人源化异种肝组织移植小鼠模型中调节性T淋巴细胞归巢的成像研究

Imaging study on the homing of regulatory T cells in humanized heterologous liver tissue transplantation mouse model

目的探讨DiR荧光染料标记的调节性T淋巴细胞(Tregs)在异种肝组织移植小鼠体内的归巢特征.方法检测不同温育时间和不同质量浓度DiR染料标记的Tregs荧光强度,结合新型四唑化合物(MTS)实验检测细胞活力,从而确定最佳温育时间及染料质量浓度.采用流式细胞术验证DiR染料对Tregs功能及表型的影响.构建异种肝组织移植小鼠模型并免疫重建,回输Tregs后不同时间点进行小动物活体荧光成像.通过免疫组织化学分析免疫重建以及体内淋巴细胞分布情况.多组间比较采用单因素方差分析和Dunnett-t检验.结果随着DiR浓度和温育时间增加,Tregs荧光强度增强,3d达到高峰后逐渐减弱,5.00μg/ml及20.00μg/ml组相比对照组在各时间点的细胞活力显著下降(F=120.142~182.025,t=9.969~19.329,均P<0.05),温育30min及60min后,Tregs活力相较对照组也明显下降(F=21.826~301.968,t=6.897~40.016,均P<0.05),结合MTS实验结果得出最优标记条件为2.50μg/mlDiR染料温育5min.免疫表型流式细胞术检测表明DiR染料不影响Tregs表型及功能.小动物荧光成像系统检测到Tregs可以汇聚于移植物区域.免疫组织化学分析结果表明Tregs能改善免疫重建后诱导的淋巴细胞浸润情况.结论荧光成像可直接观察DiR染料标记的Tregs在小鼠体内的分布,是一种有前景的Tregs示踪的影像学手段.

Objective To explore the homing of DiR labeled regulatory T cells(Tregs)in humanized heterologous liver tissue transplantation mouse model.Methods The fluorescence intensities of Tregs labeled with different concentrations of DiR dye and different incubation times were measured,and the cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assay to determine the optimal incubation time and dye concentration.The effect of DiR dye on the function and phenotype of Tregs was verified by flow cytometry.The xenogeneic liver tissue transplantation mouse model was constructed and the immune system was reconstituted.Small animal fluorescence imaging was performed at different time points after infusion of Tregs.Immunohistochemistry analysis was used to analyze immune reconstitution and lymphocyte distribution in vivo.One-way analysis of variance and Dunnett-t test were used to analyze the data.Results With the increase of DiR concentration and incubation time,the fluorescence intensity of Tregs increased and gradually weakened after reaching the peak at 3 d.The cell viability of the 5.00μg/ml and 20.00μg/ml groups was significantly lower than that of the control group(culture medium)at various time points(F=120.142-182.025,t=9.969-19.329,all P<0.05).After incubation for 30 min and 60 min,the activity of Tregs was also significantly lower than that of the control group(F=21.826-301.968,t=6.897-40.016,all P<0.05).Tregs were finally co-incubated with DiR dye at a concentration of 2.50μg/ml for 5 min,which was used further in vivo experiments.The flow cytometry showed that DiR dye did not affect the phenotype or the function of Tregs.The small animal fluorescence imaging showed that Tregs could locate in the graft area of mouse model.Immunohistochemical analysis showed that Tregs could improve lymphocyte infiltration induced by immune reconstitution.Conclusion After labeling Tregs with DiR dye,the distribution of Tregs can be directly observed by fluorescence imaging,which is a promising imaging method for Tregs tracer.