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

发布时间：2018-11-02 17:10

【摘要】：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。
[Abstract]:Objective to establish and optimize an absorbance method for rapid evaluation of the immunomodulatory function of human mesenchymal stem cells (human mesenchymal stem cells,h MSCs) based on the activity of indoleamine 2n 3 dioxygenase 1 (IDO 1). Methods the supernatants of hMSCs were collected from interferon 纬 (IFN 纬) activated hMSCs, medium and reacted with trichloroacetic acid (trichloroacetic acid,TCA) and p-dimethylaminobenzaldehyde (paradimethylaminobenzaldehyde,PDAB). The content of canine uric acid (kynurenine), a tryptophan metabolite, was calculated by standard curve to reflect the enzyme metabolic activity of IDO1 secreted by h MSCs, and the incubation time of), IFN 纬 in the culture system of the method (612121248896 well plate) was studied. The amount of culture medium (150200250300350400450 and 500 渭 l) and the concentration of IFN 纬 (0.20.40.60.81.0,5.0 and 10.0 ng/ml) were optimized. At the same time, the specificity of the method was verified, and the effects of preservation conditions and different resuscitation time of liquid nitrogen on the concentration of kynurenine were examined. The optimized method was used to analyze the ability of hMSCs to secrete IDO1 from different sources and generations. The results showed that the optimal detection conditions were as follows: the culture system was 48 well, the treatment time of IFN 纬 was 24 h, the volume of culture medium was 200 渭 l / L IFN- 纬 and the concentration of IFN- 纬 was 10.0 ng/ml.. This method can specifically detect the activity of IDO1, and the preservation conditions of other samples have little effect on the test results except at room temperature. The kynurenine. which is comparable to the pre-freezing level can be detected after 24 hours of recovery after cryopreservation with liquid nitrogen. This method can effectively judge the difference of IDO1 secretion function of hMSCs from different donor sources. Conclusion this method can be used to detect the level of IDO1 secreted by hMSCs quickly, specifically and stably. It is easy to operate and can be used to evaluate the immune regulation function of h MSCs.
【作者单位】：中国食品药品检定研究院生物制品检定所细胞保藏及研究中心卫生部生物技术产品检定方法及其标准化重点实验室;
【基金】：国家自然科学基金面上项目(81172102) 中国科学院干细胞与再生医学研究战略性先导科技专项(XDA01030508) 国家重点研发计划(2016YFA0101501)
【分类号】：R392

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