创伤弧菌对血管内皮细胞损伤作用的初步研究
|
摘要
研究背景和目的：
创伤弧菌(Vibrio vulnificus, Vv)是弧菌属第五群细菌,革兰染色阴性,弯曲、杆状、多形性、运动活泼、有荚膜的细菌,主要生长于海水中的鱼类及贝母类等体内,为嗜盐性海生弧菌。可分为3种生物型(Ⅰ、Ⅱ、Ⅲ型),其中Ⅰ型是导致人类创伤感染和原发性败血症的主要致病菌,一般通过外伤接触含病菌的海水或生食海鲜两种途径感染。因此创伤弧菌感染是海上创伤医学中的重要问题,也是海产品食品卫生中的重要问题。其感染后可引起人体严重感染、脓毒败血症,其引发的多器官功能障碍综合症是脓毒败血症患者死亡的主要原因,病死率高达50%以上。创伤弧菌脓毒败血症的形成主要是大量的创伤弧菌侵入到血液之中,而血管内皮细胞是阻挡创伤弧菌进入血管的重要屏障。因此,创伤弧菌对血管内皮细胞的损伤作用及机制是创伤弧菌败血症形成机制中的重要问题。
为揭示创伤弧菌对血管内皮细胞的损伤作用,阐明创伤弧菌感染时对人血管内皮细胞结构的影响,我们进行了本项研究,为临床创伤弧菌感染的诊治提供一定的理论依据。
方法：
1.创伤弧菌一般特性观察和对血管内皮细胞的影响：
1.1创伤弧菌标准株ATCC27562,购自中国科学院微生物研究所普通微生物保藏管理中心。复苏培养后,行革兰氏染色,光镜观察,划线接种于硫代硫酸盐—枸橼酸盐——胆盐—蔗糖琼脂(thiosulfate-cifrae-bile salt-sucrose, TCBS)平板及普通营养琼脂平板观察其菌落形态,并接种于弧菌属生化鉴定管及生化条培养24h后,机读细菌鉴定结果。
1.2人脐静脉血管内皮细胞(Human umbilical vein endothelial cell, HU),由广州军区广州总医院实验科提供,对其进行VIII因子免疫荧光鉴定。我们通过本课题组前期的实验结果,对创伤弧菌对血管内皮细胞的攻击浓度进行了界定,并通过96孔板培养实验以及MTT法对不同浓度的创伤弧菌侵袭过的血管内皮细胞进行增值抑制率的测定；细胞划痕实验测量不同浓度的创伤弧菌对细胞迁移能力的影响；台盼蓝拒染法检测细胞侵袭后即刻及24h细胞存活率；流式细胞仪测定不同浓度Vv对HU细胞凋亡的影响,选择攻击实验Vv的最高的实验终浓度103cfu/ml。通过Hoechest 33342荧光染色观察HU细胞核的变化；平板克隆形成实验检测细胞集落形成能力的变化,以及50个高倍视野下正常细胞与侵袭后细胞核分裂数的变化,观察极限杀伤浓度下细胞生物学行为的变化。对正常组和创伤弧菌攻击组(104cfu/ml)的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、胞浆面积(Acp)、核浆比(Rnp)以及细胞与核的长轴(dmaxc, dmaxn),细胞与核的短轴(dminc dminn),轴比(Rac、Ran),细胞与核的形状因子PE (PEc、PEn),细胞与核的形状因子AR (ARc、ARn),细胞与核的规划形状因子RFF (RFFc、RFFn),形状不规则指数FⅡ(FⅡc、FⅡn)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌攻击下增殖能力和细胞形态的改变。
2、创伤弧菌内毒素的提取、鉴定以及对血管内皮细胞的影响
2.1取新鲜培养的创伤弧菌,利用酚水法提取细菌内毒素(LPS),并对LPS进行离心纯化,以及根据2005版《中国药典》规定用内毒素测定方法“鲎试剂”对其进行鉴定,并绘制浓度曲线,确定所提取的细菌LPS的浓度。
2.2根据预实验选择创伤弧菌内毒素对细胞的半数致死浓度,选择其上下浓度域共4个浓度对人脐静脉内皮细胞进行MTT,平板克隆,划痕实验测定细胞迁移能力的改变,以及流式细胞仪对细胞凋亡率的测定。对正常组和内毒素10000EU/ml攻击组的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、周长(Cn)、细胞的体积(V),以及细胞的体积密度(Vv)、表面积密度(Sv)、数密度(Nv)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌攻击下增殖能力和细胞以及细胞核形态的改变,以判断细菌内毒素对人脐静脉内皮细胞损伤程度。
3、创伤弧菌外毒素的鉴定和浓度的测定,以及其对血管内皮细胞的影响
3.1本课题组前期已重组大肠杆菌pET-32a-vvhA/E.coliBL21(DE3),行蓝白斑筛选,挑选白色菌斑进行PCR扩增得到创伤弧菌溶细胞毒素(rVVC),对其进行SDS-PAGE电泳验证,采用BCA (bicichoninicacid)法进行蛋白定量,绘制标准曲线,计算蛋白浓度。对SPF级BALB/c小鼠进行腹腔皮下多点注射,50mg/kg,小鼠处死后对其立即解剖,取主要损伤脏器及组织固定、石蜡包埋后按常规方法制片、HE染色,光镜下观察病理变化。
3.2根据预实验选择创伤弧菌溶细胞毒素对细胞的半数致死浓度,选择其浓度域上下共4个浓度对人脐静脉内皮细胞进行MTT,平板克隆,划痕实验测定细胞迁移能力,并用流式细胞仪对细胞凋亡率的测定。对正常组和溶细胞毒素3EU／ml攻击组的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、周长(Cn)以及细胞的体积密度(Vv)、表面积密度(Sv)、数密度(Nv)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌溶细胞毒素(rVVC)攻击下增殖能力和细胞形态的改变,以判断细菌溶细胞毒素对人脐静脉内皮细胞损伤程度。
结果：
创伤弧菌1.1758株在TCBS平板上生长为绿色菌落,有光泽,为典型的粘液菌落；普通营养琼脂平板上生长为黄白色粘液半透明菌落。生化鉴别结果：“创伤弧菌%id(符合率)=98probablitity"。
1、创伤弧菌对人脐静脉内皮细胞增殖能力以及细胞形态的影响
(1)通过96孔板培养法和MTT法对不同浓度创伤弧菌侵袭过的血管内皮细胞生长曲线以及吸光率的测定,10cfu/ml组、102cfu/ml组、103cfu/ml组、104cfu/ml组细胞24h增殖抑制率分别为：(10.27±7.17)%,(16.13±14.33)%,(26.01±6.45)%,(37.46±5.06)%；48h增殖抑制率分别为：(13.12±8.60)%,(26.63±5.42)%,(34.20±7.92)%,(42.63±3.58)%；72h增殖抑制率分别为：(16.50±5.92)%,(28.93±5.48)%,(38.93±4.94)%,(49.62±20.35)%：96h增殖抑制率分别为：(25.444±8.15)%,(36.41±15.31)%,(45.37±14.51)%,(52.81±10.51)%。(2)台盼蓝拒染法检测对照组和不同浓度创伤弧菌攻击后的4组HU细胞的即刻存活率和24h存活率,即刻存活率分别为100％、100%、99.48%、99.38%、98.55%；24h细胞存活率分别为100%、96.90％、88.48％、76.78%、57.65%。(3)流式细胞仪测定不同浓度Vv对HU细胞凋亡的影响,创伤弧菌攻击组细胞凋亡率明显高于对照组；(4) Hoechest 33342荧光染色观察HU细胞核,空白对照组血管内皮细胞胞核呈均匀的蓝色荧光,加入创伤弧菌24h后,低浓度组10cfu/ml时开始出现凋亡细胞,到创伤弧菌达到104cfu/ml时凋亡细胞迅速增多,呈圆形或椭圆形的高亮蓝色荧光,可见明显的染色质凝集。(5)平板克隆形成实验中对照组克隆形成率为63.73%,10cfu/ml组为39.60%,比Control组下降37.86%；104cfu/ml组为15.53%,比Control组下降91.17%；比10cfu/ml组下降了60.79%。(6)50个高倍视野下正常细胞核分裂数平均为0.640±0.563,与103cfu/ml组细菌侵袭后细胞核分裂数为4.740±0.751。(7)正常组和104cfu/ml细菌攻击组细胞的形态结构测试结果如下：长轴(μm)分别为：36.17±14.34和30.244±8.21,短轴(μm)分别为：15.644±7.32和22.37±5.73,细胞面积(μm2)分别为：431.26±296.37和537.644±239.285,细胞周长(μm)分别为：82.20±26.10和132.69±30.66,轴比分别为：2.61±1.19和1.39±0.38,PE分别为：0.33±0.11和0.444±0.13,AR分别为：0.90±0.12和0.97±0.05,RFF分别为：0.644±0.12和0.66±0.10,FII分别为：1.80±0.30和1.56±0.28,细胞核面积(μm2)分别为：133.70±26.23和143.07±27.85,胞浆面积(μm2)分别为：312.72±268.04和371.34±237.87,核浆比分别为：0.25±0.10和0.75±0.55。(8)正常组HUVEC细胞大部分呈梭形,胞浆粉染,核圆形或卵圆形,核仁较明显。创伤弧菌104cfu/ml攻击组HUVEC细胞结构模糊不清,胞浆淡染,可见微小空泡结构形成；核染色变浅,核仁碎裂,呈消溶态。
2、创伤弧菌内毒素(LPS)的提取、鉴定以及对血管内皮细胞的影响
运用酚水法提取的创伤弧菌内毒素(LPS),经高速离心纯化,鲎试剂鉴定,以及酶标仪单波长405nm处测量吸光度,根据比色算出所提取的内毒素浓度为20434EU/ml.
