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Title:: Isoprinosine enhances T cells proliferation through activation of ERK regulated by Src/Ras and calcium signaling

Author(s):: LI Yaqin; DENG Linhong; OUYANG Mingxing; Institute of Biomedical Engineering and Health Sciences, Changzhou University，Changzhou 213164，China

Keywords:: T lymphocytes; Extracellular regulated protein kinases; Fluorescence resonance energy transfer; Isoprinosine; Src kinase; Calcium signaling

摘要:: 异丙肌苷能促进T淋巴细胞的增殖，但具体信号分子调控机制尚不清楚。本研究利用荧光共振能量转移（FRET）技术，探索了异丙肌苷对人急性T细胞白血病细胞系（Jurkat）的细胞外调节蛋白激酶（extracellular regulated protein kinases,ERK）活性的影响，以及其上游相关信号调控机制。通过细胞计数检测试剂盒-8（CCK8）方法筛选出异丙肌苷促进Jurkat细胞增殖的最佳药物浓度为80 μg/mL。将能实时检测ERK激酶活性变化的FRET分子探针转染进入细胞，在异丙肌苷作用下，ERK活性显著升高。进一步研究证实，细胞内非受体酪氨酸激酶Src/小G蛋白Ras和钙离子（Ca2+）信号起到了上游调控作用，Src的抑制剂PP1、Ras的抑制剂Salirasib和Ca2+信号通路抑制剂U71322均有效抑制了异丙肌苷诱导的ERK活性升高及细胞增殖，可认为异丙肌苷通过Src/Ras和Ca2+信号通路上调Jurkat 细胞中的ERK活性，促进了细胞增殖。基于以上研究结果，期望可为靶向T细胞的免疫治疗药物提供信号调控机制方面的新思路。

Abstract:: Isoprinosine can promote the proliferation of T lymphocytes, but the specific regulation mechanism of signal molecules is still unclear. We used fluorescence resonance energy transfer (FRET) technology to explore the effect of isoprinosine on the extracellular regulatory protein kinase (ERK) activity and its upstream related signal regulation mechanism of human acute T-cell leukemia cell line (Jurkat).The optimal concentration of Isoprinosine for promoting the proliferation of Jurkat cells was 80 μg/mL by using the cell count assay kit -8 (CCK8). The FRET probe, which can be used to detect the activity of ERK kinase in real time, was transfected into the cells. The ERK activity was significantly increased in the cells under isoproinosine stimulation. Further studies showed that non-receptor tyrosine kinase Src/Ras and calcium (Ca2+) signaling played important roles in upstream regulation. Src inhibitor PP1, Ras inhibitor Salirasib, and Ca2+ signaling inhibitor U71322 all effectively inhibited the increased ERK activity and cell proliferation induced by isoprinosine. Therefore, isoprinosine promoted the proliferation of Jurkat cells by up-regulating ERK activity, which was mediated through upstream Src/Ras and Ca2+ signaling pathways. Based on the above research results, it is expected that this can provide a new idea on the signal regulation mechanism of immunotherapy drugs targeting T cells.