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摘要： 目的：探讨兔房水中是否能够分离得到具有脂质双分子层结构的纳米级小囊泡exosomes，并检测其是否表达免疫抑制相关分子，参与眼球免疫耐受状态的维持。 方法：收集40只健康新西兰大白兔的房水，分级分离，超速离心制备exosomes，透视电镜对其形态进行鉴定；Western blot检测exosomes标志性蛋白以及免疫抑制相关分子TGF-β的表达情况；采用CCK-8细胞增殖检测方法判断兔房水来源的exosomes对经ConA诱导的兔子T淋巴细胞增殖情况的抑制功能。 结果：40只新西兰大白兔收集到约8 ml的房水，从中获得200 μg exosomes。经透射电镜鉴定：从房水中分离获得的exosomes直径主要集中在50~100 nm，具有典型的脂质双分子层结构；Wester blot结果表明，该exosomes表达热休克蛋白Hsp70、四次跨膜蛋白CD9以及内体相关蛋白Alix，不表达内质网蛋白Grp94，具有典型的exosomes蛋白表达谱。此外，该exosomes还表达免疫抑制相关分子TGF-β。体外抑制淋巴细胞增殖实验表明，房水来源的exosomes能够明显抑制T淋巴细胞的增殖。 结论：兔房水中能够分离出具有免疫抑制功能的exosomes；该exosomes很有可能参与了眼球免疫耐受的维持。
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| 关键词：
眼房水/免疫学； 纳米粒子； 转化生长因子β
/药理学； 免疫耐受； 免疫抑制法； T淋巴细胞
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Abstract: Objective： To isolate exosomes from rabbit aqueous humor and to investigate their immunosuppression function. Methods： Aqueous humor was collected from 40 New Zealand rabbits and exosomes were isolatied by fractional separation and ultracentrifugation methods; the morphology was studied with electron microscopy.The immunosuppressive-related proteins of exosomes were detected with Western blotting; their inhibitory effect on ConA-induced proliferation of T lymphocyte was estimation with CCK-8 cells proliferation assay. Results： Eight mililiters of aqueous humor were collected from 40 New Zealand rabbits and 200 μg exosomes was yielded.Under electron microscope，the exosomes had typical structure of lipid bi-layer with a diameter of 50-100 nm.The results of Western bloting showed that these exosomes expressed Hsp70，CD9 and Alix but not Grp94，presenting a typical exosomes protein profile.Moreover，exosomes expressed high level of TGF-β and significantly inhibited the proliferation of T lymphocytes. Conclusions： Immunosuppressive exosomes can be isolated from rabbit aqueous humor，which may be involved in immunotolerance of the eye. |
| Key words:
Aqueous humor/immunology
Nanoparticles
Transforming growth factor beta/pharmacology
Immune tolerance
Immunosuppression
T-lymphocytes
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出版日期: 2012-05-25
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