(1)用MTT法对不同浓度创伤弧菌内毒素侵袭过的血管内皮细胞吸光率进行测定,得出不同浓度细菌对HU细胞的增殖抑制率2500EU/ml、5000EU/ml、7500EU/ml、10000EU/ml24h的增殖抑制率分别为(1.554±0.409)％、(4.762±0.690)%、(4.028±0.743)%、(47.440±4.716)%；48h的增殖抑制率分别为(1.262±0.494)%、(2.893±1.166)%、(15.498±6.786)%、(59.591±5.696)%；72h的增殖抑制率分别为(1.459±0.458)%、(6.649±0.588)%、(14.074±4.088)%、(48.525±2.159)%；96h的增殖抑制率分别为：(4.248±1.312)%、(8.977±2..089)%、(22.929±3.696)％、(56.342±9.336)％。(2)平板克隆形成实验中对照组克隆形成率为72.73%,10000EU/ml组为40.13%,比Control组下降44.82%。(3)流式细胞仪测定LPS对HU细胞凋亡的影响,LPS攻击组细胞凋亡率明显高于正常对照组。(4)正常组和终浓度10000EU／m的细菌内毒素攻击组细胞的形态结构测试结果如下：细胞核的面积(μm2)分别为：135.80±26.37和150.43±32.69,细胞核周长(μm2)分别为：32.00±6.08和70.67±13.87,细胞面积(μm2)分别为：415.43±72.00和442.26±69.74,细胞周长(μm)分别为：141.06±30.75和133.10±17.55,胞浆面积(μm2)分别为：93.55±20.31和303.50±61.33,核浆比分别为：0.44±0.09和0.65±0.15,体积密度分别为：0.35±0.44和0.30±0.04,数密度分别为：0.00016±0.000042和0.00014±0.000045,表面积密度分别为：39.05±8.21和81.93±16.17。
3、创伤弧菌溶细胞毒素的鉴定和浓度的测定及其对血管内皮细胞的影响
对本课题组前期所合成的创伤弧菌外毒素进行SDS-PAGE电泳验证融合蛋白rWC,在凝胶上70KD时出现蛋白条带(创伤弧菌溶细胞毒素分子量约为50KD,载体蛋白约为20KD)。根据标准蛋白浓度及其相应的吸光度值,得到标准曲线,根据所测得的稀释样品的吸光度值,计算出相应的蛋白样品的浓度为4000.282ug/ml。选择50mg/kg浓度的VVC对BALB/c小鼠进行腹腔皮下多点注射,24h后用断椎法处死小鼠,取主要损伤脏器及组织固定、石蜡包埋后按常规方法制片、HE染色,光镜下观察小鼠的肺脏出现出血性损伤；肝脏组织局部细胞呈现灶状、片状坏死,有脓肿样改变；其余脏器几无损伤,表明我们所合成的VVC具有明确的生物活性,用其作用于培养的血管内皮细胞,结果表明：(1)用MTT法对不同浓度创伤弧菌溶细胞毒素(VVC)侵袭过的血管内皮细胞生长曲线以及吸光率进行测定(我们将创伤弧菌溶细胞毒素换算成国际单位),2EU/ml、2.5EU/ml、3EU/ml、3.5EU/ml24h的增殖抑制率分别为(3.31±5.61)%、(3.61±8.22)%、(50.31±2.82)%、(55.11±4.52)%；48h的增殖抑制率分别为(3.22±14.21)%、(3.74±7.91)%、(50.7±1.52)%、(58.71±50.71)%；72h的增殖抑制率分别为(6.33±1.91)％、(9.14±10.72)%、(54.23±5.71)%、(55.7±2.13)%；96h的增殖抑制率分别为(4.21±4.23)％、(5.92±11.82)%、(50.11±0.7)%、(54.33±0.9)%。(2)平板克隆形成实验中对照组克隆形成率为67.87％,3EU/ml组为33.47%,比Control组下降52.10%。(3)流式细胞仪测定细菌外毒素对HU细胞凋亡的影响,VVC攻击组细胞凋亡率高于正常对照组。正常对照组、：3EU/ml和3.5EU/ml创伤弧菌溶细胞毒素攻击组正常细胞指数为：97.43％、47.98％、35.07%；凋亡细胞指数分别为：1.77%、51.21%、61.30%。(4)正常组和3EU/ml细菌外毒素(VVC)攻击组细胞的参数值分别为：细胞核的面积(μm2)分别为：135.80±26.37和147.77±40.62,细胞核周长(μm)分别为：32.00±6.08和91.99±18.30,细胞面积(μm2)分别为：415.43±72.00和240.80±77.77,细胞周长(μm)分别为：141.06±30.75和125.83±26.77,胞浆面积(μm2)分别为：93.55±20.31和70.62±30.11,核浆比分别为：0.42±0.08和2.40±3.59,体积密度分别为：0.35±0.04和0.66±0.16,数密度分别为：0.00016±0.000042和0.00029±0.00013,表面积密度分别为：39.05±8.21和214.19±62.76。
结论
创伤弧菌对体外培养的人脐静脉内皮细胞具有明显的杀伤作用,其杀伤浓度阈值为103cfu/ml。创伤弧菌、创伤弧菌内毒素(LPS)、创伤弧菌溶细胞毒素(VVC)对体外培养的人脐静脉内皮细胞系(HU)的增殖有明显的抑制作用,其抑制能力与创伤弧菌作用的浓度和时间有关,与创伤弧菌内毒素(LPS)、创伤弧菌溶细胞毒素(VVC)作用的浓度有关；均能改变血管内皮细胞的形态并可损伤血管内皮细胞。且通过动物实验可以观察到：溶细胞毒素可以明显损伤小鼠的肝脏和肺脏组织。
Background:
Vibrio vulnificus (Vibrio vulnificus, Vv) is the fifth group of Vibrio bacteria which is gram-stain negative, curved, rod-shaped, pleomorphic, activist and with capsules halophilic. Mainly grows inside the bodies of fritillaria and fish in seawater . It can be divided into three kinds of biological type (Ⅰ、Ⅱ、Ⅲ) while type I is the major pathogens of human wound infections and primary septicemia. It usually infected through an open wound contact with bacteria-containing seawater or raw seafood. So Vibrio vulnificus infection is not only an important issue in trauma medicine at sea but also plays a significant role in the hygiene of seafood. Vv infection can cause serious human infections and sepsis. It also caused multiple organ dysfunction syndrome which is the leading cause of death in patients . The mortality rate of Vv infection is more than 50%. Vibrio vulnificus septicemia sepsis is mainly cause by a large number of Vibrio vulnificus invade into the blood while vascular endothelial cell is the first barrier to resist Vibrio vulnificus entering the blood stream . To reveal the pathogenicity of Vibrio vulnificus against the blood vessel and clarify the influence of the basic form of human vascular endothelial cell and biological behavior juring Vibrio vulnificus infection, we conducted this study for clinical diagnosis and treatment of Vibrio vulnificus infection.
Methods:
The standard strains of Vibrio vulnificus is ATCC27562 which is purchased from the Chinese Academy of Sciences Institute of Microbiology General Microbiology collections management center. Gram stained and observed under light microscopy after recovery culture. Then crossed inoculated on thiosulfate-citrate-bile salts-sucrose agar (thiosulfate-cifrae-bile salt-sucrose, TCBS) agar plate and general nutrition to observe the colony morphology. Finally, cultured the Vv in biochemical identification tube and biochemical strip, machine-readable identification of the results after 24 hours.
First, observe the general characteristics of Vibrio vulnificus and the effects on vascular endothelial cell:
(1) Human umbilical vein endothelial cells was provides by the Guangzhou General Hospital of Guangzhou Military Region. Immunofluorescence and factor VIII identification on it. Through the previous experimental results of our research group ^ we have defined the growth curve of vascular endothelial cell through the 96-well plate culture experiments and MTT method after the attack of Vibrio vulnificus. We also use cell scratch experiment to measure the influence of cell migration after attack by different concentrations of Vibrio vulnificus. (2) Trypan blue staining was used to detect the cell survival immediately and after 24h after invasion. (3) Flow cytometry was used to determine the influence of the apoptosis rate after different concentration Vv attack on the HU cell. The highest Vv attack experimental concentration is 103cfu /ml. (4) Using hoechest 33342 fluorescent staining to detect the change of HU nucleus and (5) plate colony forming assay to detect the change of cell colony-forming ability. (6) Using 50 high magnification views to detect the separatist number of the normal cells and cells after invasion. Observing the changes in the biological behavior of cells under anti-concentration limit. (7) Measuring the stereological parameters of the cell area (Ac), the perimeter (Cc), the nucleus area (An), cytoplasmic area (Acp) of nuclear cytoplasm ratio (Rnp) and cell and nuclear's long axis (dmaxc, dmaxn), cell and nuclear minor axis (dminc, dminn), axial ratio (Rac, Ran), cell and nuclear shape factor PE (PEc, PEn), cell and nuclear shape factor AR (ARc, ARn), the planning cell and nuclear shape factor RFF (RFFc, RFFn) and irregular shape index of FII (FⅡic, FⅡn) in normal group and Vv attack group (104cf/ml) to test proliferation (8) Morphological changes of the human umbilical vein endothelial cells under the attack of Vv.
Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.
To take fresh culture of Vibrio vulnificus and use phenol-water to extract endotoxin then centrifugation it. Ues TAL to identify endotoxin according to the 2005-published "Chinese Pharmacopoeia" and draw the concentration curve.
Select the median lethal concentration of intestinal bacteria endotoxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and endotoxin attack group (104EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of endotoxin and judge the damage of HU.
Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.
Our research team has been pre-recombinanted pET-32a-vvhA/E.coliBL21 (DE3) and for blue-white screening. We select the white plaque for PCR amplification of Vibrio vulnificus cytolysin toxin (rVVC). We also use SDS-PAGE for verification and bicichoninicacid (BCA) to quantitative protein, then render the standard curve and calculate protein concentration.50mg/kg injected into the SPF level BALB/c mice. Execute the mouse and then anatomy immediately. Take the main damage organs and tissues to produced pathological slice and observe it under light microscope.
Select the median lethal concentration of cytolytic toxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and cytolytic toxin attack group (3EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of cytolytic toxin and judge the damage of HU.
Result:
Vibrio vulnificus ATCC27562 grows in TCBS plate green colonies, shiny and shows a typical mucous colony. It also appears yellow white translucent colony mucus in ordinary nutrient agar plates. Biochemical Identification Results:The Vibrio vulnificus%id (coincidence rate) is 98probablitity.
First:The proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of Vv
(1) Through 96 plate culture method and the MTT method to detect the cell growth curve and the determination of absorbance of vascular endothelial under the attack of different concentrations of Vibrio vulnificus invasion. The cells growth inhibition rates after 24h of 10/ml group,102cfu/ml group,103cfu/ml group and 104 cfu/ml were:(10.27±7.17)%, (16.13±14.33)%, (26.01±6.45)%, (37.46±5.06) % and after 48h were:(13.12±8.60)%, (26.63±5.42)%, (34.20±7.92)%, (42.63±3.58)%while after 72h were:(16.50±5.92)%, (28.93±5.48)%, (38.93±4.94)%, (49.62±20.35)% and after 96h were:(25.44±8.15)%, (36.41±15.31)%, (45.37±14.51)%, (52.8U10.51)%. (2) Trypan blue staining method detects the cell survival rate immediately and after 24h after invasion of different concentration. The immediately survival rates were 100%,100%,99.48%, 99.38%,98.55% and the 24h cell survival rates were 100%,96.90%,88.48%,76.78%, 57.65%. (3) Flow cytometry detect result shows the apoptosis rate in Vv attack group is significantly higher than the control group. (4) Hoechest 33342 fluorescent staining of HU nucleus shows the endothelial cell nuclei of blank control group appears uniform blue fluorescence. The cells in low concentration group 10cfu/ml begins to apoptosis 24h after Vv attack while the 104cfu/ml group has the most apoptotic cells and appears round or oval-shaped bright blue fluorescence. We can see a clear chromatin condensation. (5) Colony formation assay cloning efficiency is 63.73% in the control group and 39.60% in the 10/ml group.37.86% decrease compared with Control group. The 104cfu/ml group is 15.53%,91.17% decrease compared with Control group and 60.79% decrease compared with 10cfu/ml group. (6) Under 50 high-power field of vision the division of nuclear in normal cells were 1.48±0.182 and the 104cfu/ml group was 3.02±0.638. (7) The cell parameters of the control group and the Vv attack group whose concentration is 104cfu/ml were as follows:long-axis(μm) are 36.17±14.34 and 30.24±8.21, short-axis(μm) are 15.64±7.32 and 22.37±5.73, cell area(μm2) are 431.26±296.37 and 537.64±239.285, cell circumference (μm) are 82.20±26.10 and 132.69±30.66, axial ratio are 2.61±1.19 and 1.39±0.38, PE are 0.33±0.11 and 0.44±0.13, AR are 0.90±0.12 and 0.97±0.05, RFF are 0.64±0.12 and 0.66±0.10, FII are 1.80±0.30 and 1.56±0.28, nuclear area(μm2) are 133.70±26.23 and 143.07±27.85, cytoplasmic area (μm2) are 312.72±268.04 and 371.34±237.87, nuclear cytoplasm ratio are 0.25±0.10 and 0.75±0.55. (8)The human umbilical vein endothelial cells in normal group appear spindle, cytoplasm stained pink, round or oval nucleus and clear nucleolus. But the cells in Vv attack group appear unclear structure, lightly stained cytoplasm, small vacuoles formed, light nuclear staining, nucleolar fragmentation.
Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.
Detect the extracted endotoxin under a single wavelength of 405nm. According to the standard curve lgT= y-xlgC, (T is the color, C is the concentration), the concentration of the LPS is 20434EU/ml.
(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of LPS with different concentrations. The inhibition rate in groups of 2500EU/ml,5000EU/ml,7500EU/ml and 10000EU/ml after 24hwere (1.554±0.409)%, (4.762±0.690)%, (4.028±0.743)%and (47.440±4.716)%. After 48h were (1.262±0.494)%, (2.893±1.166)%, (15.498±6.786)% and (59.591±5.696)%. After 72h were (1.459±0.458)%, (6.649±0.588)%, (14.074±4.088)% and (48.525±2.159)% while after 96h were (4.248±1.312)%, (8.977±2..089)%, (22.929±3.696)% and (22.929±3.696)%. (2) Colony formation assay cloning efficiency is 72.73% in the control group and 40.13% in the 104EU/ml group. 44.82% decrease compared with Control group. (3) We use FCM to detect the affect of LPS attack over HU and the result shows the apoptosis rate in LPS attack group is higher than control group. (4)The cell parameters of the control group and the LPS group whose final concentration is 104EU/ml were as follows:An(μm2) are 135.80±26.37 and 150.43±32.69, Cn(μm) are 32.00±6.08 and 70.67±13.87, Ac(μm2) are 415.43±72.00 and 442.26±69.74, Cc(μm) are 141.06±30.75 and 133.10±17.55, cytoplasmic area(μm2) are 93.55±20.31 and 303.50±61.33, nuclear cytoplasm ratio are 0.44±0.09 and 0.65±0.15, Vv are 0.35±0.44 and 0.30±0.04, Nv are 0.00016±0.000042 and 0.00014±0.000045, Sv are 39.05±8.21 and 81.93±16.17.
Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.
Put the pre-synthesized exotoxin of Vibrio vulnificus under SDS-PAGE electrophoresis to verify the fusion protein rVVC. It shows protein bands on 70KD of the gelatin (Vibrio vulnificus cytolysin toxin molecular weight of about 50KD, carrier protein is about 20KD). According to the standard protein concentration and its corresponding absorbance values we obtained the standard calculated the corresponding concentration of the protein samples is 4000.282ug/ml. Observing of pathological results under optical microscope we can see there is hemorrhagic injury in the lung of mouse. We can also find part of spotty or sheet necrosis abscess-like change in the liver. But there is no injury in other organs.
(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of VVC with different concentrations. The growth inhibition rates in 2EU/ml group,2.5EU/ml group,3EU/ml group and 3.5EU/ml group after 24h were (3.31±5.61)%, (3.61±8.22)%, (50.31±2.82)% and (55.11±4.52)%. After 48h were (3.22±14.21)%, (3.74±7.91)%, (50.72±1.52)% and (58.71±50.71)%. After 72h were(6.33±1.91)%, (9.14±10.72)%, (54.23±5.71)% and (55.74±2.13)% while after 96h were (4.21±4.23)%, (5.92±11.82)%, (50.11±0.7)% and (54.33±0.9)%. (2)Colony formation assay cloning efficiency is 67.87% in the control group and 33.47% in the 3EU/ml group.52.10% decrease compared with Control group. (3)We use FCM to detect the affect of WC attack over HU and the result shows the apoptosis rate in VVC attack group is higher than control group. The normal cells rate in control group,3EU/ml group and 3.5EU/ml group were 97.43%, 47.98% and 35.07% and the apoptosis index were 1.77%,51.21% and 61.30%. (4)The cell parameters of the control group and the WC group whose concentration is 3EU/ml were as follows:Nucleus area (μm2) are 135.80±26.37 and 147.77±40.62, nuclear perimeter(μm) are 32.00±6.08 and 91.99±18.30, cell area(μm2) are 415.43±72.00 and 240.80±77.77, cell circumference (μm) are 141.06±30.75 and 125.83±26.77, cytoplasmic area (μm2) are 93.55±20.31 and 70.62±30.11, the nuclear cytoplasm ratio are 0.42±0.08 and 2.40±3.59, bulk density are 0.35±0.04 and 0.66±0.16, number density are 0.00016±0.000042 and 0.00029±0.00013, surface density are 39.05±8.21 and 214.19±62.76.
Conclusion:
All Vibrio vulnificus, Vibrio vulnificus lipopolysaccharide (LPS) and cytolytic toxin of Vibrio vulnificus (VVC) have significantly inhibition on proliferation of human umbilical vein endothelial cell line (HU). Its inhibitory ability has something to do with the concentration and action time of Vibrio vulnificus. It also has something to do with the concentration of LPS and WC. And all of them go through the change of the structure of vascular endothelial cells as well as the injury through the vessel wall into the bloodstream then cause severe infection and sepsis.
创伤弧菌(Vibrio vulnificus, Vv)是弧菌属第五群细菌,革兰染色阴性,弯曲、杆状、多形性、运动活泼、有荚膜的细菌,主要生长于海水中的鱼类及贝母类等体内,为嗜盐性海生弧菌。可分为3种生物型(Ⅰ、Ⅱ、Ⅲ型),其中Ⅰ型是导致人类创伤感染和原发性败血症的主要致病菌,一般通过外伤接触含病菌的海水或生食海鲜两种途径感染。因此创伤弧菌感染是海上创伤医学中的重要问题,也是海产品食品卫生中的重要问题。其感染后可引起人体严重感染、脓毒败血症,其引发的多器官功能障碍综合症是脓毒败血症患者死亡的主要原因,病死率高达50%以上。创伤弧菌脓毒败血症的形成主要是大量的创伤弧菌侵入到血液之中,而血管内皮细胞是阻挡创伤弧菌进入血管的重要屏障。因此,创伤弧菌对血管内皮细胞的损伤作用及机制是创伤弧菌败血症形成机制中的重要问题。
为揭示创伤弧菌对血管内皮细胞的损伤作用,阐明创伤弧菌感染时对人血管内皮细胞结构的影响,我们进行了本项研究,为临床创伤弧菌感染的诊治提供一定的理论依据。
方法：
1.创伤弧菌一般特性观察和对血管内皮细胞的影响：
1.1创伤弧菌标准株ATCC27562,购自中国科学院微生物研究所普通微生物保藏管理中心。复苏培养后,行革兰氏染色,光镜观察,划线接种于硫代硫酸盐—枸橼酸盐——胆盐—蔗糖琼脂(thiosulfate-cifrae-bile salt-sucrose, TCBS)平板及普通营养琼脂平板观察其菌落形态,并接种于弧菌属生化鉴定管及生化条培养24h后,机读细菌鉴定结果。
1.2人脐静脉血管内皮细胞(Human umbilical vein endothelial cell, HU),由广州军区广州总医院实验科提供,对其进行VIII因子免疫荧光鉴定。我们通过本课题组前期的实验结果,对创伤弧菌对血管内皮细胞的攻击浓度进行了界定,并通过96孔板培养实验以及MTT法对不同浓度的创伤弧菌侵袭过的血管内皮细胞进行增值抑制率的测定；细胞划痕实验测量不同浓度的创伤弧菌对细胞迁移能力的影响；台盼蓝拒染法检测细胞侵袭后即刻及24h细胞存活率；流式细胞仪测定不同浓度Vv对HU细胞凋亡的影响,选择攻击实验Vv的最高的实验终浓度103cfu/ml。通过Hoechest 33342荧光染色观察HU细胞核的变化；平板克隆形成实验检测细胞集落形成能力的变化,以及50个高倍视野下正常细胞与侵袭后细胞核分裂数的变化,观察极限杀伤浓度下细胞生物学行为的变化。对正常组和创伤弧菌攻击组(104cfu/ml)的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、胞浆面积(Acp)、核浆比(Rnp)以及细胞与核的长轴(dmaxc, dmaxn),细胞与核的短轴(dminc dminn),轴比(Rac、Ran),细胞与核的形状因子PE (PEc、PEn),细胞与核的形状因子AR (ARc、ARn),细胞与核的规划形状因子RFF (RFFc、RFFn),形状不规则指数FⅡ(FⅡc、FⅡn)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌攻击下增殖能力和细胞形态的改变。
2、创伤弧菌内毒素的提取、鉴定以及对血管内皮细胞的影响
2.1取新鲜培养的创伤弧菌,利用酚水法提取细菌内毒素(LPS),并对LPS进行离心纯化,以及根据2005版《中国药典》规定用内毒素测定方法“鲎试剂”对其进行鉴定,并绘制浓度曲线,确定所提取的细菌LPS的浓度。
2.2根据预实验选择创伤弧菌内毒素对细胞的半数致死浓度,选择其上下浓度域共4个浓度对人脐静脉内皮细胞进行MTT,平板克隆,划痕实验测定细胞迁移能力的改变,以及流式细胞仪对细胞凋亡率的测定。对正常组和内毒素10000EU/ml攻击组的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、周长(Cn)、细胞的体积(V),以及细胞的体积密度(Vv)、表面积密度(Sv)、数密度(Nv)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌攻击下增殖能力和细胞以及细胞核形态的改变,以判断细菌内毒素对人脐静脉内皮细胞损伤程度。
3、创伤弧菌外毒素的鉴定和浓度的测定,以及其对血管内皮细胞的影响
3.1本课题组前期已重组大肠杆菌pET-32a-vvhA/E.coliBL21(DE3),行蓝白斑筛选,挑选白色菌斑进行PCR扩增得到创伤弧菌溶细胞毒素(rVVC),对其进行SDS-PAGE电泳验证,采用BCA (bicichoninicacid)法进行蛋白定量,绘制标准曲线,计算蛋白浓度。对SPF级BALB/c小鼠进行腹腔皮下多点注射,50mg/kg,小鼠处死后对其立即解剖,取主要损伤脏器及组织固定、石蜡包埋后按常规方法制片、HE染色,光镜下观察病理变化。
3.2根据预实验选择创伤弧菌溶细胞毒素对细胞的半数致死浓度,选择其浓度域上下共4个浓度对人脐静脉内皮细胞进行MTT,平板克隆,划痕实验测定细胞迁移能力,并用流式细胞仪对细胞凋亡率的测定。对正常组和溶细胞毒素3EU／ml攻击组的细胞爬片进行细胞面积(Ac)和周长(Cc)、细胞核的面积(An)、周长(Cn)以及细胞的体积密度(Vv)、表面积密度(Sv)、数密度(Nv)等参数的体视学测量以检验人脐静脉内皮细胞在创伤弧菌溶细胞毒素(rVVC)攻击下增殖能力和细胞形态的改变,以判断细菌溶细胞毒素对人脐静脉内皮细胞损伤程度。
结果：
创伤弧菌1.1758株在TCBS平板上生长为绿色菌落,有光泽,为典型的粘液菌落；普通营养琼脂平板上生长为黄白色粘液半透明菌落。生化鉴别结果：“创伤弧菌%id(符合率)=98probablitity"。
1、创伤弧菌对人脐静脉内皮细胞增殖能力以及细胞形态的影响
(1)通过96孔板培养法和MTT法对不同浓度创伤弧菌侵袭过的血管内皮细胞生长曲线以及吸光率的测定,10cfu/ml组、102cfu/ml组、103cfu/ml组、104cfu/ml组细胞24h增殖抑制率分别为：(10.27±7.17)%,(16.13±14.33)%,(26.01±6.45)%,(37.46±5.06)%；48h增殖抑制率分别为：(13.12±8.60)%,(26.63±5.42)%,(34.20±7.92)%,(42.63±3.58)%；72h增殖抑制率分别为：(16.50±5.92)%,(28.93±5.48)%,(38.93±4.94)%,(49.62±20.35)%：96h增殖抑制率分别为：(25.444±8.15)%,(36.41±15.31)%,(45.37±14.51)%,(52.81±10.51)%。(2)台盼蓝拒染法检测对照组和不同浓度创伤弧菌攻击后的4组HU细胞的即刻存活率和24h存活率,即刻存活率分别为100％、100%、99.48%、99.38%、98.55%；24h细胞存活率分别为100%、96.90％、88.48％、76.78%、57.65%。(3)流式细胞仪测定不同浓度Vv对HU细胞凋亡的影响,创伤弧菌攻击组细胞凋亡率明显高于对照组；(4) Hoechest 33342荧光染色观察HU细胞核,空白对照组血管内皮细胞胞核呈均匀的蓝色荧光,加入创伤弧菌24h后,低浓度组10cfu/ml时开始出现凋亡细胞,到创伤弧菌达到104cfu/ml时凋亡细胞迅速增多,呈圆形或椭圆形的高亮蓝色荧光,可见明显的染色质凝集。(5)平板克隆形成实验中对照组克隆形成率为63.73%,10cfu/ml组为39.60%,比Control组下降37.86%；104cfu/ml组为15.53%,比Control组下降91.17%；比10cfu/ml组下降了60.79%。(6)50个高倍视野下正常细胞核分裂数平均为0.640±0.563,与103cfu/ml组细菌侵袭后细胞核分裂数为4.740±0.751。(7)正常组和104cfu/ml细菌攻击组细胞的形态结构测试结果如下：长轴(μm)分别为：36.17±14.34和30.244±8.21,短轴(μm)分别为：15.644±7.32和22.37±5.73,细胞面积(μm2)分别为：431.26±296.37和537.644±239.285,细胞周长(μm)分别为：82.20±26.10和132.69±30.66,轴比分别为：2.61±1.19和1.39±0.38,PE分别为：0.33±0.11和0.444±0.13,AR分别为：0.90±0.12和0.97±0.05,RFF分别为：0.644±0.12和0.66±0.10,FII分别为：1.80±0.30和1.56±0.28,细胞核面积(μm2)分别为：133.70±26.23和143.07±27.85,胞浆面积(μm2)分别为：312.72±268.04和371.34±237.87,核浆比分别为：0.25±0.10和0.75±0.55。(8)正常组HUVEC细胞大部分呈梭形,胞浆粉染,核圆形或卵圆形,核仁较明显。创伤弧菌104cfu/ml攻击组HUVEC细胞结构模糊不清,胞浆淡染,可见微小空泡结构形成；核染色变浅,核仁碎裂,呈消溶态。
2、创伤弧菌内毒素(LPS)的提取、鉴定以及对血管内皮细胞的影响
运用酚水法提取的创伤弧菌内毒素(LPS),经高速离心纯化,鲎试剂鉴定,以及酶标仪单波长405nm处测量吸光度,根据比色算出所提取的内毒素浓度为20434EU/ml.
