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伯克霍尔德氏菌在植物病害生物防治中的研究进展马白鸽，魏喜红，孟祥佳，孙正祥*（长江大学农学院，湖北省农林病虫害预警与调控工程技术研究中心，湖北荆州434025）摘要：伯克霍尔德氏菌（Burkholderia ）是一类革兰氏阴性细菌，随着与植物相关的伯克霍尔德氏菌的研究不断增加，越来越多的结果表明，该属细菌可作为一类重要的生防有益微生物。
本文综述了伯克霍尔德氏菌的分类和生理生化特征；在植物病害生物防治上的应用及作用机制，主要包括嗜铁素产生及生存空间竞争，拮抗作用中抗生素产生，诱导植物产生抗病性等；还综述了伯克霍尔德氏菌的固氮、解磷、植物激素产生等促生长特性。
本论文为伯克霍尔德氏菌的生防机制研究和应用开发提供了理论依据。
关键词：伯克霍尔德氏菌；生物防治；机制；诱导抗病性；促生中图分类号：S182文献标志码：AAdvancements in Study on Burkholderia for PlantDisease BiocontrolMA Baige,WEI Xihong,MENG Xiangjia,SUN Zhengxiang *(Engineering Technology Research Center for Pest Early Warning and Control in Agriculture and Forestry,College ofAgriculture,Yangtze University,Jingzhou,Hubei 434025,China)Abstract:Burkholderia is a group of Gram-negative bacteria.As plant-related research on Burkholderia incrementally came out,more and more evidence indicated that this bacterial genus could serve as an important beneficial microorganism in biocontrol.This paper provides an overview of the classification and physiological and biochemical characteristics of Burkholderia ;application and mechanisms of Burkholderia in plant disease biocontrol,including siderophore production and spatial competition for survival,antibiotic production in antagonistic action,and induction of plant disease resistance;moreover,it reviewed the growth-promoting traits of Burkholderia ,such as nitro ‐gen fixation,phosphate solubilization,and production of plant hormones.This paper contributes a theoretical foundation to the research and application development of Burkholderia biocontrol mechanisms.基金项目：中国烟草总公司重大科技项目（110202201023LS-07）；大学生创新创业训练计划项目（Yz2022193）。
