大鼠肠系膜淋巴结和外周淋巴结固有exosome的制备及其对T细胞增殖的影响

Purification of exosomes from rat mesenteric and peripheral lymph nodes and their effect on T cell proliferation

目的从大鼠肠系膜淋巴结、外周淋巴结中分离纯化exosome,鉴定其对T细胞增殖的影响。方法取出BN(RT1n)大鼠的外周淋巴结及肠系膜淋巴结并研磨、离心以获得非细胞组织上清,采用超滤密度梯度离心法提取exosome。透射电镜对收集的exosome进行形态学鉴定,BCA法进行蛋白定量,SDS-PAGE电泳分离蛋白,Western blot检测IL-10的表达,并通过混合淋巴细胞培养检测其对T细胞增殖的影响。结果两种淋巴组织来源的非细胞组织上清均含有exosome,肠系膜淋巴结源性exosome蛋白浓度[(1.92±0.10)μg/μL]显著高于外周淋巴结源性exosome[(1.57±0.06)μg/μL,P<0.05],SDS-PAGE显示两种组织源性exosome所含蛋白种类和数量有明显差异,Western blot显示肠系膜淋巴结固有exosome中IL-10的表达显著高于外周淋巴结固有exosome,混合淋巴细胞培养结果显示肠系膜淋巴结固有exosome和外周淋巴结固有exosome对T细胞的增殖均有促进作用(P<0.05),外周淋巴结源性exosome的促增殖作用显著大于肠系膜淋巴结源性exosome(P<0.05)。结论肠系膜淋巴结、外周淋巴结均含有组织固有exosome,两种组织来源exosome有蛋白浓度和蛋白种类的差异,且均能促进淋巴细胞增殖,促进程度有显著差异。

tissues of rats and d Objective To investigate the purification methods of the exosomes derived form lymphoid etermine their effect on T cell proliferation. Methods mesenteric lymph nodes from BN （ RT1 n） rats were separated, and their impun Then, the exosomes were purified from the acellular supernatants by means o The peripheral lymph nodes and ty were removed by centrifugation. ff differential uhracentrifugation or uhrafiltration and sucrose gradient centrifugation, and confirmed by transmission electron microscopy. The protein expression of the exosomes was analyzed by SDS-PAGE eleetrophoresis. Western blot analysis was used to test the expression of IL-10. Finally, mixed lymphocyte culture was used to evaluate the immune function of the exosomes from peripheral lymph nodes and mesenteric lymph nodes. Results The protein concentration of the exosomes purified from mesenteric lymph nodes were higher than that from peripheral lymph nodes （ 1.92±0.10 vs 1.57 ± 0.06 μg/μL, between the exosomes from 2 stronger in the exosomes from P 〈 0. 05）. SDS-PAGE displayed the kinds of lymph nodes. Western blotting mesenteric lymph nodes than those from differences in protein contents and types indicated that the expression of IL-10 was peripheral lymph nodes. The two kinds of exosomes both promoted lymphocyte proliferation （P 〈 0. 05 ）, while the exosomes from peripheral lymph nodes exosomes stronger than from mesenteric lymph nodes （P 〈 0. 05 ）. Conclusion There are exosomes in both peripheral and mesenteric lymph nodes, but with significant differences in protein contents and types. The exosomes from 2 kinds of lymph nodes promote lymphocyte proliferation, though with significant differences.