猪ECR1-Like免疫黏附功能对PAMs捕获GFP-E. coli的影响

doi:10.3864/j.issn.0578-1752.2023.19.016

摘要/Abstract

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256 301.56±9 208.85（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（136 666±8 818）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2 892.18±47.76降至2 407.43±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。

关键词: 猪, 红细胞, 猪肺泡巨噬细胞, 类Ⅰ型补体受体, 免疫黏附

Abstract:

【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 256 301.56±9 208.85 (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 (136 666±8 818) 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 2 892.18±47.76 before circulation to 2 407.43±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.

Key words: porcine, erythrocyte, PAMs, CR1-like, immune adherence

张峥, 凌小雅, 范阔海, 孙娜, 孙盼盼, 孙耀贵, 李宏全, 尹伟. 猪ECR1-Like免疫黏附功能对PAMs捕获GFP-E. coli的影响[J]. 中国农业科学, 2023, 56(19): 3905-3916.

ZHANG Zheng, LING XiaoYa, FAN KuoHai, SUN Na, SUN PanPan, SUN YaoGui, LI HongQuan, YIN Wei. Effect of Porcine ECR1-Like Immune Adhesion on PAMs Capturing GFP-Escherichia coli[J]. Scientia Agricultura Sinica, 2023, 56(19): 3905-3916.

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https://www.chinaagrisci.com/CN/Y2023/V56/I19/3905

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基因 Gene	引物序列 Primers sequence（5′-3′）
β-D-galactosidase	F: CGTTATGTGATGCTGCACGG R: CGGATCGTTCGGGTAGTGAG
GAPDH	F: TTGGCTACAGCAACAGGGTG R: CAGGAGATGCTCGGTGTGTT

组别 Groups	阳性细胞率 Positive cell rate (%)	平均荧光强度 Mean fluorescence intensity
A（空白对照组Blank cell group）	86.49±1.53	207488.60±1405.65
B（红细胞黏附组Erythrocyte adhesion group）	90.74±1.07	297655.94±14833.96
C（ECR1-like阻断组ECR1-like blocking group）	88.32±0.92	256301.56±9208.85
D（同型对照组Isotype control group）	91.82±3.12	300211.32±8001.76

组别 Groups	CFU (mL)
A（空白对照组Blank cell group）	111666±25927
B（红细胞黏附组Erythrocyte adhesion group）	203333±4713
C（ECR1-like阻断组ECR1-like blocking group）	136666±8818
D（同型对照组Isotype control group）	186666±28284

组别 Groups		阳性细胞率 Positive cell rate (%)	平均荧光强度 Mean fluorescence intensity
Ⅰ（空白对照组 Blank control group）	循环前免疫黏附 Pre-circulating immune adhesion	20.06±0.44	2846.64±26.84
Ⅰ（空白对照组 Blank control group）	循环后免疫黏附 Post-circulatory immune adhesion	19.07±1.32	2729.63±49.82
Ⅱ（试验组 Test group）	循环前免疫黏附 Pre-circulating immune adhesion	20.58±0.36	2892.18±47.76
Ⅱ（试验组 Test group）	循环后免疫黏附 Post-circulatory immune adhesion	17.39±0.23	2407.43±141.78
Ⅲ（阴性对照组 Negative control group）	循环前免疫黏附Pre-circulating immune adhesion	20.83±0.23	2835.38±18.99
Ⅲ（阴性对照组 Negative control group）	循环后免疫黏附 Post-circulatory immune adhesion	21.25±0.01	2877.65±19.61

组别 Groups	阳性细胞率 Positive cell rate (%)	平均荧光强度 Mean fluorescence intensity
a（正常对照组Normal control group）	30.20±1.24	344.33±37.92
b（Ⅰ处理组Treatment group Ⅰ）	29.79±0.21	333.58±15.60
c（Ⅱ处理组Treatment group Ⅱ）	28.27±0.64	291.56±11.99
d（Ⅲ处理组Treatment group Ⅲ）	30.07±1.15	341.49±34.80
e（同型对照组Isotype control group）	15.27±1.91	141.10±10.30
f（空白细胞组Blank cell group）	/	37.88±0.33