35-37 kDa形式可溶性MHC I释放机制研究

目的:探讨35-37 k Da形式的可溶性MHC I释放机制,为开展造血系统恶性肿瘤免疫干预治疗研究奠定理论基础。方法:以细胞表面标记、免疫沉淀、免疫印迹和增强化学发光法探讨EGTA和蔗糖对THP1细胞释放43和35-37 k Da可溶性MHC I的影响;以超速离心法纯化外排小体,并用免疫沉淀、免疫印迹和增强化学发光法检测43和35-37 k Da可溶性MHC I;用Quantity One软件对43和35-37 k Da可溶性MHC I进行相对定量分析。结果:EGTA同时显著抑制43和35-37 k Da可溶性MHC I产生;蔗糖同时显著促进43和35-37 k Da可溶性MHC I产生;43 k Da可溶性MHC I存在于外排小体上,而35-37 k Da可溶性MHC I在外排小体上检测不到。结论:35-37 k Da可溶性MHC I与外排小体都来源于细胞内多泡小体同质膜的溶合后释放,但35-37 k Da可溶性MHC I并不包含在外排小体的泡囊中,而是独立于外排小体释放。

Release Mechanismof 35-37 kDa of Soluble MHC I

Objective:To investigate the release mechanism of 35-37 kDa of soluble MHC I and to lay a foundation for theimmune intervention of hematopoietic malignancies.Methods:Cell surface labeling, immunoprecipitation, Western blot and enhancedchemiluminescence methods were used to investigate the effect of EGTA and sucrose on the release of 43 and 35-37 kDa of solubleMHC I in THP1 cells respectively. Exosomes were separated from the supernatant of cell culture by ultracentrifugation, and then solubleMHC I was detected with immunoprecipitation, Western blot and enhanced chemiluminescence methods. The quantity of soluble MHC Irelease was analyzed with Quantity One software after chemiluminescence.Results:EGTA could inhibit the release of 43 and 35-37 kDaof soluble MHC I simultaneously, and sucrose could increase the release of 43 and 35-37 kDa of soluble MHC I simultaneously. No35-37 kDa of soluble MHC I was found in exosomes, contrary to 43 kDa of soluble MHC I.Conclusion:Both 35-37 kDa of solubleMHC I and exosomes were released by fusing of multivesicular bodies with plasma membrane, but while being released, 35-37 kDa ofsoluble MHC I was independent of exosomes, while 43 kDa of soluble MHC I was located in exosomes and released with exosomes.