钙调蛋白磷酸酶（Calcienuirn，CN）是迄今发现的唯一一种活性受Ca2+和钙调素（Calmodulin, CaM）调节的丝氨酸/苏氨酸蛋白磷酸酶，参与免疫系统、神经系统和心血管系统的生长发育，在生物体中发挥重要功能。CN活力调节异常将直接影响其功能的发挥，可能参与多种病理生理过程。因此，研究细胞内与CN相关的内源性调节因子及其对CN活性的调控具有非常重要的意义。RCANs (regulators of calcineurin，RCANs）家族蛋白是目前研究较为广泛的CN内源性调节因子，在神经系统中有着重要的功能，可以调控CN酶活。本文研究发现293T细胞中过表达RCAN1-1L可抑制细胞内CN磷酸酶活力，抑制率达49%，并用RT-PCR法验证过表达模型成功建立。利用细胞免疫荧光实验对GFP-RCAN1-1L以及CNA在细胞中定位进行了观察，结果发现RCAN1-1L均匀分布在细胞质和细胞核，而CNA则主要分布在细胞质。此外，为进一步研究RCAN1-1L对CN/NFAT通路的影响，在1μM Ionomycin（钙离子载体） 和50ng/ml PMA（佛波醇酯）作用下，CN被激活，检测细胞核中NFAT的蛋白含量以及NFAT的转录活性，结果显示RCAN1-1L可以抑制NFAT进入细胞核及其相应的下游IL-2基因的转录活性，抑制效果与CsA相似。活化的T细胞核因子（nuclear factor of activated T-cells, NFAT）是细胞信号转导通路中一类非常重要的转录因子，NFAT的活化主要是通过Ca2+/Calcineurin的刺激启动，转移入核参与多种特定基因表达。本文研究了已构建的的6个NFATc3剪切体在293T细胞中的定位，发现N端在细胞质定位中起着非常重要的作用，且NFATc3中DNA结合区（RHD）中620-700位氨基酸序列以及C端影响其在细胞核中分布状态。α-synuclein蛋白是一种在神经细胞中广泛表达的突触核蛋白，参与神经系统中多种生理过程，有研究发现α-synuclein与细胞内Ca2+浓度及CaM有关，因此本文初步探索了CNA与α-synuclein的关系。首先在体外条件下检测到α-synuclein对CN酶活具有一定的激活作用，α-synuclein蛋白浓度1mg/ml时对CN酶活的激活率可达到130%，然后分别在293T细胞和稳转α-synuclein的293T细胞中利用免疫共沉淀法研究α-synuclein与CNA的相互作用，结果显示这两种细胞细胞中，α-synuclein和CNA存在一定的相互作用。总之，本论文利用酶活测定，免疫共沉淀，细胞免疫荧光等实验方法在293T细胞系中，研究了CN/NFAT信号通路中与CN相互作用的三个内源蛋白，这些基础工作的研究为今后对该通路的研究提供了一定的依据。

Calcineurin is a serine/threonine-specific protein phosphatase which is regulated by calcium and calmodulin. It plays an important role in immune system, nervous system and cardiovascularthe. CN activity dysregulation will directly affect a variety of pathophysiological processes.Therefore, the study of the endogenous regulatory factors of CN and the regulation of CN activity has a very important significance.RCANs (regulators of calcineurin, RCANs) family proteins which is kowm as CN endogenous regulatory factor, is very important in nervous system. Furthermore, RCANs can inhibit CN enzyme activity. In our study, CN activity in HEK293 cells was inhibited by 49% after transfection of GFP-RCAN1-1L. The over-expression model was confirmed by quantitative PCR method. We detected nucleus NFAT protein content and NFAT transcriptional activity after incubation of 1μM ionomycin and 50ng/ml PMA which would activate CN activity. These observations indicate that RCAN1-1L suppresses NFAT-dependent transcriptional activity and expression of IL-2 by inhibiting CN, which is similar to CsA. Activated T cell nuclear factor (nuclear factor of activated T-cells, NFAT) is an important transcription factor in the cell signal transduction pathway. NFAT is activated mainly by Ca2+/ calcineurin. Activated-NFAT translocates into nucleus, interacts with DNA and regulats the expression of target genes by working together with other transcription factors. In this thesis, we used six constructed GFP-NFATc3 plasmids, observed their localization in 293T cells. Thus, we propose that the N-terminal plays a very important role in the cytoplasmic localization. Beside, amino acid 620-700 in NFATc3 RHD(Rel-homology domain) and the C-terminal are critical impormant in their nucleus distribution.Alpha-synuclein protein is widely expressed in nervous cells, involved in a variety of physiological processes in nervous system. It is reportered that α-synuclein may affected by intracellular Ca2+ and CaM. Here, we have explored the interaction between CNA and α-synuclein. The results demonstrate that α-synuclein significantly up-regulate CN activity in vitro and the largest activition ratio reaches 130%. Moreover, by using co-IP method we found that CNA can interact with α-synuclein in some conditions in 293T cells or stable transfection α-synuclein 293T cells.In summary, we use enzymatic assays, co-IP, immunofluorescence experiments to study several proteins related with CN/NFAT signaling pathway in HEK 293T cells. The study of these basic works provides a foundation for future study of CN/NFAT signaling pathway.