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Molecular Pharmacological research on the roles of lymphocytic cholinergic system in T cell function

淋巴细胞胆碱能系统对T细胞功能作用的分子药理学研究

基本信息

批准号：
16590060
负责人：
FUJII Takeshi
金额：
$ 2.18万
依托单位：
Doshisha Women's College of Liberal Arts (2005)Kyoritsu University of Pharmacy (2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Human Leukemic T cell lines CCRF-CEM and MOLT-3 were used.1.Effects of small interfering RNA (siRNA) on Ca^<2+> signaling in T cells.Transfection of anti-M_3, anti-M_5 and anti-α7 small interfering RNA significantly down-regulated respective mRNA expression, while no changes were observed in gene expression of other mAChR subtypes or nAChR subunits. Ca^<2+> signals evoked by oxotremorine-M (Oxo-M), a non-selective mAChR agonist, were reduced by anti-M_3 or anti-M_5 siRNA. Ca^<2+> signals evoked by nicotine were reduced by anti-α7 siRNA. These findings indicate that M_3, M_5 mAChR and α7 nAChR subtypes play major roles in Ca^<2+> signals to acetylcholine in T cells, and suggest that these receptors are involved in regulation of immune function.2.Effects of anti-CD11a monoclonal antibody on lymphocytic cholinergic activity in T cells.Anti-CD11a mAb significantly increased both the ACh content of MOLT-3 cells and its release into the culture medium, whereas simvastatin had no effect on ACh content or its release. On the other hand, simvastatin completely blocked the enhancement of ACh content and release evoked by anti-CD11a mAb.Similarly, anti-CD11a mAb significantly up-regulated ChAT mRNA expression, whereas simvastatin had no effect on the expression of ChAT mRNA. But when added to the culture medium along with anti-CD11a mAb, simvastatin completely blocked anti-CD11a mAb-induced ChAT mRNA expression. Likewise, simvastatin significantly diminished anti-CD11a mAb-induced expression of M_5 mAChR mRNA. By contrast, neither simvastatin nor anti-CD11a mAb had any effect on expression of M_3 or M_4 mAChR mRNA.Consistent with its effects on ChAT expression and ACh content and release, anti-CD11a mAb significantly enhanced ChAT-catalyzed ACh synthesis in MOLT-3 cells, as compared to control. And consistent with all of the results summarized above, simvastatin alone did not affect basal ChAT activity, but did abolish the anti-CD11a mAb-induced enhancement of that activity.

1.小干扰RNA（siRNA）对人白血病T细胞系CCRF-CEM和MOLT-3细胞内Ca^<2+>信号转导的影响：抗M3、抗M5和抗α7小干扰RNA转染后，可显著下调相应mRNA的表达，而对其他mAChR亚型和nAChR亚基的基因表达无明显影响。抗M_3或抗M_5的siRNA可抑制非选择性mAChR激动剂氧化震颤素M（oxotremorine-M，Oxo-M）诱发的Ca^<2+>信号。尼古丁诱发的Ca^<2+>信号可被抗α7 siRNA抑制。这些结果表明，M_3、M_5和α7 nAChR亚型在T细胞乙酰胆碱的Ca^2+信号中起主要作用，抗CD 11 a单克隆抗体对T淋巴细胞胆碱能活性的影响：抗CD 11 a单克隆抗体能显著增加MOLT-1细胞内ACh含量，并能显著降低MOLT-1细胞内ACh含量，提示MOLT-1细胞内ACh含量与MOLT-1细胞内ACh含量呈负相关。3细胞及其释放到培养基中，而辛伐他汀没有影响ACh含量或其释放。辛伐他汀可完全阻断抗CD 11 a单抗引起的ACh含量和释放的增加，抗CD 11 a单抗可显著上调ChAT mRNA的表达，而辛伐他汀对ChAT mRNA的表达无影响。但当沿着抗CD 11 a mAb加入培养液中时，辛伐他汀完全阻断抗CD 11 a mAb诱导的ChAT mRNA表达。同样，辛伐他汀也能显著降低抗CD 11 a单抗诱导的M_5 mAChR mRNA的表达。与对照组相比，辛伐他汀和抗CD 11 a单抗对MOLT-3细胞M_3和M_4 mAChR mRNA的表达无影响，抗CD 11 a单抗对ChAT表达和ACh含量及释放的影响与对照组一致，显著增强了MOLT-3细胞ChAT催化的ACh合成。与以上总结的所有结果一致，单独使用辛伐他汀不影响基础ChAT活性，但确实消除了抗CD 11 a mAb诱导的该活性增强。