摘要： 目的 体外研究神经肽P物质(SP)对自然杀伤(NK)细胞的活化作用,并探讨SP受体NK-1R在SP调控NK细胞活性中的作用及可能的信号分子机制。方法 采用MTT释放法检测SP对NK92-MI细胞增殖和杀伤活性的影响,荧光定量PCR和流式细胞术检测NK-1R的mRNA表达水平和膜表达,Fura-2/AM荧光探针法测定NK92-MI细胞胞质钙浓度,Western blotting测定环磷酸腺苷(cAMP)反应元件结合蛋白(CREB)磷酸化水平。结果 10^-12 mol/L SP可促进NK92-MI细胞的增殖和杀伤活性,NK-1R拮抗剂可完全或大部分阻断SP的促进作用。10^-12 mol/L SP作用下,NK92-MI细胞的NK-1R表达上调,胞质钙浓度升高,提示SP通过增加NK-1R的表达及激活Ca²⁺信号通路发挥对NK92-MI细胞活性的调节作用。10^-12 mol/L SP使NK92-MI细胞CREB磷酸化水平明显增高,表明SP可诱导CREB在Ser133位点磷酸化而激活。结论 cAMP信号通路参与了NK-1R介导的SP对NK92-MI细胞的活化作用,且Ca²⁺和CREB是关键信号分子。

神经肽P物质通过Ca²⁺和CREB诱导自然杀伤细胞的活化作用

赵蔓嘉, 范世光, 赵爱农, 梁再赋, 傅炜昕

中国医科大学科学实验中心, 沈阳 110122

收稿日期:2021-06-23出版日期:2022-06-30发布日期:2022-06-09
通讯作者:傅炜昕E-mail:wxfu@cmu.edu.cn
作者简介:赵蔓嘉(1988-),女,助理实验师,本科.
基金资助:辽宁省自然科学基金(2013021085)


关键词: 神经肽P物质, 自然杀伤细胞, NK-1R
Abstract: Objective The aims of this study were to investigate the effects of substance P(SP) on natural killer(NK)cell activation in vitro and to explore the role of neurokinin-1 receptor (NK-1R) in the regulation of NK cell activity by SP and the associated signaling mechanism. Methods An MTT release assay was used to determine the effects of SP on the proliferation and killing activity of NK92-MI cells. The mRNA levels of NK-1R were measured by real-time polymerase chain reaction and the membrane localization of NK-1R was analyzed by flow cytometry. A Fura-2/AM fluorescent probe was used to determine the concentration of intracellular Ca²⁺. Levels of phosphorylated cyclic adenosine monophosphate (cAMP) response element binding protein(CREB)were measured using Western blotting. Results SP,at a concentration of 10^-12 mol/L,significantly increased the proliferation and killing activity of NK92-MI cells. NK-1R antagonists completely or mostly blocked these effects of SP. The expression levels of NK-1R in NK92-MI cells were significantly upregulated and the cytoplasmic Ca²⁺ concentration also significantly increased after treatment with 10^-12 mol/L SP. These data indicated that SP regulated the activity of NK92-MI cells by increasing the expression levels of NK-1R and activating the Ca²⁺ signaling pathway. The phosphorylation levels of CREB also significantly increased in NK92-MI cells treated with 10^-12 mol/L SP,indicating that SP induced the phosphorylation of CREB at Ser133. Conclusion The cAMP signaling pathway is involved in the SP-induced activation of NK92-MI cells through NK-1R. Ca²⁺ and CREB are the key signaling molecules in this process.
Key words: neuropeptide substance P, natural killer cell, NK-1R
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