(1)用MTT法对不同浓度创伤弧菌内毒素侵袭过的血管内皮细胞吸光率进行测定,得出不同浓度细菌对HU细胞的增殖抑制率2500EU/ml、5000EU/ml、7500EU/ml、10000EU/ml24h的增殖抑制率分别为(1.554±0.409)％、(4.762±0.690)%、(4.028±0.743)%、(47.440±4.716)%；48h的增殖抑制率分别为(1.262±0.494)%、(2.893±1.166)%、(15.498±6.786)%、(59.591±5.696)%；72h的增殖抑制率分别为(1.459±0.458)%、(6.649±0.588)%、(14.074±4.088)%、(48.525±2.159)%；96h的增殖抑制率分别为：(4.248±1.312)%、(8.977±2..089)%、(22.929±3.696)％、(56.342±9.336)％。(2)平板克隆形成实验中对照组克隆形成率为72.73%,10000EU/ml组为40.13%,比Control组下降44.82%。(3)流式细胞仪测定LPS对HU细胞凋亡的影响,LPS攻击组细胞凋亡率明显高于正常对照组。(4)正常组和终浓度10000EU／m的细菌内毒素攻击组细胞的形态结构测试结果如下：细胞核的面积(μm2)分别为：135.80±26.37和150.43±32.69,细胞核周长(μm2)分别为：32.00±6.08和70.67±13.87,细胞面积(μm2)分别为：415.43±72.00和442.26±69.74,细胞周长(μm)分别为：141.06±30.75和133.10±17.55,胞浆面积(μm2)分别为：93.55±20.31和303.50±61.33,核浆比分别为：0.44±0.09和0.65±0.15,体积密度分别为：0.35±0.44和0.30±0.04,数密度分别为：0.00016±0.000042和0.00014±0.000045,表面积密度分别为：39.05±8.21和81.93±16.17。
3、创伤弧菌溶细胞毒素的鉴定和浓度的测定及其对血管内皮细胞的影响
对本课题组前期所合成的创伤弧菌外毒素进行SDS-PAGE电泳验证融合蛋白rWC,在凝胶上70KD时出现蛋白条带(创伤弧菌溶细胞毒素分子量约为50KD,载体蛋白约为20KD)。根据标准蛋白浓度及其相应的吸光度值,得到标准曲线,根据所测得的稀释样品的吸光度值,计算出相应的蛋白样品的浓度为4000.282ug/ml。选择50mg/kg浓度的VVC对BALB/c小鼠进行腹腔皮下多点注射,24h后用断椎法处死小鼠,取主要损伤脏器及组织固定、石蜡包埋后按常规方法制片、HE染色,光镜下观察小鼠的肺脏出现出血性损伤；肝脏组织局部细胞呈现灶状、片状坏死,有脓肿样改变；其余脏器几无损伤,表明我们所合成的VVC具有明确的生物活性,用其作用于培养的血管内皮细胞,结果表明：(1)用MTT法对不同浓度创伤弧菌溶细胞毒素(VVC)侵袭过的血管内皮细胞生长曲线以及吸光率进行测定(我们将创伤弧菌溶细胞毒素换算成国际单位),2EU/ml、2.5EU/ml、3EU/ml、3.5EU/ml24h的增殖抑制率分别为(3.31±5.61)%、(3.61±8.22)%、(50.31±2.82)%、(55.11±4.52)%；48h的增殖抑制率分别为(3.22±14.21)%、(3.74±7.91)%、(50.7±1.52)%、(58.71±50.71)%；72h的增殖抑制率分别为(6.33±1.91)％、(9.14±10.72)%、(54.23±5.71)%、(55.7±2.13)%；96h的增殖抑制率分别为(4.21±4.23)％、(5.92±11.82)%、(50.11±0.7)%、(54.33±0.9)%。(2)平板克隆形成实验中对照组克隆形成率为67.87％,3EU/ml组为33.47%,比Control组下降52.10%。(3)流式细胞仪测定细菌外毒素对HU细胞凋亡的影响,VVC攻击组细胞凋亡率高于正常对照组。正常对照组、：3EU/ml和3.5EU/ml创伤弧菌溶细胞毒素攻击组正常细胞指数为：97.43％、47.98％、35.07%；凋亡细胞指数分别为：1.77%、51.21%、61.30%。(4)正常组和3EU/ml细菌外毒素(VVC)攻击组细胞的参数值分别为：细胞核的面积(μm2)分别为：135.80±26.37和147.77±40.62,细胞核周长(μm)分别为：32.00±6.08和91.99±18.30,细胞面积(μm2)分别为：415.43±72.00和240.80±77.77,细胞周长(μm)分别为：141.06±30.75和125.83±26.77,胞浆面积(μm2)分别为：93.55±20.31和70.62±30.11,核浆比分别为：0.42±0.08和2.40±3.59,体积密度分别为：0.35±0.04和0.66±0.16,数密度分别为：0.00016±0.000042和0.00029±0.00013,表面积密度分别为：39.05±8.21和214.19±62.76。
结论
创伤弧菌对体外培养的人脐静脉内皮细胞具有明显的杀伤作用,其杀伤浓度阈值为103cfu/ml。创伤弧菌、创伤弧菌内毒素(LPS)、创伤弧菌溶细胞毒素(VVC)对体外培养的人脐静脉内皮细胞系(HU)的增殖有明显的抑制作用,其抑制能力与创伤弧菌作用的浓度和时间有关,与创伤弧菌内毒素(LPS)、创伤弧菌溶细胞毒素(VVC)作用的浓度有关；均能改变血管内皮细胞的形态并可损伤血管内皮细胞。且通过动物实验可以观察到：溶细胞毒素可以明显损伤小鼠的肝脏和肺脏组织。
Background:
Vibrio vulnificus (Vibrio vulnificus, Vv) is the fifth group of Vibrio bacteria which is gram-stain negative, curved, rod-shaped, pleomorphic, activist and with capsules halophilic. Mainly grows inside the bodies of fritillaria and fish in seawater . It can be divided into three kinds of biological type (Ⅰ、Ⅱ、Ⅲ) while type I is the major pathogens of human wound infections and primary septicemia. It usually infected through an open wound contact with bacteria-containing seawater or raw seafood. So Vibrio vulnificus infection is not only an important issue in trauma medicine at sea but also plays a significant role in the hygiene of seafood. Vv infection can cause serious human infections and sepsis. It also caused multiple organ dysfunction syndrome which is the leading cause of death in patients . The mortality rate of Vv infection is more than 50%. Vibrio vulnificus septicemia sepsis is mainly cause by a large number of Vibrio vulnificus invade into the blood while vascular endothelial cell is the first barrier to resist Vibrio vulnificus entering the blood stream . To reveal the pathogenicity of Vibrio vulnificus against the blood vessel and clarify the influence of the basic form of human vascular endothelial cell and biological behavior juring Vibrio vulnificus infection, we conducted this study for clinical diagnosis and treatment of Vibrio vulnificus infection.
Methods:
The standard strains of Vibrio vulnificus is ATCC27562 which is purchased from the Chinese Academy of Sciences Institute of Microbiology General Microbiology collections management center. Gram stained and observed under light microscopy after recovery culture. Then crossed inoculated on thiosulfate-citrate-bile salts-sucrose agar (thiosulfate-cifrae-bile salt-sucrose, TCBS) agar plate and general nutrition to observe the colony morphology. Finally, cultured the Vv in biochemical identification tube and biochemical strip, machine-readable identification of the results after 24 hours.
