摘要
【目的】探讨猪红细胞类Ⅰ型补体受体(erythrocyte complement receptor type 1-like,ECR1-like)免疫黏附功能是否能够促进猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs)捕获致敏基因工程菌(GFP-Escherichia coli,GFP-E.coli),以期阐释猪红细胞免疫的分子机理及红细胞免疫在机体先天免疫中的作用。【方法】利用流式细胞术、菌落平板计数及RT-PCR技术检测PAMs捕获的GFP-E.coli的水平,分析猪ECR1-like免疫黏附对PAMs捕获GFP-E.coli的影响;运用流式细胞术、细胞免疫荧光化学技术检测猪ECR1-like免疫黏附的致敏GFP-E.coli被PAMs移除后,猪红细胞免疫黏附功能的变化及猪ECR1-like数量的变化。【结果】流式细胞术检测发现猪红细胞黏附组较空白对照组中的PAMs平均荧光强度显著提高(P<0.001),且PAMs阳性细胞率也显著提高(P<0.05);菌落涂板计数发现红细胞黏附组较空白对照组PAMs捕获GFP-E.coli情况显著增强(P<0.05);RT-PCR检测发现,红细胞黏附组PAMs中GFP-E.coli的相对数量显著高于空白对照组(P<0.01);进一步阻断猪红细胞表面的CR1-like,流式细胞术检测发现PAMs平均荧光强度降低至256301.56±9208.85(P<0.001),PAMs阳性细胞率降低至(88.32±0.92)%(P>0.05),菌落平板计数发现PAMs捕获GFP-E.coli情况减弱为(136666±8818)CFU/mL(P<0.05),RT-PCR检测发现PAMs中GFP-E.coli的相对数量显著减少(P<0.01);利用细胞流动循环互作技术发现:猪红细胞免疫黏附致敏GFP-E.coli的平均荧光强度由循环前2892.18±47.76降至2407.43±141.78(P<0.05),阳性细胞率由循环前(20.58±0.36)%降至(17.39±0.23)%(P<0.001),黏附水平显著低于循环前,与此同时,间接免疫荧光试验结果显示:试验组循环后猪ECR1-like平均荧光强度由循环前344.33±37.92降低至291.56±11.99(P<0.05),阳性细胞率由(30.20±1.24)%减少至(28.27±0.64)%(P<0.05)。【结论】猪ECR1-like通过免疫黏附功能促进了PAMs对致敏GFP-E.coli的捕获。PAMs移除猪红细胞表面黏附的致敏GFP-E.coli后,猪红细胞的活性CR1-like减少,免疫黏附功能下降。
【Objective】The aim of this study was to investigate whether the immune adherence function of porcine erythrocyte complement receptor type 1-like(ECR1-like)could promote porcine alveolar macrophages(PAMs)to capture sensitized genetic engineering bacteria GFP-E.coli,in order to explain the molecular mechanism of porcine erythrocyte immunity and its role in innate immunity.【Method】The level of GFP-E.coli captured by PAMs was detected by flow cytometry,colony plate counting and RT-PCR,and the effect of porcine ECR1-like immune adherence on the capture of GFP-E.coli by PAMs was analyzed.Flow cytometry and cellular immunofluorescence technique were used to detect the changes of immune adherence function of porcine erythrocytes,and the number of porcine ECR1-like after the sensitized GFP-E.coli with ECR1-like immune adherence was removed by PAMs.【Result】Flow cytometry showed that the average fluorescence intensity of PAMs in porcine erythrocyte adhesion group was significantly higher than that in blank control group(P<0.001),while the positive rate of PAMs cells in porcine erythrocyte adhesion group was significantly higher than that in blank control group(P<0.05).Colony smear count showed that the capture of GFP-E.coli by PAMs in erythrocyte adhesion group was significantly higher than that in blank control group(P<0.05).RT-PCR showed that the relative quantity of GFP-E.coli in PAMs of erythrocyte adhesion group was significantly higher than that of blank control group(P<0.01).Further blocking CR1-like on the surface of porcine erythrocyte,flow cytometry showed that the average fluorescence intensity of PAMs decreased to 256301.56±9208.85(P<0.001),and the positive cell rate of PAMs decreased to(88.32±0.92)%(P>0.05).Colony count showed that the capture of GFP-E.coli in PAMs decreased to(136666±8818)CFU/mL(P<0.05),and RT-PCR showed that the relative quantity of GFP-E.coli in PAMs decreased significantly(P<0.01).Using cell flow and circulation interaction technique,it was found that the average fluorescence intensity of GFP-E.coli sensitized by porcine erythrocyte immune adherence decreased from 2892.18±47.76 before circulation to 2407.43±141.78(P<0.05),and the positive cell rate decreased from(20.58±0.36)%before circulation to(17.39±0.23)%(P<0.05).The adhesion level was significantly lower than that before circulation.Meanwhile,the results of indirect immunofluorescence test showed that the average fluorescence intensity of porcine ECR1-like decreased from 344.33±37.92 before to 291.56±11.99(P<0.05),and the positive cell rate decreased from(30.20±1.24)%before to(28.27±0.64)%(P<0.05).【Conclusion】Porcine ECR1-like promoted the capture of sensitized GFP-E.coli by PAMs through its immune adhesion function.After PAMs removed sensitized GFP-E.coli adhered to the surface of porcine erythrocyte,the activity of CR1-like of porcine erythrocyte decreased,and the immune adhesion function decreased too.
作者
张峥
凌小雅
范阔海
孙娜
孙盼盼
孙耀贵
李宏全
尹伟
ZHANG Zheng;LING XiaoYa;FAN KuoHai;SUN Na;SUN PanPan;SUN YaoGui;LI HongQuan;YIN Wei(Shanxi Key Laboratory for Modernization of TCVM/College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,Shanxi;Shanxi Key Laboratory for Modernization of TCVM/Animal Experimental Center,Shanxi Agricultural University,Taigu 030801,Shanxi)
出处
《中国农业科学》
CAS
CSCD
北大核心
2023年第19期3905-3916,共12页
Scientia Agricultura Sinica
基金
山西省自然科学基金面上项目(20210302123407)
山西省科技创新人才团队专项(202204051001021)
山西省高等学校科技创新项目(2019L0364)
山西农业大学博士科研启动项目(2021BQ77)
中兽医药现代化重点实验室建设项目(202104010910015)。
