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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 E依赖性肥大细胞的激活
Objective To establish an experimental research system of mast cell activation and histamine measurement. Methods The resected human lung, skin, tonsil and synovial tissue were dissected and digested with collagenase and hyaluronidase at 37 ℃. After washing and mast cell counting, the suspended cells are homogenized in tubes containing stimulant, inhibitor or buffer and incubated for 15 minutes at 37 ° C. The reaction is stopped by cold HBSS buffer and centrifugation , Take the supernatant set - stored at 37 ℃. Histamine was measured by fluorescence spectroscopy based on glass fibers specific for histamine. Results Mast cells in lung, skin, tonsil and synovial tissues accounted for about 4%, 5%, 0.5% and 4% of the total number of suspension cells in the corresponding tissues respectively. The amount of spontaneous histamine release from mast cells in each tissue accounted for less than 9% of its total histamine. Optimal concentrations of sheep anti-human IgE and calcium ionophores were 1% and 1 [mu] mol / L, respectively, which induced up to 19% and 56% of the mast cell histamine release, respectively. Mast cell tryptase inhibitor leupeptin (leupeptin) inhibits IgE-dependent lung and tonsil mast cell activation. Conclusions Enzymatic digestion of mast cells provides a rapid and reproducible experimental basis for mast cell activation assays. The histamine measurement method based on glass fiber-specific histamine binding has good reproducibility, small sample dosage, and saves manpower and financial resources. Sheep anti-human Ig E and calcium ionophore are both potent activators of mast cells. Leupeptin inhibits IgE-dependent mast cell activation