First, observe the general characteristics of Vibrio vulnificus and the effects on vascular endothelial cell:
(1) Human umbilical vein endothelial cells was provides by the Guangzhou General Hospital of Guangzhou Military Region. Immunofluorescence and factor VIII identification on it. Through the previous experimental results of our research group ^ we have defined the growth curve of vascular endothelial cell through the 96-well plate culture experiments and MTT method after the attack of Vibrio vulnificus. We also use cell scratch experiment to measure the influence of cell migration after attack by different concentrations of Vibrio vulnificus. (2) Trypan blue staining was used to detect the cell survival immediately and after 24h after invasion. (3) Flow cytometry was used to determine the influence of the apoptosis rate after different concentration Vv attack on the HU cell. The highest Vv attack experimental concentration is 103cfu /ml. (4) Using hoechest 33342 fluorescent staining to detect the change of HU nucleus and (5) plate colony forming assay to detect the change of cell colony-forming ability. (6) Using 50 high magnification views to detect the separatist number of the normal cells and cells after invasion. Observing the changes in the biological behavior of cells under anti-concentration limit. (7) Measuring the stereological parameters of the cell area (Ac), the perimeter (Cc), the nucleus area (An), cytoplasmic area (Acp) of nuclear cytoplasm ratio (Rnp) and cell and nuclear's long axis (dmaxc, dmaxn), cell and nuclear minor axis (dminc, dminn), axial ratio (Rac, Ran), cell and nuclear shape factor PE (PEc, PEn), cell and nuclear shape factor AR (ARc, ARn), the planning cell and nuclear shape factor RFF (RFFc, RFFn) and irregular shape index of FII (FⅡic, FⅡn) in normal group and Vv attack group (104cf/ml) to test proliferation (8) Morphological changes of the human umbilical vein endothelial cells under the attack of Vv.
Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.
To take fresh culture of Vibrio vulnificus and use phenol-water to extract endotoxin then centrifugation it. Ues TAL to identify endotoxin according to the 2005-published "Chinese Pharmacopoeia" and draw the concentration curve.
Select the median lethal concentration of intestinal bacteria endotoxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and endotoxin attack group (104EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of endotoxin and judge the damage of HU.
Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.
Our research team has been pre-recombinanted pET-32a-vvhA/E.coliBL21 (DE3) and for blue-white screening. We select the white plaque for PCR amplification of Vibrio vulnificus cytolysin toxin (rVVC). We also use SDS-PAGE for verification and bicichoninicacid (BCA) to quantitative protein, then render the standard curve and calculate protein concentration.50mg/kg injected into the SPF level BALB/c mice. Execute the mouse and then anatomy immediately. Take the main damage organs and tissues to produced pathological slice and observe it under light microscope.
Select the median lethal concentration of cytolytic toxin to HU cells. Select the domain of its upper and lower concentration of total 4 concentrations on the human umbilical vein endothelial cells for MTT, plate cloning and scratches to determine the migration changes of the cells. Use flow cytometry to determine the apoptosis rate. Measuring the stereological parameters of the cell area (Ac), perimeter (Cc), the nucleus area (An), circumference (Cn), cell volume (V), cell volume density (Vv), surface density (Sv) and numerical density (Nv) in normal group and cytolytic toxin attack group (3EU/ml) to test proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of cytolytic toxin and judge the damage of HU.
Result:
Vibrio vulnificus ATCC27562 grows in TCBS plate green colonies, shiny and shows a typical mucous colony. It also appears yellow white translucent colony mucus in ordinary nutrient agar plates. Biochemical Identification Results:The Vibrio vulnificus%id (coincidence rate) is 98probablitity.
First:The proliferation and morphological changes of the human umbilical vein endothelial cells under the attack of Vv
(1) Through 96 plate culture method and the MTT method to detect the cell growth curve and the determination of absorbance of vascular endothelial under the attack of different concentrations of Vibrio vulnificus invasion. The cells growth inhibition rates after 24h of 10/ml group,102cfu/ml group,103cfu/ml group and 104 cfu/ml were:(10.27±7.17)%, (16.13±14.33)%, (26.01±6.45)%, (37.46±5.06) % and after 48h were:(13.12±8.60)%, (26.63±5.42)%, (34.20±7.92)%, (42.63±3.58)%while after 72h were:(16.50±5.92)%, (28.93±5.48)%, (38.93±4.94)%, (49.62±20.35)% and after 96h were:(25.44±8.15)%, (36.41±15.31)%, (45.37±14.51)%, (52.8U10.51)%. (2) Trypan blue staining method detects the cell survival rate immediately and after 24h after invasion of different concentration. The immediately survival rates were 100%,100%,99.48%, 99.38%,98.55% and the 24h cell survival rates were 100%,96.90%,88.48%,76.78%, 57.65%. (3) Flow cytometry detect result shows the apoptosis rate in Vv attack group is significantly higher than the control group. (4) Hoechest 33342 fluorescent staining of HU nucleus shows the endothelial cell nuclei of blank control group appears uniform blue fluorescence. The cells in low concentration group 10cfu/ml begins to apoptosis 24h after Vv attack while the 104cfu/ml group has the most apoptotic cells and appears round or oval-shaped bright blue fluorescence. We can see a clear chromatin condensation. (5) Colony formation assay cloning efficiency is 63.73% in the control group and 39.60% in the 10/ml group.37.86% decrease compared with Control group. The 104cfu/ml group is 15.53%,91.17% decrease compared with Control group and 60.79% decrease compared with 10cfu/ml group. (6) Under 50 high-power field of vision the division of nuclear in normal cells were 1.48±0.182 and the 104cfu/ml group was 3.02±0.638. (7) The cell parameters of the control group and the Vv attack group whose concentration is 104cfu/ml were as follows:long-axis(μm) are 36.17±14.34 and 30.24±8.21, short-axis(μm) are 15.64±7.32 and 22.37±5.73, cell area(μm2) are 431.26±296.37 and 537.64±239.285, cell circumference (μm) are 82.20±26.10 and 132.69±30.66, axial ratio are 2.61±1.19 and 1.39±0.38, PE are 0.33±0.11 and 0.44±0.13, AR are 0.90±0.12 and 0.97±0.05, RFF are 0.64±0.12 and 0.66±0.10, FII are 1.80±0.30 and 1.56±0.28, nuclear area(μm2) are 133.70±26.23 and 143.07±27.85, cytoplasmic area (μm2) are 312.72±268.04 and 371.34±237.87, nuclear cytoplasm ratio are 0.25±0.10 and 0.75±0.55. (8)The human umbilical vein endothelial cells in normal group appear spindle, cytoplasm stained pink, round or oval nucleus and clear nucleolus. But the cells in Vv attack group appear unclear structure, lightly stained cytoplasm, small vacuoles formed, light nuclear staining, nucleolar fragmentation.
Second:Vibrio vulnificus endotoxin extraction, identification, as well as affect on vascular endothelial cells.
Detect the extracted endotoxin under a single wavelength of 405nm. According to the standard curve lgT= y-xlgC, (T is the color, C is the concentration), the concentration of the LPS is 20434EU/ml.
(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of LPS with different concentrations. The inhibition rate in groups of 2500EU/ml,5000EU/ml,7500EU/ml and 10000EU/ml after 24hwere (1.554±0.409)%, (4.762±0.690)%, (4.028±0.743)%and (47.440±4.716)%. After 48h were (1.262±0.494)%, (2.893±1.166)%, (15.498±6.786)% and (59.591±5.696)%. After 72h were (1.459±0.458)%, (6.649±0.588)%, (14.074±4.088)% and (48.525±2.159)% while after 96h were (4.248±1.312)%, (8.977±2..089)%, (22.929±3.696)% and (22.929±3.696)%. (2) Colony formation assay cloning efficiency is 72.73% in the control group and 40.13% in the 104EU/ml group. 44.82% decrease compared with Control group. (3) We use FCM to detect the affect of LPS attack over HU and the result shows the apoptosis rate in LPS attack group is higher than control group. (4)The cell parameters of the control group and the LPS group whose final concentration is 104EU/ml were as follows:An(μm2) are 135.80±26.37 and 150.43±32.69, Cn(μm) are 32.00±6.08 and 70.67±13.87, Ac(μm2) are 415.43±72.00 and 442.26±69.74, Cc(μm) are 141.06±30.75 and 133.10±17.55, cytoplasmic area(μm2) are 93.55±20.31 and 303.50±61.33, nuclear cytoplasm ratio are 0.44±0.09 and 0.65±0.15, Vv are 0.35±0.44 and 0.30±0.04, Nv are 0.00016±0.000042 and 0.00014±0.000045, Sv are 39.05±8.21 and 81.93±16.17.
Third:Cytolytic toxin of Vibrio vulnificus identification and determination of the concentration and its effects on vascular endothelial cells.
Put the pre-synthesized exotoxin of Vibrio vulnificus under SDS-PAGE electrophoresis to verify the fusion protein rVVC. It shows protein bands on 70KD of the gelatin (Vibrio vulnificus cytolysin toxin molecular weight of about 50KD, carrier protein is about 20KD). According to the standard protein concentration and its corresponding absorbance values we obtained the standard calculated the corresponding concentration of the protein samples is 4000.282ug/ml. Observing of pathological results under optical microscope we can see there is hemorrhagic injury in the lung of mouse. We can also find part of spotty or sheet necrosis abscess-like change in the liver. But there is no injury in other organs.
(1)Through MTT and pre-test we determine the cell growth curve and absorbance of HU under the attack of VVC with different concentrations. The growth inhibition rates in 2EU/ml group,2.5EU/ml group,3EU/ml group and 3.5EU/ml group after 24h were (3.31±5.61)%, (3.61±8.22)%, (50.31±2.82)% and (55.11±4.52)%. After 48h were (3.22±14.21)%, (3.74±7.91)%, (50.72±1.52)% and (58.71±50.71)%. After 72h were(6.33±1.91)%, (9.14±10.72)%, (54.23±5.71)% and (55.74±2.13)% while after 96h were (4.21±4.23)%, (5.92±11.82)%, (50.11±0.7)% and (54.33±0.9)%. (2)Colony formation assay cloning efficiency is 67.87% in the control group and 33.47% in the 3EU/ml group.52.10% decrease compared with Control group. (3)We use FCM to detect the affect of WC attack over HU and the result shows the apoptosis rate in VVC attack group is higher than control group. The normal cells rate in control group,3EU/ml group and 3.5EU/ml group were 97.43%, 47.98% and 35.07% and the apoptosis index were 1.77%,51.21% and 61.30%. (4)The cell parameters of the control group and the WC group whose concentration is 3EU/ml were as follows:Nucleus area (μm2) are 135.80±26.37 and 147.77±40.62, nuclear perimeter(μm) are 32.00±6.08 and 91.99±18.30, cell area(μm2) are 415.43±72.00 and 240.80±77.77, cell circumference (μm) are 141.06±30.75 and 125.83±26.77, cytoplasmic area (μm2) are 93.55±20.31 and 70.62±30.11, the nuclear cytoplasm ratio are 0.42±0.08 and 2.40±3.59, bulk density are 0.35±0.04 and 0.66±0.16, number density are 0.00016±0.000042 and 0.00029±0.00013, surface density are 39.05±8.21 and 214.19±62.76.
Conclusion:
All Vibrio vulnificus, Vibrio vulnificus lipopolysaccharide (LPS) and cytolytic toxin of Vibrio vulnificus (VVC) have significantly inhibition on proliferation of human umbilical vein endothelial cell line (HU). Its inhibitory ability has something to do with the concentration and action time of Vibrio vulnificus. It also has something to do with the concentration of LPS and WC. And all of them go through the change of the structure of vascular endothelial cells as well as the injury through the vessel wall into the bloodstream then cause severe infection and sepsis.
引文
[1]Roland FP. Leg gangrene and endotoxin shock due to vibrio parahaemolyticus-an infection acquired in New England coastal waters [J]. N Engl J Med,1970,28(2):1306-9.
[2]Hollis DG, Weaver RE, Baker CN, et al. Halophilic Vibrio species isolated from blood cultures [J]. Clin Microbiol,1976,3(5):425-428.
