人原代肥大细胞体外诱导和组织分离方法的建立

目的: 建立获取和培养人原代肥大细胞的方法,为探讨肥大细胞的免疫调节作用及其机制奠定基础。方法: 用重组人源干细胞因子、IL-6和IL33刺激CD34+造血干细胞使之分化成肥大细胞;Percoll密度梯度离心结合免疫磁珠分选法,分离肠道黏膜肥大细胞。应用免疫荧光染色技术分析所获得的原代肥大细胞表面分子CD117和FcεRⅠ,甲苯胺蓝染色或间接免疫荧光染色检测胞内类胰蛋白酶含量。结果: 黏膜肥大细胞及CD34+造血干细胞刺激分化而来的肥大细胞均表达CD117和FcεRⅠ,胞内均含有类胰蛋白酶。结论: 这两种方法均可以有效地获得大量纯化的原代肥大细胞,且各有优势,所得细胞适用于肥大细胞在机体免疫调节和病原防御等生理功能的后续研究。

Establishment of techniques for generation in vitro and isolationex vivo for human primary mast cells

Objective: To establish the technique for generation or isolation of human primary mast cells,and for further studying their roles in regulating host immunity and elucidating immunopathogenesis. Methods: CD34+hemopoietic stem cells were cultured in presence of stem cell factor, IL 6, and IL 3 to generate primary mast cells.Mast cells allocated in human intestinal tissues were isolated through digestion, density gradient centrifugation and purification with specific antibodies coated magnetic beads. Cell phenotype was monitored by immuostaining of surface markers of CD117 and FcεRⅠ and intracellular tryptase, or cells were identified with Toluidine blue staining. Results: Both of the CD34+cell derived and intestinal mast cells expressed CD117 and FcεRⅠ， the specific markers for mast cells, and contained intracellular tryptase. Conclusion: Both methods could provide sufficient and purified primary mast cells for further study in regulating host immune response and defence against pathogenic microorganism.