基于吲哚胺2,3-双加氧酶1活性评价人间充质干细胞免疫调控功能的吸光光度法的建立及优化

Development and optimization of an indoleamine 2, 3-dioxygenase 1 activity-based absorption photometry for evaluation of immunomodulatory activity of human mesenchymal stem cells

目的建立一种基于吲哚胺2,3-双加氧酶1(indoleamine 2,3-dioxygenase 1,IDO1)的活性快速评价人间充质干细胞(human mesenchymal stem cells,h MSCs)免疫调控功能的吸光光度法,并进行优化。方法以含干扰素γ(interferonγ,IFNγ)的完全培养基激活hMSCs,收集hMSCs培养上清,与三氯乙酸(trichloroacetic acid,TCA)和对二甲氨基苯甲醛(paradimethylaminobenzaldehyde,PDAB)反应,检测A492值,通过标准曲线计算样品中色氨酸代谢产物犬尿氨酸(kynurenine)的含量,以反映h MSCs所分泌IDO1的酶代谢活性,并对方法中的培养体系(6、12、24、48、96孔板)、IFNγ处理时间(4、8、12、24 h)、培养基使用量(150、200、250、300、350、400、450及500μl)及IFNγ作用浓度(0.2、0.4、0.6、0.8、1.0、5.0及10.0 ng/ml)进行优化。同时验证方法的特异性,并检测样品保存条件及液氮冻存后不同复苏时间对kynurenine浓度的影响。采用优化后的方法分析不同来源、不同代次的hMSCs分泌IDO1的能力。结果确定最佳检测条件为:培养体系为48孔体系,IFNγ处理时间为24 h,培养基使用体积为200μl,IFNγ作用浓度为10.0 ng/ml。该方法可特异性检测IDO1的活性;除常温外,其他样品保存条件对检测结果影响较小;液氮冻存后复苏24 h可检测到与冻存前水平相当的kynurenine。该方法可有效判断不同供体来源的hMSCs在分泌IDO1功能上的差异。结论该方法可快速、特异、稳定地检测hMSCs分泌IDO1的水平,且操作简便,可用于快速评价h MSCs的免疫调控功能。

Objective To develop and optimize an indoleamine 2, 3-dioxygenase 1（IDO1） activity-based absorption photometry for rapid evaluation of immunomodulatory activity of human mesenchymal stem cells（hMSCs）. Methods The hMSCs were activated with complete medium containing interferon γ（IFNγ）, of which the culture supernatant was collected and reacted sequentially with trichloroacetic acid（TCA） and paradimethylaminobenzaldehyde（PDAB）, and determined for absorbance at a wave length of 492 nm. The content of kynurenine, an immediate metabolite of tryptophan,was calculated according to the standard curve, by which the metabolic activity of IDO1 was reflected. The culture system（6-, 12-, 24-, 48- and 96-well plates）, time for treatment with IFNγ（4, 8, 12 and 24 h）, volume of medium used（150,200, 250, 300, 350, 400, 450 and 500 μl） and IFNγ concentration for treatment（0. 2, 0. 4, 0. 6, 0. 8, 1. 0, 5. 0 and10. 0 ng / ml）in the method were optimized. Meanwhile, the method was verified for specificity, and the effects of condition for storage of samples and time for resuscitation of samples stored in liquid nitrogen on kynurenine concentration were determined. The IDO1 activities of hMSCs from various origins and of various passages were determined by the optimized method. Results The optimal culture system, time for treatment with IFNγ, volume of medium used and IFNγ concentration for treatment of the developed method were 48-well plate, 24 h, 200 μl and 10. 0 ng / ml respectively. The method was suitable for specific determination of IDO1 activity. The effect of condition for storage of samples, except room temperature, showed little effect on the determination result. The kynurenine level in samples resuscitated for 24 h after storage in liquid nitrogen was equivalent to that before storage. The difference of hMSCs from various origins in the function of secreting IDO1 was effectively judged by the method. Conclusion The absorption photometry proved to be a rapid, easyoperating method with sufficient stability and specificity for evaluation of the immunomodulatory activity of hMSCs.