[3]Farmer JJ. Ⅲ. Vibrio ('Beneckea') vulnificus. The bacterium associated with sepsis, Septicaemia, and the sea [J]. Lancet,1979,12(9):902-5.
[4]韩怀忠,张丽菊,郑艳菊,等.创伤弧菌食物中毒[J].中国食品卫生杂志,1997,9(6)：32-34.
[5]Haq SM, Dayal HH. Chronic Liver Disease and Consumption of Raw Oysters: A Potentially Lethal Combinaton-A review of Vibrio vulnificus Septicemia [J]. Am J Gastroenterol,2005,100(5):1195-9.
[6]M Y Sie, P C Ip-Yam, L L E Oon. Vibrio vulnificus septicaemia [J]. Anaesthesia and Intensive Care,2002,30(1):77-79.
[7]Chang JJ, Sheen IS, Peng SM, et al. Vibrio vulnificus infection-report of 8 cases and review of cases[J]. Changgeng Yi Xue Za Zhi,1994,17(4):339-346.
[8]Patel VJ, Gardner E, Burton CS. Vibrio vulnificus septicemia and leg ulcer [J]. Am Acad Dermatol.2002,46(5):144-5.
[9]Hlady Wg, Klontz Kc. The epide miology of Vibrio infections in Florida, 1981～1993 [J]. Infect Dis,1996,173(5):1176-1183.
[10]Klontz KC,Lieb B,Schreiber M, et al. Syndromes of Vibrio vulnificus infection-clinical and epide miologic features in Florida cases,1981～1987[J]. Ann Intern Med,1988,109(12):318-323.
[11]韦兴,侯树勋.海水浸泡火器伤的特点与救治原则[J].解放军预防医学杂志,2003,21(1)：76-79.
[12]卜海激,申洪,朱天岭等.犬海水中火器伤细菌感染定量研究[J]..第一军医大学学报.2003,23(6)：598-601.
[13]Johnson RW, Arnett FC. A fatal case of Vibrio vulnificus presenting as septic arthritis [J]. Arch Intern Med,2001,161(21):2616-8.
[14]Sato T, Inatomi Y, Yonehara T, et al. A patient with Vibrio Vulnificus meningoencephalitis [J]. Rinsho Shinkeigaku,2005,45(1):18-21.
[15]Hsueh, P.R., C. Y. Lin, et al. Vibrio vulnificus in Taiwan [J]. Emerg Infect Dis, 2005,10(9):1363-1368.
[16]Osaka, K., M. Komatsuzaki, H. Takahashi, et al.2004. Vibrio vulnificus septicaemia in Japan:an estimated number of infections and physicians' knowledge of the syndrome [J]. Epidemiol Infect,2009,13(2):993-996.
[17]江维平.创伤弧菌肠道感染一例[J].中华医学检验杂志,1990,13(5)：255.
[18]Tsai YH, Huang TJ, Hsu RW et al. Necrotizing soft-tissue infections and primary sepsis caused by Vibrio vulnificus and Vibrio cholerae non-O1 [J] Trauma,2009,66(3):899-905.
[19]陈龙、洪建军、陈星隆等以骨筋膜室综合征首发的创伤弧菌败血症1例[J].浙江创伤外科,2004,6(9)：418-418.
[20]Klontz K.C, Lieb S, Schreiber M, et al. Syndromes of Vibrio vulnificus infections:Clinical and epidemiologic features in Florida cases,1981-1987 [J]. Ann Intern Med,1988,109(1988):318.
[21]Koenig K.L, Mueller J, Rose T. Hazard on the half shell [J]. West J Med, 1991,155 (15):400-406.
[22]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005；13(7)：860-863.
[23][23]Dalsgaard A, Frimodt-Moller N, Bruun B, et al. Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark [J]. Clin Microbiol Infect Dis,2009,15(7):227-230.
[24]Liu M, Naka H, Crosa JH. HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6 [J]. Mol Microbiol,2009,72(2):491-505.
[25]Mark S. Strom, Rohinee N. Paranjpye:Epidemiology and pathogenesis of Vibrio vulnificus [J]. Microbes and Infection,2000, (2):177-188
[26]Zhao JF, Sun AH, Ruan P.Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway [J]. Microb Pathog,2009,46(4):194-200.
[27]Chen YC, Chang MC, Chuang YC, et al. Characterization and virulence of hemolysin III from Vibrio vulnificus[J]. Curr Microbiol,2004,49(3):175-179.
[28]Gray L.D, Kreger A.S, Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus [J]. Infect Immun,1998,48(5):62-67.
[29]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion initiated apoptotic signaling pat hway in human ECV304 cells [J]. Biol Chem,2001,276(50):475182-47523.
[30]Kang MK, J hee EC, Koo BS, et al. Induction of nitric oxide synt hase expression by Vibrio vulnificus cytolysin [J]. Biochem Biophys Res Commun, 2002,290(3):1090-1095.
[31]Viriyakosol S, Kirkland T. Knowledge of cellular receptors for bacterial endotoxin 1995[J]. Clin Infect Dis,1995,21(2):190-195.
[32]罗向东,杨宗城,黎鳌.家兔烧伤早期内脏血管内皮细胞损伤的变化[J].第三军医大学学报,1995,17(4)：279-283.
[33]黎鳌.创伤后脏器功能不全[M].河北科学技术出版社,1999.560-584.
[34]李主一.火器伤外科学[M].人民军医出版社,第一版.1982.114-120.
[35]贾小明,郭振荣,盛志勇,等.肉芽创面组织细菌定量培养与植皮成活的关系[J].解放军医学杂志,1985,17(5)：102-122.
[1]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[2]王贵明,创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白主动免疫的动物实验研究[M].南方医科大学硕士论文,2009.
[3]彭慧如,吴赛珠.睾酮对血管内皮细胞衰老的干预作用[J].中国老年病杂志,2008,28(10)：940-942.
[4]H Shen. A new concept and calculating method for quantitative form description. Quantitative Cytology and Histology,2000
[5]申洪,沈忠英.实用生物体视学技术[M].广州：中山大学出版社,1991.08.
[6]Parvizi J, Parpura V, Greenleaf JF, et al.Calcium signaling is required for μltrasound-stimμlated aggrecan synthesis by rat chondrocytes [J]. J Orthop Res,2002,20(1):51-57.
[7]Hsueh PR, Lin CY, Tang HJ, et al. Vibrio vulnificus in Taiwan [J]. Emerg Infect Dis,2004,10(8):1363-1368.
[8]Kang, In-hye, Ju-Sim kim, Jeong K. LEE. The Virulence of Vibrio vulnificus is Affected by the Cellular Level of Superoxide Dismutase Activity [J]. Microbiol. Biotechnol,2007,17(8):1399-1402.
[9]Man Hwan, Hee Gon Jeong, and Sang Ho Choi. Proteomic Identification and Characterization of Vibrio vulnificus Proteins Induced upon Exposure to INT-407 Intestinal Epithelial Cells[J]. Microbiol Biotechnol,2008,18(5): 968-974.
[10]Zuppardo AB, Siebeling RJ. An epimerase gene essential for capsule synthesis in Vibrio vulnificus [J]. Infect Immun,1998,66(6):2601-2606.
[11]Wright A.C, Simpson L.M, Oliver J.D, et al. Phenotypic evaluation of acapsular transposon mutants ofVibrio vulnificus [J]. Infect Immun,2008,58 (1990):1769-1773.
[12]Yoshida S., Ogawa M, Mizuguchi Y. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus [J]. Infect Immun,1985,47 (5):446-9.
[13]Valiente E, Jimenez N, Merino S, Tomas JM. Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence [J]. Infect Immun,2008,76(4):1628-38.
[14]Oliver JD,Wear JE,Thimas MB,et al. Production of extracellular enzymes and cytotoxicity by Vibrio Vulnificus[J]. Diagn Microbiol Infect Dis,1986,5(2): 99-111.
[15]Vinogradov E, Wilde C, Anderson EM. Structure of the lipopolysaccharide core of Vibrio vulnificus type strain[J]. Carbohydrate Research,2009,344(4): 484-490.
[16]Gray L.D., Kreger A.S., Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus [J]. Infect Immun,1985,48 (5): 62-68.
[17]Zhao JF, Sun AH, Ruan P.Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway[J]. Microb Pathog,2009,46(4):194-200.
[18]Chen YC, Chang MC, Chuang YC, Jeang CL.Characterization and virulence of hemolysin III from Vibrio vulnificus [J]. Curr Microbiol.2004, 49(3):175-179.
[19]Kang-Beom Kwon, Jeong-Yeh Yang, Do-Gon Ryu. Vibrio vulnificus Cytolysin Induces Superoxide Anion-initiatedApoptotic Signaling Pathway in Human ECV304 Cells [J].The journal of biological chemistry,2001,276(50): 47518-47523.
[20]Fan JJ, Shao CP, Ho YC. Isolation and Characterization of a Vibrio vulnificus Mutant Deficient in Both Extracellular Metalloprotease and Cytolysin [J]. Infect Immun,2001,69(9):5943-5948
[21]chung-ping shao,lien-I Hor.Metalloprotease Is Not Essential for Vibrio vulnificus Virulence in Mice [J].Infection and immunity,2000,15(24): 3569-3573.
[22]Bisharat N,Agmon V,Finkelstein R,etal.Clinical, epide miological, and microbiological features of Vibrio vulnificus biogroups causing outbreaks of wound infection and bacteraemia in Israel.Israel Vibrio Study Group [J]. Lancet,1999,354(9188):1421-8.
[23]M Y Sie, P C Ip-Yam,L L E Oon. Vibrio Vulnificus sepeicamia and Intensive Care[J].2002,30(1):77-79.
[24]Choi HJ,Lee DK, Lee MW, Moon KC,Koh JK. Vibrio vulnificus septicaemia presenting as purpura fulminans[J]. dermatol,2005,32(1):46-51.
[25]Cohen AL, Oliver JD, DePaola A, et al. Emergence of a virulent clade of Vibrio vulnificus and correlation with the presence of a 33-kilobase genomic island [J]. Appl Environ Microbiol,2007,73(17):5553-65.
[26][26]Russell AD.Radiation, Russell AD, et al.Principles and Practice of Disinfection. Preservation and Sterilization[J] Blackwell Scien.tific Publication,1999,49(6):688-702.
[27]吴斌,卢中秋创伤弧菌感染的致病机制研究现状[J]. Chin J Crit Care Med, 2004,24(11):834.
[28]Luder CG, Gross U, Lopes MF. Int racellular protozoan parasites and apoptosis:diverse st rategies to modulate parasite2host inter2 actions[J]. Trends Parasitol,2001,17(10):480-486.
[29]郑晶.创伤弧菌溶细胞毒素及金属蛋白酶的基因克隆、蛋白表达、抗体制备及其致病性的研究[J].广州：南方医科大学博士学位论文,2008.18
[1]王贵明.创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白抗原主动免疫的动物实验研究[M].南方医科大学硕士毕业论文,2009.
[2]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[3]宋宏新,刘晓阳,李宏.改良热酚水法制备大肠杆菌0157：H7脂多糖抗原的研究[J].食品科学,2006,27(10)：273-275.
[4]Dejana E, Corada M, Lampugnani MG. Endothelial cell to cell junction[J]. FASEB J,1995,9(14):910-918.
[5]张小燕,梁朝.细菌内毒素作用血管内皮细胞的机制及其临床疾病[J].中华检验医学杂志,2004,27(111)：811-813.
[6]国家药典委员会编著.2005版,中国药典第二部[M],化学药品,212-214.
