海分枝杆菌培养及致病性研究
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摘要
海分枝杆菌(Mycobacterium marinum，M．marinum)为一种腐生性非典型分枝杆菌，其分布遍及世界各国，它存在于海水、淡水中，为接触感染。随着对海洋的开发利用，人、鱼共患的疾病存在潜在的危险性，目前已有许多关于海分枝杆菌对人类感染的病例报道。海水养殖人员、海洋捕捞人员、渔民以及观赏鱼养殖人员等，感染的几会明显增加。本文从感染的病变组织中分离到分枝杆菌菌株，通过对其培养和药物敏感试验、再次感染斑马鱼和实验鼠以及分离细菌致病性蛋白的实验，进行致病机理的研究。
(1)分枝杆菌的分离试验：从人感染的脓液组织中，分离培养出的分枝杆菌(R1)，以及从活体的蟹体表分离到的分枝杆菌(Y10)，分别与标准海分枝杆菌菌株进行对照研究发现，培养菌落的对光反应，即产生黄色反应菌落、对9种药物的敏感性试验结果、抗酸染色特性、化学反应特性，以及构成菌株壁蛋白质的分子量大小均表现出一致性，说明这三种分枝杆菌菌株的基因来源有共性。
(2)斑马鱼的感染试验：选择健康的幼斑马鱼60条，随机选取30条，每10条为一组，分别给予腹腔内注射0．1 mL液体的Y10、R1和标准海分枝杆菌菌液，分别放置于28℃恒温的水箱中养殖，观察斑马鱼的生存情况。腹腔内注射0．1 mL海分枝杆菌菌液，30条斑马鱼相继在7天内死亡。从死亡的斑马鱼尸体上可以看到受感染的病变组织，病变组织变成暗棕红色，打开腹腔可以看到坏死组织，从病变组织中已经分离、培养到海分枝杆菌的致病菌株。
(3)小鼠和大鼠的感染模型建立：分别选取12只健康鼠，随机分成实验组和对照组。根据感染途径的不同，实验组又分成实验组A、B和实验组C。实验组A：经鼠的尾静脉注射分枝杆菌菌液；实验组B：经鼠的腹腔注射分枝杆菌菌液；实验组C经消化道感染。对照组鼠给予经尾静脉注射同等剂量的生理盐水做对照。感染第1周后，重复感染一次，第8周采取其尾静脉血分别进行细胞因子(白细胞介素、干扰素-gamma和肿瘤坏死因子-alpha)、免疫球蛋白(IgA、IgG和IgM)、C-反应蛋白和补体(C3和C4)的检测。细胞因子检测采用酶联免疫吸附试验(ELISA)；免疫球蛋白、C-反应蛋白和补体的检测方法采用全自动生化分析仪测定。
海分枝杆菌对昆明鼠有致病性，从尸体解剖病理切片标本中可以看到明显的肉芽肿斑块，肉芽肿包含大量的颗粒状物质，即干酪样坏死物。感染2周后的小鼠外周静脉血中白细胞介素检测结果，实验组A和B小鼠在感染期间血清中白细胞介素IL-2、IL-4、IL-10、IL-12差异无统计学意义(p＞0．05)，与实验组C和对照组小鼠比较有明显的增加，IL-4和IL-10增加的更加明显。IL-2和IL-12与对照组差异有统计学意义(p＜0．05)；IL-4和IL-10与实验组C和对照组差异有统计学意义(p＜0．01)。
大鼠感染第8周后外周静脉血清中，细胞因子检测：感染8周后血清中sIL-2R和IFN-γ的分泌明显的增加，实验组与对照组之间差异有统计学意义(p＜0．05)，实验组A、B和C组之间差异无统计学意义(p＞0．05)；TNF-α在感染8周后尽管分泌有增加的趋势，但实验组和对照组之间差异无统计学意义(p＞0．05)。补体检测：实验组A、B和C组之间，C3、C4、CRP的含量差异无统计学意义(p＞0．05)，实验组A、B和C组与对照组之间的差异有统计学意义(t=3．88-2．58，p＜0．05)。免疫球蛋白检测：实验组A、B和C组之间的差异无统计学意义(p＞0．05)，对照组与实验组之间的差异有统计学意义(p＜0．05)。
(4)分离分枝杆菌菌体蛋白，电泳分离，并进行分析。分离到分子量65kD左右的蛋白抗原，为主要的致病性蛋白。双向电泳实验表明分枝杆菌菌株细胞壁检测到490个蛋白质斑点。分子质量范围均处于10～100kD。蛋白的pI值主要分布于4．5～6．0，其中在pH4．5～5．5之间蛋白分布比较密集，5．5～6．0之间的蛋白点比较稀散，即偏碱性侧的蛋白几乎没有，蛋白主要集中在偏酸性侧。两株分离株和标准海分枝杆菌的致病性蛋白存在同一性。
Mycobacterium marinum (M.marinum)is a pythogenesis and atypicalMycobacterium,which is distributed all over the world.It resides in salt water andfresh water.It can infect fish and human beings by contacting.With the developmentand utilization of ocean,the disease incidence of co-suffering diseases shared byhuman and fish has a latent risk.At present,there were many reports aboutMycobacterium marinum infected human.The infecting incidence increasesobviously for mariculture staff,catching fish staff,fishmen and beautiful fishescultivation staff,etc.We have isolated M.marinum from pathological tissue,whichwas cultivated and drug sensitivity tested,and infected zebra fish and experimentmice.And we also have isolated pathological proteins from it and carried outpathopoiesis research.
(1)Isolated experiment for Mycobacterium.We have isolated two Mycobacteriathat they were come from human'infected abscessus tissue(R1)and intravital bodysurface of crab (Y10),respectively.We have studyed them compare with typicalMycobacterium marinum in light reaction of cultivated colony,namely bring aboutyellow colony,and in drug sensitive test for nine drugs,and in acid-fast staining andchemical reaction,as well as the molecular weight of the strain wall-held protein.Wefound that they have consistency in above aspects,which can explain that they havecommon genes origin.
(2)Zebra fish infectional tests:60 healthy immature zibra fish were chose,select30 at random,which were divided into three groups:A,B and C group.Every grouphas ten zebra fish.0.1 bacteria liquid which come from Y10,R1 and Mycobacteriummarinum were injected in intraperitoneal and cultivation in aquarium tank at 28℃respectively,to observe the living subsistence of zebra fish.Ten zebra fishes in trial group were died one after another in seven days by intraperitoneal injection 0.1mLMycobacterium marinum liquid.Pathological dim-Brownish red color tissue can beobserved in the dead zebra fish corpses when opened the abdominal cavity.Pathogenic strains of Mycobacterium marinum were isolated and cultivated frompathological tissues.
(3)The infection modeling of mice and rates:12 healthy mice and rats werechosen respectively,divided into trial group and control group.According to thedifferent route of infection,the trial groups were divided into group A,B and C.Group A:trial mouse were given Mycobacterium liquid by vena caudalis intravenousinjection.Group B:trial mouse were given Mycobacterium liquid by intraperitonealinjection.Group C:trial mouse were given Mycobacterium liquid by alimentaryinfection.Control group mouse were given the same dosage normal sodium (NS)byvena caudalis intravenous injection.After one week infected,the trila mouse wereinfected again.Post-infected eight four weeks,venous blood from all the mouse venacaudalis were detected for cytokines (interleukin,interferon-gamma,tumour necrosisfactor-alpha),immunoglobulin(IgA、IgG and IgM),C-reaction protein andcomplements (C3 and C4)respectively.Cytokines was detected by enzyme linkedimmunosorbent assay(ELISA).Immunoglobulin,C-reaction proteiand andcomplements were assayed by automatic biochemistry analyzer.
Mycobacterium marinum has pathogenicity to Kunming mice.Granuloma wasseen in the corpse by autopsy.The granulo-material was involved in granuloma,which was cheesy necrosis.The results of interleukin (IL-2,IL-4,IL-10 and IL-12)were no difference between trial A and trial B group after infected two weeks(p＞0.05),but higher than that of group C and control group(p＜0.05).IL-4 and IL-10 were moreincreased compared with IL-2 and IL-12(p＜0.05).There were statistical significancein IL-4 and IL-10 in group A and B,C andcontrol group(p＜0.01).
The cytokine(sIL-2R and IFN-γ)from peripheral vein blood of infected eightweeks bigger rat were more enhanced in trial group A,B and C compared withcontrol group(p＜0.05).But there was no statistical difference in trial group A,B andC (p＞0.05).TNF-alpha was less increased secretion,but no difference between trial group A,B and C.The complement (C3 and C4)and C-reaction protein in trial groupA,B and C were no difference (p＞0.05).But there was statistical significancebetween trial group and control group(p＜0.05).The immunoglobulin (IgA,IgG andIgM)were no difference (p＞0.05)in trial groups A,B and C.While there wasstatistical significance between trial group and control group(p＜0.05).
