松针多糖对鸡巨噬细胞HD11的天然免疫调节

doi:10.3864/j.issn.0578-1752.2020.15.017

中国农业科学 ›› 2020, Vol. 53 ›› Issue (15): 3180-3186.doi: 10.3864/j.issn.0578-1752.2020.15.017

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

松针多糖对鸡巨噬细胞HD11的天然免疫调节

崔小珍¹(),栾艳²,李婷婷³,杨裕¹,关文超¹,张凯¹,王福传¹,宋献艺¹()

¹山西省农业科学院饲料兽药研究所,太原 030036
²湖北省宜昌市夷陵区畜牧技术推广站,湖北宜昌 443100
³山西省农业科学院畜牧兽医研究所,太原 030032

收稿日期:2019-08-23 接受日期:2020-06-30 出版日期:2020-08-01 发布日期:2020-08-06
通讯作者: 宋献艺
作者简介:崔小珍,Tel：0351-7187658;E-mail： cuiyuchan.002@163.com。
基金资助:
山西省重点研发计划项目(201903D421040);山西省农业科学院博士研究基金(YBSJJ1711);山西省重点研发计划重点项目(201603D21109-1);山西省面上青年基金项目(201701D221198)

Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11

CUI XiaoZhen¹(),LUAN Yan²,LI TingTing³,YANG Yu¹,GUAN WenChao¹,ZHANG Kai¹,WANG FuChuan¹,SONG XianYi¹()

¹Feed and Veterinary Medicine Research Institute of Shanxi Academy of Agricultural Sciences, Taiyuan 030036
²Animal Husbandry Technology Promotion Station of Yiling, Yichang 443100, Hubei
³Animal Husbandry and Veterinary Science Research Institute of Shanxi Academy of Agricultural Sciences, Taiyuan 030032

Received:2019-08-23 Accepted:2020-06-30 Online:2020-08-01 Published:2020-08-06
Contact: XianYi SONG

摘要/Abstract

摘要：

【目的】探讨松针多糖对鸡巨噬细胞HD11的天然免疫功能的影响,以期为松针多糖在肉鸡疾病防治方面应用提供科学依据。【方法】试验采用不同浓度的的松针多糖作用于巨噬细胞HD11,试验分为空白对照组、阳性对照组（终浓度为1 μg·mL^-1的LPS）和松针多糖组（终浓度分别为25、50、100、200、400 μg·mL^-1的松针多糖）。噻唑蓝（MTT）检测巨噬细胞HD11细胞活性、Griess法检测巨噬细胞HD11细胞上清液中一氧化氮（NO）分泌量、中性红吞噬试验检测巨噬细胞HD11吞噬活性,酶联免疫（ELISA）检测巨噬细胞HD11细胞上清液中干扰素-α（IFN-α）、一氧化氮合酶（iNOS）、白介素-10（IL-10）、白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的含量,荧光定量PCR技术检测HD11细胞中iNOS mRNA表达。【结果】 MTT试验结果表明：与空白对照组相比,不同浓度的松针多糖（25、50、100、200、400 μg·mL^-1）对细胞活性没有影响,说明在25-400 μg·mL^-1范围内对巨噬细胞没有毒性,可以进行免疫调节作用实验。免疫调节作用试验结果表明：与空白对照组相比,不同浓度的松针多糖极显著（P<0.01）增加了NO释放量和细胞吞噬活性,极显著（P<0.01）或显著（P<0.05）提高了细胞因子IFN-α的含量和iNOS的含量及mRNA的表达量,极显著（P<0.01）或显著（P<0.05）降低了细胞因子IL-10的分泌量。当松针多糖浓度在50、100、200、400 μg·mL^-1浓度时,细胞因子IL-6和TNF-α的含量极显著（P<0.01）高于空白对照组。与阳性对照组相比,不同浓度的松针多糖极显著（P<0.01）降低NO释放量,极显著（P<0.01）或显著（P<0.05）降低了细胞因子IFN-α的含量、iNOS的含量及mRNA表达量和IL-10含量。当松针多糖浓度在25、50、100 μg·mL^-1浓度时,细胞吞噬活性极显著（P<0.01）低于阳性对照组,细胞因子IL-6的含量极显著（P<0.01）低于阳性对照组。当松针多糖浓度在25、50、100、200 μg·mL^-1浓度时,细胞因子TFN-α的含量极显著（P<0.01）低于阳性对照组。【结论】松针多糖能显著提高巨噬细胞HD11的天然免疫调节能力,且存在剂量依赖效应。

关键词: 松针多糖, 巨噬细胞, 免疫调节, 细胞因子

Abstract:

【Objective】 The innate immunomodulatary effect of pine needle polysaccharide on chicken macrophage HD11 were examined to provide theoretical ground for broiler disease prevention and treatment.【Method】Different concentrations of pine needle polysaccharide were used to treat the macrophage HD11. The experiment was divided into blank control group, positive control group (1 μg·mL^-1 was the final concentration of LPS), and pine needle polysaccharide group (25, 50, 100, 200 and 400 μg·mL^-1 were the final concentration of pine needle polysaccharide). MTT method was used to evaluate the effect of pine needle polysaccharide on the proliferation of HD11 cells. the content of NO in the supernatant of HD11 cells was determined, phagocytic function was detected by neutral red, the release of IFN-α, iNOS, IL-10, IL-6 and TNF-α were detected by ELISA, the levels of iNOS mRNA in the HD11 cells were detected by PCR.【Result】The results of MTT show, there was no significant difference among all groups (25, 50, 100, 200, 400 μg·mL^-1) in proliferation of HD11 cells. It indicated that there was no toxicity to macrophages in the range of 25-400 μg·mL^-1, and the immunoregulation experiment could be carried out. The immunomodulatory test results showed that, the concentration of NO, phagocytic function were higher significantly (P<0.01) than control group. The release of cytokines IFN-α and iNOS were higher significantly (P<0.01 or P<0.05) than control group. The amount of cytokines IL-10 was lower significantly (P<0.01 or P<0.05) than control group. The content of cytokines IL-6 and TNF-α were higher significantly (P<0.01) than control group when the pine needle polysaccharide concentration were 50, 100, 200, 400 μg·mL^-1. the concentration of NO was lower significantly (P<0.01) than positive control group. The release of cytokines IFN-α, iNOS and IL-10 were lower significantly (P<0.01 or P<0.05) than positive control group. The phagocytic function and the content of cytokines IL-6 was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100 μg·mL^-1. The content of cytokines TNF-α was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100, 200 μg·mL^-1.【Conclusion】 The results showed that pine needle polysaccharide can activate macrophages and enhance the innate immune function of macrophages HD11, and there was a dose-dependent effect.

Key words: needles polysaccharide, macrophage, immune regulation, cytokine

崔小珍,栾艳,李婷婷,杨裕,关文超,张凯,王福传,宋献艺. 松针多糖对鸡巨噬细胞HD11的天然免疫调节[J]. 中国农业科学, 2020, 53(15): 3180-3186.

CUI XiaoZhen,LUAN Yan,LI TingTing,YANG Yu,GUAN WenChao,ZHANG Kai,WANG FuChuan,SONG XianYi. Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11[J]. Scientia Agricultura Sinica, 2020, 53(15): 3180-3186.

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参考文献 33

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基因名称 Primer name	引物名称 Primer name	引物序列 Primer sequence	退火温度 Annealing temperature (℃)	产物长度 Products size ( bp)
iNOS	iNOS-F	5′-GCTATTGGGCAAGCGAGAA-3′	58.9	150
iNOS	iNOS-R	5′-ATGACTTTCCCCACAGCCTTA-3′	58.9	150
GAPDH	GAPDH-F	5′- ATGCCATCACAGCCACACA -3′	58.9	127
GAPDH	GAPDH-R	5′-TGACCATCAGGGAGTTCATAGC-3′	58.9	127

组别 Group	浓度 Concentration (μg·mL^-1)	OD值 Value of OD
空白对照组 Control group	0	0.3984±0.02Bb
松针多糖组 Pine needles polysaccharide group	25	0.4318±0.01ABab
	50	0.4382±0.02ABabc
	100	0.4548±0.02ABbc
	200	0.4708±0.02Bbc
	400	0.4792±0.01Bbc

组别 Group	浓度 Concentration (μg·mL^-1)	OD值 Value of OD
空白对照组 Control group	0	21.36±0.59Aa
脂多糖组 LPS group	1	54.67±1.72Ff
松针多糖组 Pine needles polysaccharide group	25	28.37±1.06Bb
	50	31.07±0.28BCbc
	100	34.63±1.61CDcd
	200	37.84±1.77DEde
	400	41.48±1.77Ee

组别 Group	浓度 Concentration (μg·mL^-1)	中性红吞噬率 Neutral red phagocytosis (%)
空白对照组 Control group	0	100.00±2.31Aa
脂多糖组 LPS Group	1	151.72±3.18Dc
松针多糖组 Pine needles polysaccharide group	25	132.28±2.08Bb
	50	135.81±1.73Bb
	100	136.89±3.35BCb
	200	146.41±2.49CDc
	400	146.81±2.10CDc

组别 Group	浓度 Concentration (μg·mL^-1)	干扰素-α IFN-α (ng·L^-1)	白细胞介素-6 IL-6 (ng·L^-1)	白细胞介素-10 IL-10 (ng·L^-1)	一氧化氮合酶 INOS (ng·mL^-1)	肿瘤坏死因子-α TNF-α (ng·L^-1)
空白对照组 Control group	0	27.18±0.50Aa	22.68±0.88Aa	37.14±1.05Bd	0.63±0.01Aa	12.46±0.63Aa
脂多糖组 LPS group	1	67.93±1.28Df	38.53±1.20Cd	41.55±1.41Ce	1.60±0.04Ee	24.80±0.57Dd
松针多糖组 Pine needles polysaccharide group	25	31.61±0.88Ab	24.68±0.81Aa	33.28±0.98Bc	0.86±0.02Bb	12.90±0.53Aa
	50	41.47±1.20Bc	31.70±1.27Bb	26.54±1.30Ab	0.93±0.04BCbc	17.16±0.41Bb
	100	58.50±1.95Cd	32.14±1.22Bbc	25.89±1.03Aab	1.01±0.03CDc	18.08±0.53Bb
	200	59.60±1.90Cd	35.20±1.28BCbcd	25.51±0.97Aab	1.03±0.04CDc	21.61±0.75Cc
	400	63.63±1.15CDe	35.58±1.46BCcd	22.67±0.76Aa	1.14±0.03Dd	24.47±0.66Dd

松针多糖对鸡巨噬细胞HD11的天然免疫调节

Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11

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组别 Group	浓度 Concentration (μg·mL^-1)	mRNA相对表达量 The relative expression of mRNA
空白对照组 Control group	0	1.00±0.11Aa
脂多糖组 LPS Group	1	5.40±0.06De
松针多糖组 Pine needles polysaccharide group	25	1.22±0.01ABb
	50	1.29±0.03Bbc
	100	1.43±0.04Bc
	200	2.10±0.02Cd
	400	2.15±0.02Cd

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