摘要
目的 探讨Ras鸟苷酸释放蛋白4(RasGRP4)是否直接参与自然杀伤细胞(NK细胞)调控干扰素-γ的分泌.方法 以RasGRP4基因敲除小鼠和C57BL/6野生型小鼠为研究对象,共24只.根据随机数字法随机分为C57 BL/6+生理盐水组、C57BL/6+脂多糖组、RasGRP4-/-+生理盐水组、RasGRP4-/-+脂多糖组,每组6只小鼠.采用流式细胞术微球阵列法分析两种小鼠在脂多糖免疫激活状态下血清干扰素-γ等免疫炎性因子水平;采用流式细胞术探讨RasGRP4基因敲除对小鼠外周血和脾细胞中主要干扰素-γ分泌细胞(NK细胞、CD4+T细胞、CD8+T细胞)比例的影响,以及在脂多糖激活2.5h时干扰素-γ+NK细胞、干扰素-γ+CD4+T细胞、干扰素-γ+CD8+T细胞的比例;分别提纯两种小鼠的NK细胞,比较其在脂多糖体外激活下干扰素-γ分泌能力.结果 与C57BI/6+生理盐水组和RasGRP4-/-+生理盐水组相比,C57BL/6+脂多糖组和RasGRP4-/-+脂多糖组白细胞介素(IL)-12p70、IL-10、肿瘤坏死因子-α、单核细胞趋化蛋白-1、IL-6、干扰素-γ较脂多糖激活前均显著升高(t =2.823 7~6.310 3,P均<0.01).RasGRP4-/-+脂多糖组血清中干扰素-γ水平约是C57BL/6+脂多糖组的20%(t=5.546 1,P<0.01);尽管C57BL/6+生理盐水组和RasGRP4-/-+生理盐水组外周血、脾细胞中NK细胞、CD4+T细胞、CD8+T细胞的比例差异均无统计学意义(P均>0.05),但在脂多糖激活状态下,RasGRP4-/-+脂多糖组外周血干扰素-γ+的NK1.1细胞比例仅为C57BL/6+脂多糖组的30%(t=8.011 2,P<0.01);而脾脏细胞中干扰素-γ+NK1.1的比例约为C57BL/6+脂多糖组的50%(t=4.429 2,P<0.01).在体外实验中,RasGRP4-/-来源的NK细胞在脂多糖刺激下细胞上清中干扰素-γ的水平为C57BL/6来源的30%(t=5.522 8,P<0.01).结论 RasGRP4在NK细胞分泌干扰素-γ的过程中起重要调控作用.
Objective To explore whether Ras guanine releasing protein 4 (RasGRP4) is directly involved in the regulation of interferon-γ secretion by natural killer cell (NK cell).Methods RasGRP4 gene knockout mice and C57BL/6 wide type mice were used,and the total number is 24.These mice were randomly divided into C57BL/6 + normal saline group,C57BL/6 + lipopolysaccharide group,RasGPR4-/-+normal saline group and RasGPR4-/-+ lipopolysaccharide group according to random number.Each group had 6 mice.Cytometer beads array technique was used to compare the levels of serum inflammatory cytokines between two type of mice challenged by lipopolysaccharide.Flow cytometry was used to investigate the effects of RasGRP4 geue knockout on the ratio of primary interferon-γ secreting cells (NK cells,CD4 + T cells,and CD8 + T cells) in peripheral blood and splenocytes of mice,and the ratio of interferon-γ positive cells under stimulation of lipopolysaccharide for 2.5 h in both peripheral blood and splenocytes in the two mice strains were analyzed.NK cell was purified from both mice strains and the ability of interferon-γ production by both NK cell challenged by lipopolysaccharide were compared.Results Compared with C57BL/6 + normal saline group and RasGRP4-/-+ normal saline group,the levels of interleukin-12p70,interleukin-10,tumor necrosis factor-α,monocyte chemotactic protein-1,interleukin-6 and interferon-γ in both C57BL/6 + lipopolysaccharide group and RasGRP4-/-+ lipopolysaccharide group were all significantly increased after the activation of lipopolysaccharide (t = 2.823 7-6.310 3,all P <0.01).The levels of interferon-γ in RasGRP4-/-+ lipopolysaccharide group was only 20% of that in C57BL/6 + lipopolysaccharide group (t = 5.546 1,P < 0.01).Although there was not any significant difference of the ration of NK cell,CD4 + T cell and CD8 +T cell between C57BL/6 + normal saline group and RasGRP4-/-+ normal saline group(all P > 0.05),the interferon-γ+ NK 1.1 cells from peripheral blood of RasGRP4-/-+ lipopolysaccharide group was about 30% of that from C57BL/6 + lipopolysaccharide group (t = 8.011 2,P <0.01) under the condition of lipopolysaccharide;the interferon-γ+ NK 1.1 cells from splenocytes of RasGRP4-/-+ lipopolysaccharide group was about 50% of that from C57BL/6 + lipopolysaccharide group (t =4.429 2,P <0.01).The interferon-γlevel in NK cell supernatants from RasGRP4-/-+ normal saline groups stimulated by lipopolysaccharide was 30% of that from C57BL/6 +normal saline group (t =5.522 8,P<0.01).Conclusion RasGRP4 plays an important regulatory role in the secretion of interferon-γby NK cell.
作者
李昕
于珮
于德民
周赛君
Li Xin;Yu Pei;Yu Demin;Zhou Saijun(Department of Nephrology and Dialysis,Key Laboratory of Hormones and Development (Ministry of Health),Tianjin Key Laboratory of Metabolic Diseases,The Metabolic Diseases Hospital &Tianfin Institute of Endocrinology,Tianjin Medical University,Tianjin 300070,China)
出处
《国际内分泌代谢杂志》
2018年第6期374-380,共7页
International Journal of Endocrinology and Metabolism
基金
国家自然科学基金青年科学基金(81600643)
国家自然科学基金(91746205)
天津市自然科学基金(17JCYBJC2700).