(4)we have drown off the tropina from Mycobacterium marinum,electrophoreticseparation,and carried out analysis.We have isolated 65kD proteantigen,which wascapital pathogenicity protein.490 protein spots were detected by two-dimensionalelectrophoresis,which molecular mass range from 10 to 100kD.The PI value ofprotein were mostly in pH 4.5 6.0,which were pH 4.5--5.5 relatively intensive.While the protein spots were rather scatter in pH5.5—6.0,namely the protein spotswere mainly in meta acids side,nothing were in alkalinity side.There is identity inpathogenity protein for the two isolated Mycobacteria and Mycobacteriun marinum.
(1)分枝杆菌的分离试验：从人感染的脓液组织中，分离培养出的分枝杆菌(R1)，以及从活体的蟹体表分离到的分枝杆菌(Y10)，分别与标准海分枝杆菌菌株进行对照研究发现，培养菌落的对光反应，即产生黄色反应菌落、对9种药物的敏感性试验结果、抗酸染色特性、化学反应特性，以及构成菌株壁蛋白质的分子量大小均表现出一致性，说明这三种分枝杆菌菌株的基因来源有共性。
(2)斑马鱼的感染试验：选择健康的幼斑马鱼60条，随机选取30条，每10条为一组，分别给予腹腔内注射0．1 mL液体的Y10、R1和标准海分枝杆菌菌液，分别放置于28℃恒温的水箱中养殖，观察斑马鱼的生存情况。腹腔内注射0．1 mL海分枝杆菌菌液，30条斑马鱼相继在7天内死亡。从死亡的斑马鱼尸体上可以看到受感染的病变组织，病变组织变成暗棕红色，打开腹腔可以看到坏死组织，从病变组织中已经分离、培养到海分枝杆菌的致病菌株。
(3)小鼠和大鼠的感染模型建立：分别选取12只健康鼠，随机分成实验组和对照组。根据感染途径的不同，实验组又分成实验组A、B和实验组C。实验组A：经鼠的尾静脉注射分枝杆菌菌液；实验组B：经鼠的腹腔注射分枝杆菌菌液；实验组C经消化道感染。对照组鼠给予经尾静脉注射同等剂量的生理盐水做对照。感染第1周后，重复感染一次，第8周采取其尾静脉血分别进行细胞因子(白细胞介素、干扰素-gamma和肿瘤坏死因子-alpha)、免疫球蛋白(IgA、IgG和IgM)、C-反应蛋白和补体(C3和C4)的检测。细胞因子检测采用酶联免疫吸附试验(ELISA)；免疫球蛋白、C-反应蛋白和补体的检测方法采用全自动生化分析仪测定。
海分枝杆菌对昆明鼠有致病性，从尸体解剖病理切片标本中可以看到明显的肉芽肿斑块，肉芽肿包含大量的颗粒状物质，即干酪样坏死物。感染2周后的小鼠外周静脉血中白细胞介素检测结果，实验组A和B小鼠在感染期间血清中白细胞介素IL-2、IL-4、IL-10、IL-12差异无统计学意义(p＞0．05)，与实验组C和对照组小鼠比较有明显的增加，IL-4和IL-10增加的更加明显。IL-2和IL-12与对照组差异有统计学意义(p＜0．05)；IL-4和IL-10与实验组C和对照组差异有统计学意义(p＜0．01)。
大鼠感染第8周后外周静脉血清中，细胞因子检测：感染8周后血清中sIL-2R和IFN-γ的分泌明显的增加，实验组与对照组之间差异有统计学意义(p＜0．05)，实验组A、B和C组之间差异无统计学意义(p＞0．05)；TNF-α在感染8周后尽管分泌有增加的趋势，但实验组和对照组之间差异无统计学意义(p＞0．05)。补体检测：实验组A、B和C组之间，C3、C4、CRP的含量差异无统计学意义(p＞0．05)，实验组A、B和C组与对照组之间的差异有统计学意义(t=3．88-2．58，p＜0．05)。免疫球蛋白检测：实验组A、B和C组之间的差异无统计学意义(p＞0．05)，对照组与实验组之间的差异有统计学意义(p＜0．05)。
(4)分离分枝杆菌菌体蛋白，电泳分离，并进行分析。分离到分子量65kD左右的蛋白抗原，为主要的致病性蛋白。双向电泳实验表明分枝杆菌菌株细胞壁检测到490个蛋白质斑点。分子质量范围均处于10～100kD。蛋白的pI值主要分布于4．5～6．0，其中在pH4．5～5．5之间蛋白分布比较密集，5．5～6．0之间的蛋白点比较稀散，即偏碱性侧的蛋白几乎没有，蛋白主要集中在偏酸性侧。两株分离株和标准海分枝杆菌的致病性蛋白存在同一性。
Mycobacterium marinum (M.marinum)is a pythogenesis and atypicalMycobacterium,which is distributed all over the world.It resides in salt water andfresh water.It can infect fish and human beings by contacting.With the developmentand utilization of ocean,the disease incidence of co-suffering diseases shared byhuman and fish has a latent risk.At present,there were many reports aboutMycobacterium marinum infected human.The infecting incidence increasesobviously for mariculture staff,catching fish staff,fishmen and beautiful fishescultivation staff,etc.We have isolated M.marinum from pathological tissue,whichwas cultivated and drug sensitivity tested,and infected zebra fish and experimentmice.And we also have isolated pathological proteins from it and carried outpathopoiesis research.
(1)Isolated experiment for Mycobacterium.We have isolated two Mycobacteriathat they were come from human'infected abscessus tissue(R1)and intravital bodysurface of crab (Y10),respectively.We have studyed them compare with typicalMycobacterium marinum in light reaction of cultivated colony,namely bring aboutyellow colony,and in drug sensitive test for nine drugs,and in acid-fast staining andchemical reaction,as well as the molecular weight of the strain wall-held protein.Wefound that they have consistency in above aspects,which can explain that they havecommon genes origin.
(2)Zebra fish infectional tests:60 healthy immature zibra fish were chose,select30 at random,which were divided into three groups:A,B and C group.Every grouphas ten zebra fish.0.1 bacteria liquid which come from Y10,R1 and Mycobacteriummarinum were injected in intraperitoneal and cultivation in aquarium tank at 28℃respectively,to observe the living subsistence of zebra fish.Ten zebra fishes in trial group were died one after another in seven days by intraperitoneal injection 0.1mLMycobacterium marinum liquid.Pathological dim-Brownish red color tissue can beobserved in the dead zebra fish corpses when opened the abdominal cavity.Pathogenic strains of Mycobacterium marinum were isolated and cultivated frompathological tissues.
(3)The infection modeling of mice and rates:12 healthy mice and rats werechosen respectively,divided into trial group and control group.According to thedifferent route of infection,the trial groups were divided into group A,B and C.Group A:trial mouse were given Mycobacterium liquid by vena caudalis intravenousinjection.Group B:trial mouse were given Mycobacterium liquid by intraperitonealinjection.Group C:trial mouse were given Mycobacterium liquid by alimentaryinfection.Control group mouse were given the same dosage normal sodium (NS)byvena caudalis intravenous injection.After one week infected,the trila mouse wereinfected again.Post-infected eight four weeks,venous blood from all the mouse venacaudalis were detected for cytokines (interleukin,interferon-gamma,tumour necrosisfactor-alpha),immunoglobulin(IgA、IgG and IgM),C-reaction protein andcomplements (C3 and C4)respectively.Cytokines was detected by enzyme linkedimmunosorbent assay(ELISA).Immunoglobulin,C-reaction proteiand andcomplements were assayed by automatic biochemistry analyzer.
Mycobacterium marinum has pathogenicity to Kunming mice.Granuloma wasseen in the corpse by autopsy.The granulo-material was involved in granuloma,which was cheesy necrosis.The results of interleukin (IL-2,IL-4,IL-10 and IL-12)were no difference between trial A and trial B group after infected two weeks(p＞0.05),but higher than that of group C and control group(p＜0.05).IL-4 and IL-10 were moreincreased compared with IL-2 and IL-12(p＜0.05).There were statistical significancein IL-4 and IL-10 in group A and B,C andcontrol group(p＜0.01).
The cytokine(sIL-2R and IFN-γ)from peripheral vein blood of infected eightweeks bigger rat were more enhanced in trial group A,B and C compared withcontrol group(p＜0.05).But there was no statistical difference in trial group A,B andC (p＞0.05).TNF-alpha was less increased secretion,but no difference between trial group A,B and C.The complement (C3 and C4)and C-reaction protein in trial groupA,B and C were no difference (p＞0.05).But there was statistical significancebetween trial group and control group(p＜0.05).The immunoglobulin (IgA,IgG andIgM)were no difference (p＞0.05)in trial groups A,B and C.While there wasstatistical significance between trial group and control group(p＜0.05).
(4)we have drown off the tropina from Mycobacterium marinum,electrophoreticseparation,and carried out analysis.We have isolated 65kD proteantigen,which wascapital pathogenicity protein.490 protein spots were detected by two-dimensionalelectrophoresis,which molecular mass range from 10 to 100kD.The PI value ofprotein were mostly in pH 4.5 6.0,which were pH 4.5--5.5 relatively intensive.While the protein spots were rather scatter in pH5.5—6.0,namely the protein spotswere mainly in meta acids side,nothing were in alkalinity side.There is identity inpathogenity protein for the two isolated Mycobacteria and Mycobacteriun marinum.
引文
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