[7]Riely L W, Remis R S, Helgerson S D, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype[J]. N Eng J Med,1983,30(8):681-685.
[8]Carol G Currie, Ian R Poxton. The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-0157 verotoxin-producing E.coli[J]. FEMS Immunology and Medical Microbiology,1999,24:57-62.
[9]Nishiuchi Y, Doe M, Hotta H, et al. Structure and serologic properties of-specificpolysaccharidefrom Citrobacterfreundii possessingcrossreactivity with Escherichia coli O157:H7[J]. Immunology and Medical Microbiology, 2000,28:163-171.
[10]Williams AH, Raetz CRH. Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase[J]. Proc Natl Acad Sci USA,2007,104(34):13543-13550.
[11]冯淑霞,李金钟.硫酸链霉素细菌内毒素定量测定法的研究中国兽药杂志2006,40(3)：6-8.
[12]Coggins BE, Li X, McClerren AL, et al. Structure of the LpxC deacetylase with a bound substrate-analog inhibitor[J]. Nat Struct Biol,2003,10(8): 645-651.
[13]Yamamoto T, Harada N, Kano K, et al. The Ras TargetAF26 Interactswith ZO21 and Serves as a Peripheral Component of Tight Junctions in Ep ithelial Cells [J]. J Cell Biol,1997,139(3):785-795.
[14]Izumi Y, Hirose T, Tamai Y, et al. An Atyp ical PKC Directly Associates and Colocalizes at the Ep ithelial Tight Junction with ASIP, aMammalian Homologue of Caenorhabditis elegans Polarity Protein PAR23 [J]. J Cell Biol, 1998,143(1):95-106.
[15]Chandana S, Sanjay K, Nigam, et al. Expanding Role of G Proteins in Tight Junction Regulation:GaS Stimulates TJ Assembly [J]. Biochemical and Biophysical Research Communications,2001,285(2):250-256.
[16]申洪,沈忠英.实用生物体视学技术[M].中山大学出版社,1991.08.
[17]Hsueh PR, Lin CY, Tang HJ, et al. Vibrio vulnificus in Taiwan. Emerg Infect Dis[J].2004,10(8):1363-1368.
[18]王永堂.鲁秀敏细菌内毒素与抗内毒素治疗[J]. Chinese Journal of Biochemical Pharmaceutics,2002,23(5):266-268.
[19]闻玉梅.现代医学微生物学[M].上海医科大学出版社,1999.124-130.
[20]张小燕.梁朝细菌内毒素作用血管内皮细胞的机制及其临床疾病.Chin J Lab Med,2004,27(11):811-814.
[21]Triantafilou M, Triantafilou K, Fernandez N. Rough and smooth forms of fluorescein labelled bacterial endotoxin exhibit CD 14/LBP dependent and independent binding that is influencedby endotoxin concentration[J]. Eur J Biochem,2000,267:2218-2226.
[22]Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity[J]. J Endotoxin Res,2001,7(3):167-171.
[1]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion2initiated apoptotic signaling pat hway in human ECV304 cells[J]. J Biol Chem,2001,276(50):47518-47523.
[2]王贵明,创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白主动免疫的动物实验研究[M].南方医科大学硕士论文,导师：申洪,2009.
[3]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[4]彭慧如,吴赛珠.睾酮对血管内皮细胞衰老的干预作用[J].中国老年病杂志,2008,28(10)：940-942.
[5]顾小龙,严全能,张园.尾加压素Ⅱ对人脐静脉内皮细胞凋亡的影响[J].实用医学杂志,2006,22(11)：1228-1230.
[6]申洪,沈忠英.实用生物体视学技术[M].中山大学出版社,1991.08.
[7]Parvizi J, Parpura V, Greenleaf JF, et al.Calcium signaling is required for ultrasound-stimμlated aggrecan synthesis by rat chondrocytes.J Orthop Res, 2002,20(1):51-57.
[8]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion2initiated apoptotic signaling pat hway in human ECV304 cells[J]. J Biol Chem,2001,276(50):47518-47523.
[9]李桂军楼永良创伤弧菌细胞素致病机制的研究进展[J].Int J Lab Med, 2006,27(7):237-241.
[10]Park KH, Yang HB, Kim HG, et al. Low density lipoprotein inactivates Vibrio vulnificus cytolysin t hrough t he oligomerization of toxin monomer[J]. Med Microbiol Immunol (Berl),2005,194(3):137-141.
[11]Lee YR, Park KH, Lin ZZ, et al. A calcium2calmodulin antagonist blocks experimental Vibrio vulnificus cytolysin induced let hality in an experimental mouse model[J]. Infect Immun,2004,72(10):6157-6159.
[12]Shao CP, Hor LI. Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect Immun,2000,68(6):3569-3573.
[13]Fan JJ, Shao CP, Ho YC, et al. Isolation and characterization of a Vibrio vulnificus mutant deficient in bot h ext racellular metallopro tease and cytolysin[J]. Infect Immun,2001,69(9):5943-5948.
[14]阮国瑞,范子文,张雪萍.细菌荚膜多糖的纯化、分析及免疫原性的研究现状[J].南京铁道医学院学报,1997,16(3)：216-218.
[15]李岩,张卓然.细菌荚膜多糖的抵御对抗微生物多肽的作用.国外医学·微生物学分册,2005,28(3)：43-44.
[16]彭志齐.细菌外毒素在脓毒症发病中的意义[J].中国急救医学,2000,20(4)：253-254.
[2]Hollis DG, Weaver RE, Baker CN, et al. Halophilic Vibrio species isolated from blood cultures [J]. Clin Microbiol,1976,3(5):425-428.
[3]Farmer JJ. Ⅲ. Vibrio ('Beneckea') vulnificus. The bacterium associated with sepsis, Septicaemia, and the sea [J]. Lancet,1979,12(9):902-5.
[4]韩怀忠,张丽菊,郑艳菊,等.创伤弧菌食物中毒[J].中国食品卫生杂志,1997,9(6)：32-34.
[5]Haq SM, Dayal HH. Chronic Liver Disease and Consumption of Raw Oysters: A Potentially Lethal Combinaton-A review of Vibrio vulnificus Septicemia [J]. Am J Gastroenterol,2005,100(5):1195-9.
[6]M Y Sie, P C Ip-Yam, L L E Oon. Vibrio vulnificus septicaemia [J]. Anaesthesia and Intensive Care,2002,30(1):77-79.
[7]Chang JJ, Sheen IS, Peng SM, et al. Vibrio vulnificus infection-report of 8 cases and review of cases[J]. Changgeng Yi Xue Za Zhi,1994,17(4):339-346.
[8]Patel VJ, Gardner E, Burton CS. Vibrio vulnificus septicemia and leg ulcer [J]. Am Acad Dermatol.2002,46(5):144-5.
[9]Hlady Wg, Klontz Kc. The epide miology of Vibrio infections in Florida, 1981～1993 [J]. Infect Dis,1996,173(5):1176-1183.
[10]Klontz KC,Lieb B,Schreiber M, et al. Syndromes of Vibrio vulnificus infection-clinical and epide miologic features in Florida cases,1981～1987[J]. Ann Intern Med,1988,109(12):318-323.
[11]韦兴,侯树勋.海水浸泡火器伤的特点与救治原则[J].解放军预防医学杂志,2003,21(1)：76-79.
[12]卜海激,申洪,朱天岭等.犬海水中火器伤细菌感染定量研究[J]..第一军医大学学报.2003,23(6)：598-601.
[13]Johnson RW, Arnett FC. A fatal case of Vibrio vulnificus presenting as septic arthritis [J]. Arch Intern Med,2001,161(21):2616-8.
[14]Sato T, Inatomi Y, Yonehara T, et al. A patient with Vibrio Vulnificus meningoencephalitis [J]. Rinsho Shinkeigaku,2005,45(1):18-21.
[15]Hsueh, P.R., C. Y. Lin, et al. Vibrio vulnificus in Taiwan [J]. Emerg Infect Dis, 2005,10(9):1363-1368.
[16]Osaka, K., M. Komatsuzaki, H. Takahashi, et al.2004. Vibrio vulnificus septicaemia in Japan:an estimated number of infections and physicians' knowledge of the syndrome [J]. Epidemiol Infect,2009,13(2):993-996.
[17]江维平.创伤弧菌肠道感染一例[J].中华医学检验杂志,1990,13(5)：255.
[18]Tsai YH, Huang TJ, Hsu RW et al. Necrotizing soft-tissue infections and primary sepsis caused by Vibrio vulnificus and Vibrio cholerae non-O1 [J] Trauma,2009,66(3):899-905.
[19]陈龙、洪建军、陈星隆等以骨筋膜室综合征首发的创伤弧菌败血症1例[J].浙江创伤外科,2004,6(9)：418-418.
[20]Klontz K.C, Lieb S, Schreiber M, et al. Syndromes of Vibrio vulnificus infections:Clinical and epidemiologic features in Florida cases,1981-1987 [J]. Ann Intern Med,1988,109(1988):318.
[21]Koenig K.L, Mueller J, Rose T. Hazard on the half shell [J]. West J Med, 1991,155 (15):400-406.
[22]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005；13(7)：860-863.
[23][23]Dalsgaard A, Frimodt-Moller N, Bruun B, et al. Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark [J]. Clin Microbiol Infect Dis,2009,15(7):227-230.
[24]Liu M, Naka H, Crosa JH. HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6 [J]. Mol Microbiol,2009,72(2):491-505.
[25]Mark S. Strom, Rohinee N. Paranjpye:Epidemiology and pathogenesis of Vibrio vulnificus [J]. Microbes and Infection,2000, (2):177-188
[26]Zhao JF, Sun AH, Ruan P.Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway [J]. Microb Pathog,2009,46(4):194-200.
[27]Chen YC, Chang MC, Chuang YC, et al. Characterization and virulence of hemolysin III from Vibrio vulnificus[J]. Curr Microbiol,2004,49(3):175-179.
[28]Gray L.D, Kreger A.S, Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus [J]. Infect Immun,1998,48(5):62-67.
[29]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion initiated apoptotic signaling pat hway in human ECV304 cells [J]. Biol Chem,2001,276(50):475182-47523.
[30]Kang MK, J hee EC, Koo BS, et al. Induction of nitric oxide synt hase expression by Vibrio vulnificus cytolysin [J]. Biochem Biophys Res Commun, 2002,290(3):1090-1095.
[31]Viriyakosol S, Kirkland T. Knowledge of cellular receptors for bacterial endotoxin 1995[J]. Clin Infect Dis,1995,21(2):190-195.
[32]罗向东,杨宗城,黎鳌.家兔烧伤早期内脏血管内皮细胞损伤的变化[J].第三军医大学学报,1995,17(4)：279-283.
[33]黎鳌.创伤后脏器功能不全[M].河北科学技术出版社,1999.560-584.
[34]李主一.火器伤外科学[M].人民军医出版社,第一版.1982.114-120.
[35]贾小明,郭振荣,盛志勇,等.肉芽创面组织细菌定量培养与植皮成活的关系[J].解放军医学杂志,1985,17(5)：102-122.
[1]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[2]王贵明,创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白主动免疫的动物实验研究[M].南方医科大学硕士论文,2009.
[3]彭慧如,吴赛珠.睾酮对血管内皮细胞衰老的干预作用[J].中国老年病杂志,2008,28(10)：940-942.
[4]H Shen. A new concept and calculating method for quantitative form description. Quantitative Cytology and Histology,2000
[5]申洪,沈忠英.实用生物体视学技术[M].广州：中山大学出版社,1991.08.