多重微珠免疫法英文缩写英文回答：Multiplex bead immunoassay (MBIA) is a high-throughput technique used for the simultaneous quantification of multiple analytes in a single sample. It is based on the principle of flow cytometry, where beads coated with specific antibodies are incubated with the sample. The beads are then washed and analyzed using a flow cytometer, which measures the fluorescence intensity of each bead. The fluorescence intensity is proportional to the amount of analyte bound to the bead.MBIA has several advantages over traditional immunoassays. First, it is highly multiplexed, allowing for the simultaneous quantification of up to 100 analytes in a single sample. Second, it is sensitive, with a lower limit of detection in the picomolar range. Third, it is fast and efficient, with results available in less than an hour.MBIA is used in a wide variety of applications, including:Clinical diagnostics.Drug discovery.Food safety testing.Environmental monitoring.中文回答：多重微珠免疫法（MBIA）是一种高通量技术，用于在单个样品中同时定量分析多种分析物。
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Keywords hypertension;phlegm-damp accumulation;animal models;diet induction;blood lipids;experimental study基金项目国家自然科学基金项目(No.81872972)作者单位 1.北京中医药大学(北京100029);2.中国医学科学院北京协和医学院北京协和医院(北京100730)通讯作者王灵芝,E-mail:***************.cn;徐钟慧,E-mail:*******************引用信息朱甫臻,尹艳燕,梁劲杰,等.痰湿壅盛型高血压大鼠模型的建立[J].中西医结合心脑血管病杂志,2023,21(24):4519-4523.高血压是一种以主动脉收缩压升高为主要特征的常见慢性病[1],严重危害着人类健康㊂2019年世界卫生组织统计,全世界约14亿例高血压病人,控制率仅为14%[2]㊂在我国,大中型城市如北京㊁上海等地高血压发病率约为35.9%和29.1%[3],给我国带来沉重的医疗负担㊂高血压病在中医学上归属于 眩晕 头痛 范畴,按照证候分为痰湿壅盛型㊁肝火亢盛型㊁阴虚阳亢型和阴阳两虚型[4]㊂其中,痰湿壅盛型占比最高,约为17.96%[5]㊂痰湿壅盛型高血压的主要临床表现为头重如裹㊁胸脘痞闷㊁纳呆恶心㊁呕吐痰涎㊁身重困倦㊁少食多寐㊁苔腻㊁脉滑等症状㊂‘景岳全书“提到: 脾为生痰之源 [6],其病机主要为脾运化不利,故生痰浊㊂‘金匮要略“记载: 心下有痰饮,胸胁支满,目眩,苓桂尤甘汤主之 ㊂张仲景提出对痰饮 当以温药和之 ,对饮停中焦之证,应用苓桂术甘汤法健脾温阳利水㊂‘丹溪心法㊃头眩“中云: 头眩,痰挟气虚并火,治痰为主,挟补气药及降火药,无痰则不作眩 ,主张清化痰热㊁补充正气㊂动物模型在研究人类疾病的发病机制㊁发展规律及药物研发中发挥着重要作用㊂目前,常用高血压动物模型主要有:1)自发性高血压动物模型,即自发性高血压大鼠(spontaneous hypertension rats,SHR),由日本京都医学院利用有明显高血压症状的Wistar Kyoto雄性大鼠与带有轻微高血压症状雌性大鼠连续交配培育而成,成年后血压高达200 mmHg以上,高血压自发率为100%㊂2)药物诱导法,常用药物包括醋酸去氧皮质酮(deoxycorticosterone acetate,DOCA)㊁亚硝基左旋精氨酸甲酯(nitro L arginine methyl ester,L-NAME)和血管紧张素Ⅱ(AngⅡ)㊂20世纪70年代,DOCA-盐模型首次用于高血压研究,皮下注射DOCA同时配合高盐饮食,饮用盐水可促进动物高血压的发展[7]㊂Leo等[8]采用L-NAME饲喂Wistar大鼠诱导高血压模型;Cao等[9]采用AngⅡ灌注C57BL6小鼠诱导高血压㊂3)手术诱导法,主要包括一肾一夹[10]㊁两肾一夹[11]㊁两肾两夹法[12]等㊂近年来,痰湿壅盛型高血压动物模型的建立逐渐引起人们关注,该模型的建立常采用陈腾蛟等[13]研究方法,即饲喂Wistar大鼠高脂饲料,该方法存在着造模周期时间长㊁成功率较低等不足,且造模评价指标为血压升高和行为学变化㊂该中医证型的临床特点是低密度脂蛋白胆固醇(LDL-C)升高,高密度脂蛋白胆固醇(HDL-C)降低[14],应进一步完善造模评价指标,以提高造模效率㊂本研究采用不同方法建立快速㊁稳定的痰湿壅盛型高血压大鼠模型,为该病的作用机制和药物研发提供合理的研究基础㊂1材料与方法1.1实验动物Wistar大鼠5只,无特定病原体(SPF)级,雄性,体质量120~150g;购自北京维通利华实验动物技术有限公司,动物合格证号:SCXK(京)2021-0006㊂SHR 共20只,SPF级,雄性,体质量75~105g,由北京维通利华实验动物技术有限公司提供,动物合格证号为SCXK(京)2021-0006㊂所有动物均饲养于北京中医药大学动物房㊂1.2实验饲料动物饲料:普通大鼠维持饲料㊁高脂饲料1(蛋黄粉10%,蔗糖10%,猪油15%,胆固醇1.2%,胆酸钠0.2%,适量的酪蛋白㊁磷酸氢钙㊁石粉等);高脂饲料2(蛋白质24%,碳水化合物41%,猪油24%,酪蛋白㊁糊精㊁蔗糖㊁纤维素㊁豆油㊁多矿㊁多维㊁胆碱);高脂饲料3(蔗糖20%,猪油15%,胆固醇1.2%,胆酸钠0.2%,适量的酪蛋白,磷酸氢钙,石粉)㊂所有饲料均由北京华阜康生物科技股份有限公司提供㊂1.3实验方法1.3.1动物分组及造模SPF级SHR(20只)和Wistar大鼠(5只),适应性喂养1周后,将SHR随机分为SHR对照组㊁模型Ⅰ组㊁模型Ⅱ组和模型Ⅲ组,每组5只;将Wistar大鼠作为正常对照组㊂其间,SHR对照组及正常对照组喂食普通饲料,模型Ⅰ组㊁模型Ⅱ组和模型Ⅲ组分别饲喂高脂饲料1㊁高脂饲料2和高脂饲料3,连续饲喂8周㊂1.3.2观察指标建模期间,每日观察记录大鼠毛色㊁活动程度㊁二便及饮食㊁进水等情况,每周测量体质量㊂血压:采用尾袖法测定大鼠血压[15],无创血压仪型号:BP2000(美国Visitech systems公司);血压测定台37ħ预热5 min,将大鼠放入固定器内适应3~5min,测量安静状态下尾动脉血压,连续测量20次,取平均值,每2周测量1次㊂血脂:眼眶取血,置于无抗凝剂离心管内,室温放置1h,1000ˑg(重力加速度)离心15min,取上清备用㊂采用全自动生化仪(型号:AU480,美国Beckman Coulter公司)测定大鼠血清总胆固醇(TC)㊁三酰甘油(TG)㊁HDL-C㊁LDL-C水平㊂实验结束后,冰上分离大鼠心㊁肝㊁脾㊁肺㊁肾,并称重,按照如下公式计算:脏器指数(%)=脏器质量大鼠体质量ˑ100%1.3.3造模评价标准造模评价标准参照相关文献[13],痰湿型高血压病人常患有高脂血症[14],故将血脂纳入评价标准:1)身型肥胖㊁嗜睡懒动㊁胃纳呆滞㊁不思饮水㊁大便不成形㊁肛门不洁;2)收缩压ȡ140mmHg和(或)舒张压ȡ90mmHg;3)血脂水平变化符合4种高脂血症中的1种,高胆固醇血症,血清TC水平上升;高三酰甘油血症,血清TG水平升高;混合型高脂血症,血清TC㊁TG 