信号蛋白RasGRP4经介导NK/DC 交叉对话调控糖尿病NK细胞IFN-γ分泌的作用机制研究

The high risk of infection and difficulty in control infection in patients with type 2 diabetes mellitus (DM) is a major project faced by the clinical; The damage of the IFN – gamma secretion by Natural killer (NK) cell is important reason for the declining defense capabilities of DM patients. Our research team previously identified that the production of IFN – gamma by NK cell depends on the signaling protein Ras guanylic acid release protein 4 (RasGRP4) and we also found out bnormal RasGRP4 signal existed in dendritic cells (DC) in DM patients. On these basis, we proposed the hypothesis :the damage of IFN – gamma by NK cell in diabetic patients has something to do with bnormal RasGRP4 signal which regulates IFN gamma secretion by NK cell via mediating NK - DC cross talk. To test this hypothesis, This study will adopt RasGRP4 knockout DM mice and DM patients to reveal the effect of DM on the signaling protein of ability of IFN -gamma secretion by NK cells from the overall level.We will further use CRISPR/Cas9 technology to reduce and activate RasGRP4 signal respectively to elucidate the mechanism of R asGRP4 regulating IFN- gamma production. And cell co-culture, antibody blocking experiments will further carried out to clarify the way of NK-DC cross-talk and the effective moleculars. This study will reveal the mechanism of defensive ability in diabetic patients from the perspective of NK - DC cell communication barried. the results of the study is expected to provide new ideas for prevention and cure of immunity decline in DM patients.

2型糖尿病（DM）患者感染的高度易感性和难控制性是困扰临床的一大难题。NK细胞IFNγ分泌受损是DM患者防御能力下降的重要原因，其机制不详。前期研究证实NK细胞IFNγ分泌依赖于信号蛋白Ras鸟苷酸释放蛋白4（RasGRP4），且DM患者树突状细胞（DC）内该信号蛋白异常，据此提出2型DM患者DC内异常的RasGRP4信号介导NK/DC通讯障碍，导致IFNγ分泌受损的假说。为验证该假说，本研究拟采用RasGRP4基因敲除DM小鼠和DM人群，从整体水平揭示RasGRP4信号的变化与NK细胞IFNγ分泌能力的关系；采用CRISPR/Cas9技术敲减和激活RasGRP4，从细胞分子水平证实其调控IFN-γ分泌的作用；通过共培养、抗体阻断等方法阐明其介导NK/DC对话方式及效应分子。从NK/DC细胞通讯障碍的角度揭示DM患者防御能力下降的分子机制，研究结果可望为防治DM患者免疫能力的下降提供新思路

天然杀伤细胞IFN-γ分泌受损是2型糖尿病患者防御能力减低的重要表现，其机制不详。.本研究采用RasGRP4基因敲除DM小鼠和DM人群，从整体水平揭示RasGRP4信号的变化与NK细胞IFNγ分泌能力的关系；采用CRISPR/Cas9技术敲减和激活RasGRP4，从细胞分子水平证实其调控IFN-γ分泌的作用；通过共培养、抗体阻断等阐明其介导NK/DC对话方式及效应分子人群研究，收集不同DM病程的患者外周血，采用流式细胞术分析DM病程对NK细胞以及RasRGP4的表达和变异体。.研究结果.1.RasGRP4-/-小鼠脾脏cDCs比例较WT小鼠明显减少；WT小鼠血浆中IFN-γ水平较RasGRP4-/-小鼠升高5-10倍。RasGRP4-/-小鼠，外周血和脾脏中IFN-γ+的NK1.1显著减少至WT小鼠的30%和50%；IFN-γ水平较WT小鼠明显降低。.2.CD117+CD11C+CD11b+cDCs亚群是小鼠脾细胞中CD117+细胞主要亚群，也是表达RasGRP4的DC亚群； CD117+DCs以浓度依赖和时间依赖的方式促进NK细胞分泌IFN-γ。.3.CD117+DCs促进NK细胞分泌IFN-γ不依赖于DC-NK细胞间的直接接触；RasGRP4促进CD117+DCs分泌IL-1α、GM-CSF调节NK细胞分泌IFN-γ；RasGRP4上调CD117+DCs 中Pydc3基因、IL-1家族相关基因，增加干扰素效应相关基因2'-5' oligoadenylate synthetase 1A等表达。.4.DM外周血单个核细胞中RasGRP4变异体表达量明显增加，且与DM病程和HbAC1相关.本研究证实了RasGRP4信号蛋白通过NK-CD117+DCs间接通讯的方式调控NK1.1分泌IFN-γ的作用机制，揭示2型糖尿病患者外周血中RasGRP4变异体的出现可能是NK细胞IFN-γ分泌受损的原因。