类糜蛋白酶抑制剂对人大肠肥大细胞类胰蛋白酶释放的影响

Effect of chymase inhibitors on tryptase release from human colon mast cells

目的:研究类糜蛋白酶抑制剂对人大肠肥大细胞释放类胰蛋白酶的影响。方法:人大肠组织经酶消化后,细胞成份有全HBSS重新悬浮。激发过程在LP4试管中、37℃条件下完成。类胰蛋白酶水平用酶联免疫吸附试验(ELISA)方法测定。结果:促分泌剂抗IgE抗体和CI在培养15分钟和35分钟时可明显刺激人大肠肥大细胞类胰蛋白酶的释放,在相同实验体系中,类糜蛋白酶抑制剂ZIGPFM、TPCK和α1抗胰蛋白酶无明显刺激人大肠肥大细胞释放类胰蛋白酶的作用。类糜蛋白酶抑制剂均可以剂量依赖性的方式抑制抗IgE抗体诱导的类胰蛋白酶的释放,最大浓度的ZIGPFM(1μmolml)、TPCK(80μmolml)和α1抗胰蛋白酶(30μmolml)可分别抑制37%、40%和36.6%的类胰蛋白酶释放。在37℃条件下同大肠细胞预培养20分钟与无预培养相比,ZIGPFM和TPCK对抗IgE抗体诱导的类胰蛋白酶释放的抑制作用略增强。Amastatin对抗IgE抗体诱导的类胰蛋白酶的释放无作用。类糜蛋白酶抑制剂均可以剂量依赖性的方式抑制CI诱导的类胰蛋白酶的释放,抑制范围在23%～35.3%。在37℃条件下同大肠细胞预培养20分钟与无预培养相比,ZIGPFM对CI诱导的类胰蛋白酶释放的抑制作用有增强,TPCK则无此特点。结论:类糜蛋白酶抑制剂可抑制人大肠肥大细胞IgE依赖性和非依赖性类胰蛋白酶的释放,提示类糜蛋白酶抑制剂可望成为炎症性肠病或其它肥大细胞相关疾病的一个新的治疗途径。

Objective：To investigate the ability of chymase inhibitors on tryptase release from human colon mast cells. Methods：Human mast cells were dispersed from colon tissue with collagenase and hyaluronidase, and were challenged with stimulus for 15 min at 37℃. Tryptase assay performed following previous procedures. In brief, a 96-well microfitre plate was coated with antiserum to human tryptase. The tryptase levels in the samples were detected with a monoelonal antibody specific to tryptase and the reaction was visualized by addition of OPD. Results：At 15 min and 35 min following incubation,anti-IgE and calcium ionophore were able to provoke significant tryptase release from human colon mast cells. Chymase inhibitors ZIGPFM, TPCK and α1-antitrypsin had no sfimulatory effect on colon mast cells at both 15 min and 35 min incubation periods. All the chymase inhibitors were able to inhibit anti-IgE induced tryptase release in a concentration dependent manner with a maximum of 37 %, 40 % and 36.6 % inhibition being achieved with 1 μmol/mL of ZIGPFM, 80 μmol/mL of TPCK, 30 μmol/mL of α1-anfitrypsin,respeetively. Preincubation of inhibitors of ZIGPFM and TPCK with cells for 20 min at 37℃ before challenging with anfi-IgE was able to slightly enhance their inhibitory actions. Amastatin, a specific inhibitor of aminopeptidase, had no effect on anti-IgE induced tryptase release. All the chymase inhibitors were able to inhibit calcium ionophore induced tryptase release, the maximum inhibition were 23%-35.3%. And the extent of inhibition by ZIGPFM was increased when colon mast cells were preincubated for 20 min before calcium ionophore being added. However,the same treament failed to improve the action of TPCK. Conclusion： We found for the first time that inhibitors of chymase were able to inhibit anti-IgE and calcium ionophore induced tryptase release from human colon mast cells,which may indicated a potential of a novel therapy for the treatment of inflammatory bowel disease or other mast cell related diseases.