水平均增高;低高密度脂蛋白血症,血清HDL-C水平降低㊂1.3.4心脏结构变化大鼠麻醉后,使用超高分辨小动物超声影像系统VevoTM2100(加拿大Visual Sonics公司)测量左室收缩末期内径(left ventricular internal diameter at end-systole,LVIDs)㊁左室舒张末期内径(left ventricular internal diameter at end-diastole,LVIDd)㊁左室收缩末期后壁厚度(left ventricular posterior wall at end-systole,LVPWs)㊁左室舒张末期后壁厚度(left ventricular posterior wall at end-diastole,LVPWd)㊁左室收缩末期前壁厚度(left ventricular anterior wall at end-systole,LVAWs)㊁左室舒张末期前壁厚度(left ventricular anterior wall at end-diastole,LVAWd)㊁左心室平均质量(LV Mass AW)㊁左室射血分数(left ventricular ejection fraction,LVEF)㊁左室短轴缩短率(left ventricular fraction shortening,LVFS)㊂1.4统计学处理采用SPSS19.0分析软件进行数据处理,符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,两组间比较采用t检验,多组间比较采用单因素方差分析ANOVA法㊂采用GraphPad Prism8进行绘图㊂以P< 0.05为差异有统计学意义㊂2结果2.1不同模型大鼠形态学指标造模期间,正常对照组和SHR对照组大鼠毛发洁白㊁柔顺有光泽,精神状态良好,活动度正常,大便黏软有形,肛周洁净㊂各模型组大鼠在造模期间均呈痰湿证表现,包括体质量增加㊁嗜睡萎靡㊁活动度下降㊁饮食饮水减少㊁便溏㊁肛周不洁等㊂从第4周开始,各模型组动物体质量均高于SHR对照组(P<0.05)㊂详见图1㊂2.2不同模型大鼠收缩压变化造模期间,正常对照组收缩压基本维持在131.8~ 150.2mmHg,SHR各组收缩压维持在167.8mmHg以上,且呈现缓慢上升趋势㊂详见图2㊂图1不同模型大鼠体质量变化图2不同模型大鼠收缩压变化2.3不同模型大鼠血脂含量变化模型Ⅰ组LDL-C从第4周开始较SHR对照组升高(P<0.05),HDL-C第8周较SHR对照组降低(P< 0.05);模型Ⅱ组第8周TC和TG均显著增高(P< 0.05),模型Ⅲ组第8周HDL-C水平较SHR对照组显著降低(P<0.05),第4周LDL-C水平较SHR对照组显著升高(P<0.05)㊂详见图3㊂根据临床报道,模型Ⅰ组更符合临床症状[14]㊂2.4不同模型大鼠心脏结构变化为确立痰湿壅盛型高血压大鼠模型,进一步对模型Ⅰ组进行心脏结构分析㊂相较于正常对照组大鼠, SHR对照组大鼠和模型Ⅰ组LVAWs㊁LV Mass AW㊁LVPWd增加(P<0.05),LVIDd降低(P<0.05),模型Ⅰ组LVIDs较SHR对照组降低㊂详见图4㊁表1㊂表明痰湿壅盛型高血压大鼠已出现心脏向心性肥大,提示心脏结构变化可作为痰湿壅盛型高血压模型的评价指标㊂2.5不同模型大鼠脏器指数比较与正常对照组比较,SHR对照组和模型Ⅰ组心脏指数均升高;模型Ⅰ组肝脏指数较SHR对照组升高(P<0.05)㊂提示该饲喂方式对肝脏结构具有一定影响㊂详见表2㊂图3不同模型大鼠血脂含量变化(A为TC;B为TG;C为HDL-C;D为LDL-C㊂与正常对照组同时间比较,*P<0.05;与SHR对照组同时间比较,#P<0.05)图4不同模型大鼠心脏超声结构(短轴)表1不同模型大鼠心脏结构变化(xʃs)组别只数LVAWd(mm)LVAWs(mm)LVIDd(mm)LVIDs(mm)正常对照组5 2.37ʃ0.43 3.43ʃ0.29 6.36ʃ0.62 2.06ʃ0.32 SHR对照组5 2.27ʃ0.18 4.17ʃ0.18① 5.45ʃ0.25① 1.72ʃ0.21模型Ⅰ组5 