髓源树突细胞源性外泌体对实验性自身免疫性重症肌无力大鼠NK及NK T细胞的影响

The effects of exosomes derived from bone marrow dendritic cells on NK and NK T cells of experimental autoimmune myasthenia gravis rats

目的探讨他汀类药物诱导髓源树突细胞(BMDCs)源性外泌体(exosomes)对实验性自身免疫性重症肌无力(EAMG)大鼠自然杀伤细胞(NK)及NK T细胞的影响。方法阿托伐他汀和DMSO分别与BMDCs共培养,采用流式细胞术分析其表型。应用梯度离心法提取不同组BMDCs分泌的外泌体(分别记为他汀Dex和对照Dex),将不同组外泌体注射于EAMG大鼠体内,采用双盲法观察EAMG临床症状;通过流式细胞术检测各组大鼠淋巴结NK、NK T和干扰素γ(IFN-γ)阳性细胞、白细胞介素-10(IL-10)阳性细胞的表达水平;采用ELISA方法检测各组血清抗R97-116IgG抗体及其亚型水平。结果他汀类药物能够明显抑制BMDCs表面CD80和CD86的表达水平(CD80:1.10%比22.80%,P<0.01;CD86:30.78%比43.93%,P<0.01),这些BMDCs可进一步分泌他汀Dex。与对照Dex治疗组比较,他汀Dex治疗组EAMG大鼠淋巴结NK细胞比例增加(2.30%比1.63%,P<0.05),淋巴结单个核细胞(MNC)中IFN-γ+细胞比例(1.05%比2.24%,P<0.05)、血清抗R97-116IgG抗体及其亚型IgG2a和IgG2b水平均明显降低(IgG:0.44比0.64,IgG2a:0.26比0.35,IgG2b:1.47比1.94;均P<0.05)。结论他汀Dex可缓解EAMG大鼠的临床症状,这种作用与上调淋巴结MNC中的NK细胞有关。

Objective To investigate the effects of exosomes derived from statin-modified bone marrow dendritic cells(BMDCs)on NK and NKT cells of experimental autoimmune myasthenia gravis(EAMG)rats.Methods BMDCs were incubated with atorvastatin or DMSO.For phenotypic analysis,statin-BMDCs and control-BMDCs were examined by flow cytometry.Exosomes were prepared from the cell culture supernatants of statin-BMDCs and control-BMDCs by differential centrifugation(regarded as statin-Dex or control-Dex,respectively).Then,EAMG rats were injected intravenously with statin-Dex or control-Dex,respectively.The severity of the disease was scored by measuring muscular weakness in a blinded fashion.The expressions of NK,NKT cells,IFN-γ+cells,and IL-10+cells were examined by flow cytometry.The levels of anti-R97-116 IgG antibody and its subtypes were detected by ELISA.Results The phenotypic analysis of BMDCs showed that the expression of CD80 and CD86 were inhibited on statin-BMDCs when compared with control-BMDCs(CD80:1.10%vs.22.80%,P<0.01;CD86:30.78%vs.43.93%,P<0.01).Exosomes derived from statin-BMDCs were regarded as statin-Dex.Statin-Dex treatment increased the percentage of NK cells in lymph node mononuclear cells(MNC)when compared with those in the control-Dex group(2.30%vs.1.63%,P<0.05).Statin-Dex treatment decreased the percentage of IFN-γ+cells in lymph node MNC when compared with those in the control-Dex group(1.05%vs.2.24%,P<0.05).Rats treated with statin-Dex had lower levels of serum anti-R97-116 IgG,IgG2a,and IgG2b antibodies compared with control-Dex group rats(IgG:0.44 vs.0.64,IgG2a:0.26 vs.0.35,IgG2b:1.47 vs.1.94;P<0.05).Conclusions The rats in the statin-Dex group exhibited milder clinical symptoms when compared with rats in the control-Dex group,which was associated with the increased number of NK cells in lymph node MNC.