[6]Parvizi J, Parpura V, Greenleaf JF, et al.Calcium signaling is required for μltrasound-stimμlated aggrecan synthesis by rat chondrocytes [J]. J Orthop Res,2002,20(1):51-57.
[7]Hsueh PR, Lin CY, Tang HJ, et al. Vibrio vulnificus in Taiwan [J]. Emerg Infect Dis,2004,10(8):1363-1368.
[8]Kang, In-hye, Ju-Sim kim, Jeong K. LEE. The Virulence of Vibrio vulnificus is Affected by the Cellular Level of Superoxide Dismutase Activity [J]. Microbiol. Biotechnol,2007,17(8):1399-1402.
[9]Man Hwan, Hee Gon Jeong, and Sang Ho Choi. Proteomic Identification and Characterization of Vibrio vulnificus Proteins Induced upon Exposure to INT-407 Intestinal Epithelial Cells[J]. Microbiol Biotechnol,2008,18(5): 968-974.
[10]Zuppardo AB, Siebeling RJ. An epimerase gene essential for capsule synthesis in Vibrio vulnificus [J]. Infect Immun,1998,66(6):2601-2606.
[11]Wright A.C, Simpson L.M, Oliver J.D, et al. Phenotypic evaluation of acapsular transposon mutants ofVibrio vulnificus [J]. Infect Immun,2008,58 (1990):1769-1773.
[12]Yoshida S., Ogawa M, Mizuguchi Y. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus [J]. Infect Immun,1985,47 (5):446-9.
[13]Valiente E, Jimenez N, Merino S, Tomas JM. Vibrio vulnificus biotype 2 serovar E gne but not galE is essential for lipopolysaccharide biosynthesis and virulence [J]. Infect Immun,2008,76(4):1628-38.
[14]Oliver JD,Wear JE,Thimas MB,et al. Production of extracellular enzymes and cytotoxicity by Vibrio Vulnificus[J]. Diagn Microbiol Infect Dis,1986,5(2): 99-111.
[15]Vinogradov E, Wilde C, Anderson EM. Structure of the lipopolysaccharide core of Vibrio vulnificus type strain[J]. Carbohydrate Research,2009,344(4): 484-490.
[16]Gray L.D., Kreger A.S., Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus [J]. Infect Immun,1985,48 (5): 62-68.
[17]Zhao JF, Sun AH, Ruan P.Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway[J]. Microb Pathog,2009,46(4):194-200.
[18]Chen YC, Chang MC, Chuang YC, Jeang CL.Characterization and virulence of hemolysin III from Vibrio vulnificus [J]. Curr Microbiol.2004, 49(3):175-179.
[19]Kang-Beom Kwon, Jeong-Yeh Yang, Do-Gon Ryu. Vibrio vulnificus Cytolysin Induces Superoxide Anion-initiatedApoptotic Signaling Pathway in Human ECV304 Cells [J].The journal of biological chemistry,2001,276(50): 47518-47523.
[20]Fan JJ, Shao CP, Ho YC. Isolation and Characterization of a Vibrio vulnificus Mutant Deficient in Both Extracellular Metalloprotease and Cytolysin [J]. Infect Immun,2001,69(9):5943-5948
[21]chung-ping shao,lien-I Hor.Metalloprotease Is Not Essential for Vibrio vulnificus Virulence in Mice [J].Infection and immunity,2000,15(24): 3569-3573.
[22]Bisharat N,Agmon V,Finkelstein R,etal.Clinical, epide miological, and microbiological features of Vibrio vulnificus biogroups causing outbreaks of wound infection and bacteraemia in Israel.Israel Vibrio Study Group [J]. Lancet,1999,354(9188):1421-8.
[23]M Y Sie, P C Ip-Yam,L L E Oon. Vibrio Vulnificus sepeicamia and Intensive Care[J].2002,30(1):77-79.
[24]Choi HJ,Lee DK, Lee MW, Moon KC,Koh JK. Vibrio vulnificus septicaemia presenting as purpura fulminans[J]. dermatol,2005,32(1):46-51.
[25]Cohen AL, Oliver JD, DePaola A, et al. Emergence of a virulent clade of Vibrio vulnificus and correlation with the presence of a 33-kilobase genomic island [J]. Appl Environ Microbiol,2007,73(17):5553-65.
[26][26]Russell AD.Radiation, Russell AD, et al.Principles and Practice of Disinfection. Preservation and Sterilization[J] Blackwell Scien.tific Publication,1999,49(6):688-702.
[27]吴斌,卢中秋创伤弧菌感染的致病机制研究现状[J]. Chin J Crit Care Med, 2004,24(11):834.
[28]Luder CG, Gross U, Lopes MF. Int racellular protozoan parasites and apoptosis:diverse st rategies to modulate parasite2host inter2 actions[J]. Trends Parasitol,2001,17(10):480-486.
[29]郑晶.创伤弧菌溶细胞毒素及金属蛋白酶的基因克隆、蛋白表达、抗体制备及其致病性的研究[J].广州：南方医科大学博士学位论文,2008.18
[1]王贵明.创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白抗原主动免疫的动物实验研究[M].南方医科大学硕士毕业论文,2009.
[2]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[3]宋宏新,刘晓阳,李宏.改良热酚水法制备大肠杆菌0157：H7脂多糖抗原的研究[J].食品科学,2006,27(10)：273-275.
[4]Dejana E, Corada M, Lampugnani MG. Endothelial cell to cell junction[J]. FASEB J,1995,9(14):910-918.
[5]张小燕,梁朝.细菌内毒素作用血管内皮细胞的机制及其临床疾病[J].中华检验医学杂志,2004,27(111)：811-813.
[6]国家药典委员会编著.2005版,中国药典第二部[M],化学药品,212-214.
[7]Riely L W, Remis R S, Helgerson S D, et al. Hemorrhagic colitis associated with a rare Escherichia coli serotype[J]. N Eng J Med,1983,30(8):681-685.
[8]Carol G Currie, Ian R Poxton. The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-0157 verotoxin-producing E.coli[J]. FEMS Immunology and Medical Microbiology,1999,24:57-62.
[9]Nishiuchi Y, Doe M, Hotta H, et al. Structure and serologic properties of-specificpolysaccharidefrom Citrobacterfreundii possessingcrossreactivity with Escherichia coli O157:H7[J]. Immunology and Medical Microbiology, 2000,28:163-171.
[10]Williams AH, Raetz CRH. Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase[J]. Proc Natl Acad Sci USA,2007,104(34):13543-13550.
[11]冯淑霞,李金钟.硫酸链霉素细菌内毒素定量测定法的研究中国兽药杂志2006,40(3)：6-8.
[12]Coggins BE, Li X, McClerren AL, et al. Structure of the LpxC deacetylase with a bound substrate-analog inhibitor[J]. Nat Struct Biol,2003,10(8): 645-651.
[13]Yamamoto T, Harada N, Kano K, et al. The Ras TargetAF26 Interactswith ZO21 and Serves as a Peripheral Component of Tight Junctions in Ep ithelial Cells [J]. J Cell Biol,1997,139(3):785-795.
[14]Izumi Y, Hirose T, Tamai Y, et al. An Atyp ical PKC Directly Associates and Colocalizes at the Ep ithelial Tight Junction with ASIP, aMammalian Homologue of Caenorhabditis elegans Polarity Protein PAR23 [J]. J Cell Biol, 1998,143(1):95-106.
[15]Chandana S, Sanjay K, Nigam, et al. Expanding Role of G Proteins in Tight Junction Regulation:GaS Stimulates TJ Assembly [J]. Biochemical and Biophysical Research Communications,2001,285(2):250-256.
[16]申洪,沈忠英.实用生物体视学技术[M].中山大学出版社,1991.08.
[17]Hsueh PR, Lin CY, Tang HJ, et al. Vibrio vulnificus in Taiwan. Emerg Infect Dis[J].2004,10(8):1363-1368.
[18]王永堂.鲁秀敏细菌内毒素与抗内毒素治疗[J]. Chinese Journal of Biochemical Pharmaceutics,2002,23(5):266-268.
[19]闻玉梅.现代医学微生物学[M].上海医科大学出版社,1999.124-130.
[20]张小燕.梁朝细菌内毒素作用血管内皮细胞的机制及其临床疾病.Chin J Lab Med,2004,27(11):811-814.
[21]Triantafilou M, Triantafilou K, Fernandez N. Rough and smooth forms of fluorescein labelled bacterial endotoxin exhibit CD 14/LBP dependent and independent binding that is influencedby endotoxin concentration[J]. Eur J Biochem,2000,267:2218-2226.
[22]Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity[J]. J Endotoxin Res,2001,7(3):167-171.
[1]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion2initiated apoptotic signaling pat hway in human ECV304 cells[J]. J Biol Chem,2001,276(50):47518-47523.
[2]王贵明,创伤弧菌消杀抗性及其菌体抗原、溶细胞毒素蛋白主动免疫的动物实验研究[M].南方医科大学硕士论文,导师：申洪,2009.
[3]郑晶,申洪,陈清.创伤弧菌经食管感染小鼠的致病性[J].世界华人消化杂志,2005,13(7)：860-863.
[4]彭慧如,吴赛珠.睾酮对血管内皮细胞衰老的干预作用[J].中国老年病杂志,2008,28(10)：940-942.
[5]顾小龙,严全能,张园.尾加压素Ⅱ对人脐静脉内皮细胞凋亡的影响[J].实用医学杂志,2006,22(11)：1228-1230.
[6]申洪,沈忠英.实用生物体视学技术[M].中山大学出版社,1991.08.
[7]Parvizi J, Parpura V, Greenleaf JF, et al.Calcium signaling is required for ultrasound-stimμlated aggrecan synthesis by rat chondrocytes.J Orthop Res, 2002,20(1):51-57.
[8]Kwon KB, Yang J Y, Ryu DG, et al. Vibrio vulnificus cytolysin induces superoxide anion2initiated apoptotic signaling pat hway in human ECV304 cells[J]. J Biol Chem,2001,276(50):47518-47523.
[9]李桂军楼永良创伤弧菌细胞素致病机制的研究进展[J].Int J Lab Med, 2006,27(7):237-241.
[10]Park KH, Yang HB, Kim HG, et al. Low density lipoprotein inactivates Vibrio vulnificus cytolysin t hrough t he oligomerization of toxin monomer[J]. Med Microbiol Immunol (Berl),2005,194(3):137-141.
[11]Lee YR, Park KH, Lin ZZ, et al. A calcium2calmodulin antagonist blocks experimental Vibrio vulnificus cytolysin induced let hality in an experimental mouse model[J]. Infect Immun,2004,72(10):6157-6159.
[12]Shao CP, Hor LI. Metalloprotease is not essential for Vibrio vulnificus virulence in mice. Infect Immun,2000,68(6):3569-3573.
[13]Fan JJ, Shao CP, Ho YC, et al. Isolation and characterization of a Vibrio vulnificus mutant deficient in bot h ext racellular metallopro tease and cytolysin[J]. Infect Immun,2001,69(9):5943-5948.
[14]阮国瑞,范子文,张雪萍.细菌荚膜多糖的纯化、分析及免疫原性的研究现状[J].南京铁道医学院学报,1997,16(3)：216-218.
[15]李岩,张卓然.细菌荚膜多糖的抵御对抗微生物多肽的作用.国外医学·微生物学分册,2005,28(3)：43-44.
[16]彭志齐.细菌外毒素在脓毒症发病中的意义[J].中国急救医学,2000,20(4)：253-254.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |