肥大细胞激活及组织胺水平测定

Challenge of Mast Cell and Measurement of Histamine

目的 建立肥大细胞激活及组织胺测量的实验研究系统。方法 将手术切除的人类肺、皮肤、扁桃体及关节滑膜组织剪碎后 ,在 37℃条件下用 型胶原酶和 型透明质酸酶进行消化。经洗净及肥大细胞计数后 ,悬浮的细胞被均匀地加入含刺激剂、抑制剂或缓冲液的试管中 ,并在 37℃条件下培养 15分钟 ,用冷 HBSS缓冲液及离心方法终止上述反应 ,取上清液置 - 37℃下保存。用与组织胺特异结合的玻璃纤维为基础的荧光显色法测量组织胺。结果 肺、皮肤、扁桃体和关节滑膜组织的肥大细胞分别占相应组织悬浮细胞总数的 4 %、5 %、0 .5 %和4 %左右。各组织中肥大细胞自发性组织胺释放量均占其组织胺总量的 9%以内。绵羊抗人 Ig E和钙离子载体的最佳浓度分别是 1%和 1μmol/ L,其分别能引起高达 19%和 5 6 %的肥大细胞组织胺释放。肥大细胞类胰蛋白酶抑制剂亮肽素 (亮肽素 )能抑制 Ig E依赖性肺及扁桃体肥大细胞的激活。结论 酶消化法分离肥大细胞为肥大细胞激发试验提供了快速、重复性好的试验基础。以玻璃纤维特异性结合组织胺为基础的组织胺测量方法重复性好、样品用量小 ,节省人力及财力。绵羊抗人 Ig E和钙离子载体均为肥大细胞的有效激活剂。亮肽素能抑制 Ig

Objective To establish a system for mast cell study including dispersion and challenge of mast cell and measurement of histamine. Methods Small tissue fragments cut from lung, skin, tonsil and synovium were digested with collagenase and hyaluronidase. Cell suspension was then divided into each tube containing stimulus, inhibitor or buffer alone. The reactions in tubes were terminated by addition of cold HBSS and immediate centrifugation. Histamine was measured by using a glass fiber based fluorescence assay. Results Mast cells consist of 4.0%, 5.0%, 0.5% and 4.0% of total dispersed cells from lung, skin, tonsil and synovial tissues, respectively. Spontaneous histamine release from the above four tissues was lower than 9% of total histamine. The optimum concentrations of anti histamine or calcium ionophore were 1% or 1 μmol/L, which could induce 19% or 56% histamine release respectively. An inhibitor of tryptase, leupeptine was able to inhibit IgE dependent tonsil mast cell activation. Conclusion Establishment of a mast cell activation system will contribute greatly to investigation of mechanisms of mast cell activation and discovery of novel mast cell stabilizers.