BCL3通过STAT3调控NK细胞的功能和机制研究

It is very important for the body health to activate and timely terminate NK function. B-cell CLL/lymphoma 3 (BCL3) is a transcriptional coregulator, but its function in NK cell activation is unclear. Our current study data shown that BCL3 was highly expressed in NK cells from mice infected with cytomegalovirus (CMV). Overexpression of BCL3 inhibited the cytotoxic activity of NK-92 cell, NK cells derived from BCL3 deficient mice secreted more Perforin and INF-γ, and it was shown that BCL3 interacted with STAT3, and inhibition of STAT3 can suppress excessive activation of NK induced by BCL3 deficient. All the data suggested that BCL3 may regulate NK activity through STAT3. The project will establish NK cell specific deletion of BCL3 mice through adoptive transfer of NK from BCL3 deficient mice to B-NSG, a series of assay including flow cytometry, cytotoxicity test assay, ELISA, Cell immunofluorescence, Co-IP, CHIP and Western blot were performed in analyzing the role and mechanism of BCL3 interacting with STAT3. The completion of the project not only reveals a new mechanism of NK cells activity regulation, but also provide potential drug targets for treatment to the disease associated with excessive activation of NK cells.

NK细胞及时活化与适时终止对于机体健康与稳定极为重要，BCL3是转录辅助调节因子，但其在NK活化中的功能还不清楚。我们前期研究发现BCL3在MCMV感染小鼠的NK中高表达，在NK细胞系中过表达BCL3可抑制NK的杀伤活性，BCL3缺失小鼠的NK能分泌更多的Perforin和IFN-γ等活性分子,并且BCL3与STAT3存在相互作用，抑制STAT3能逆转BCL3缺失小鼠的NK的过度活化，提示BCL3可能通过STAT3调控NK的生物学活性。本研究将以BCL3敲除小鼠和免疫缺陷B-NSG小鼠为研究对象，通过细胞过继转输建立NK细胞特异性缺失的BCL3小鼠，应用流式细胞数、细胞杀伤试验、ELISA、细胞免疫荧光、CHIP、免疫共沉淀和免疫印迹等实验手段，研究BCL3通过STAT3负调控NK的作用和机制。该项目的完成不仅能揭示NK活性调控新机制，还可为NK过度活化相关疾病的控制提供潜在的药物靶点。

前期的研究发现在感染MCMV病毒的小鼠体内，BCL3基因在NK细胞中上调表达，且与NK细胞的激活有关。本课题通过构建BCL3基因敲除小鼠研究BCL3对NK细胞生物学活性的调节作用，结合质谱分析、蛋白质生物化学、细胞生物学等方法与技术，对BCL3参与调节NK细胞的分子机制进行了研究。首先采用CRIPR-Cas9技术构建BCL3基因敲除小鼠，通过流式细胞仪进行免疫分型，发现BCL3基因敲除小鼠的NK细胞数量显著下降，抵抗肿瘤的能力下降。发现BCL3通过与STAT3结合，影响NK细胞活性分子的产生，从而调节NK细胞的杀伤活性，为基于NK细胞的抗病毒、抗肿瘤免疫疗法提供理论基础。项目资助发表5篇学术论文，待发表2篇。培养了3名硕士研究生。项目投入20万元，支出16.4849万元，各项支出基本与预算相符。剩余经费3.5151万元，剩余经费计划用于本项目研究的后续支出。