白酶9[18]、抑制支气管上皮细胞的离子转移[19]、刺激人类血管内皮细胞的有丝分裂[20]和血管平滑细胞增生[21]、激活人类主动脉平滑肌细胞[22]。近来的研究还表明, PAR-2缺陷鼠与野生型对照鼠相比, 前者的外科创伤后炎症发生较迟[23], 说明PAR-2参与创伤性炎症的发生过程。PAR-2激动剂的上述功能以及PAR-2所具有的介导胰蛋白酶引起嗜酸性粒细胞激活[24]的作用, 表明PAR-2还参与变态反应性炎症的发生过程。
总之, tc-LIGRLO具有的激活肥大细胞的作用, 间接证明了在肥大细胞上有PAR-2的存在。这进一步完善了我们先前提出的, 肥大细胞具有通过类胰蛋白酶对激活的信号进行自我放大的能力的假说。
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