2.35ʃ0.29 4.11ʃ0.23① 5.07ʃ0.45① 1.04ʃ0.14①②组别LVPWd(mm)LVPWs(mm)LVEF(%)LVFS(%)LV Mass AW(mg)正常对照组 2.51ʃ0.24 4.25ʃ0.1390.79ʃ0.2461.09ʃ0.14975.58ʃ109.71 SHR对照组 4.52ʃ0.28① 6.49ʃ0.37①95.29ʃ0.2872.45ʃ0.371255.49ʃ92.79①模型Ⅰ组 3.20ʃ0.36①② 4.83ʃ0.37②98.11ʃ0.4479.53ʃ1.701126.30ʃ107.27①注:与正常对照组比较,①P<0.05;与SHR对照组比较,②P<0.05㊂表2不同模型大鼠脏器指数比较(xʃs)单位:%组别只数心肝脾肺肾正常对照组50.29ʃ0.04 2.71ʃ0.180.17ʃ0.060.37ʃ0.070.35ʃ0.05 SHR对照组50.39ʃ0.02① 2.82ʃ0.130.15ʃ0.010.41ʃ0.050.36ʃ0.03模型Ⅰ组50.36ʃ0.02① 3.40ʃ0.11①②0.13ʃ0.010.36ʃ0.010.32ʃ0.02注:与正常对照组比较,①P<0.05;与SHR对照组比较,②P<0.05㊂3讨论高血压发病机制复杂多样,包括系统性代谢紊乱㊁肾脏代谢[16]㊁肥胖症[17]㊁糖尿病[18]㊁内皮功能失调[19]㊁氧化应激及炎症[20]等㊂基于中医体质学,高血压的影响因素包括平和质㊁气虚质㊁阳虚质㊁阴虚质㊁痰湿质㊁湿热质㊁血瘀质㊁气郁质㊁特禀质,其中痰湿质㊁阴虚质和气虚质为主要影响因素㊂痰湿质是由气血津液运化失司,淤积形成痰,以浊重黏滞为主要特征的一种体质[21]㊂痰湿既是高血压的病理产物,又是其致病因素,在高血压发生过程中发挥重要作用,是高血压的常见证型之一[22]㊂中医学认为血脂异常是 痰浊 重要的生理生化指标和物质基础㊂高脂血症是一种由脂质代谢异常引起的代谢性疾病,通常以TG㊁TC㊁LDL-C增加和/或HDL-C 降低进行判断㊂有研究表明,肾脏中脂质堆积阻碍肾素分泌,导致血压异常[23]㊂肥胖是高血压发生的重要因素,中医学认为 肥人多痰湿 ,肥胖㊁超重等因素可增加高血压发生的风险[24],长期高脂饮食导致SD大鼠内脏脂肪增加㊁平均动脉压升高,发生内皮功能障碍[25]㊂本研究结果表明,高脂饮食使SHR体质量显著增加,且SHR血压增加快且稳定,收缩压维持在167.8 mmHg以上,达到造模中血压标准;Wistar大鼠与SHR脂肪代谢方面存在着一定差异,SHR的HDL-C 水平低于Wistar大鼠(P<0.05),符合痰湿壅盛型高血压的模型要求,表明以SHR建立痰湿壅盛型高血压大鼠模型省时高效㊂目前常用高脂饮食诱导法建立痰湿壅盛型高血压大鼠模型,其中高脂配方中的脂肪主要来源为猪油[26-27],来源单一,存在一定的局限性;应用高脂高糖配方可诱导痰湿型高血压模型[28-29]㊂本研究采用3种不同的高脂饲料,其中高脂饲料2为经典肥胖高脂饲料配方,高脂饲料3为常用高脂高糖饲料配方,高脂饲料1中添加了10%蛋黄粉,导致SHR的HDL-C㊁LDL-C 水平升高,符合临床痰湿壅盛型的特点㊂说明丰富的脂肪种类诱导的动物模型更符合临床痰湿型,今后可进一步将HDL-C及LDL-C水平纳入建模评判标准㊂综上所述,本研究采用高脂饲料1(蛋黄粉10%㊁蔗糖10%㊁猪油15%㊁胆固醇1.2%㊁胆酸钠0.2%,适量的酪蛋白㊁磷酸氢钙㊁石粉等)饲喂SHR建立了快速稳定的痰湿壅盛型高血压大鼠模型,可为今后相关疾病的作用机制解析和药物研发提供可靠的实验基础㊂参考文献:[1]JAHANDIDEH F,WU J P.Perspectives on the potential benefitsof antihypertensive peptides towards metabolic syndrome[J].International Journal of Molecular Sciences,2020,21(6):2192. 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