海分枝杆菌培养及致病性研究
发布时间：2018-08-28 19:30
【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。
[Abstract]:Mycobacterium marinum (M. marinum) is a saprophytic atypical mycobacterium. It exists in seawater and freshwater all over the world. In order to contact with infection, Mycobacterium marinum has been widely used in the world. With the development and utilization of the ocean, there is a potential danger of human and fish co-infected diseases. Mycobacterium strains were isolated from infected pathological tissues, and were cultured and tested for drug sensitivity, re-infected zebrafish, laboratory mice and bacterial pathogenic proteins. Study on pathogenesis.
(1) Mycobacterium isolation test: Mycobacterium (R1) isolated from human infected pus tissue and Mycobacterium (Y10) isolated from the surface of living crabs were compared with the standard Mycobacterium marinum strains. The results showed that the cultured colonies were sensitive to nine drugs, that is, the Yellow reaction colony. The results showed that the characteristics of acid-fast staining, chemical reaction and the molecular weight of the wall proteins of the three strains were consistent, which indicated that the three strains had the same gene sources.
(2) Zebrafish infection test: 60 healthy juvenile zebrafish were randomly selected, 30 of them were randomly selected, and each group was given intraperitoneal injection of 0.1 mL of liquid Y10, R1 and standard Mycobacterium marinum, respectively, and cultured in a constant temperature tank at 28 C to observe the survival of zebrafish. Thirty zebrafish died within seven days. Infected tissues could be seen from dead zebrafish carcasses, which turned dark brown and red. Necrotizing tissues could be seen from the abdominal cavity. Pathogenic strains of Mycobacterium marinum were isolated from the diseased tissues.
(3) Establishment of infection model in mice and rats: 12 healthy mice were randomly divided into experimental group and control group. According to different infection routes, experimental group was divided into experimental group A, B and experimental group C. Experimental group A: Mycobacterium was injected into tail vein of rats; experimental group B: Mycobacterium was injected into abdominal cavity of rats; experimental group C: Mycobacterium was injected into experimental group C. The control group was given the same dose of normal saline via caudal vein as control group. After the first week of infection, the mice were repeatedly infected. At the eighth week, the tail vein blood was taken for cytokines (interleukin, interferon-gamma and tumor necrosis factor-alpha), immunoglobulin (IgA, IgG and IgM), C-reactive protein and complement (C3). Cytokines were detected by enzyme-linked immunosorbent assay (ELISA), immunoglobulin, C-reactive protein and complement by automatic biochemical analyzer.
Mycobacterium marinum is pathogenic to Kunming mice. Obvious granulomatous plaques can be seen in autopsy specimens. Granulomas contain a large number of granular substances, i.e. caseous necrosis. IL-2, IL-4, IL-10 and IL-12 had no significant difference (p > 0.05). Compared with experimental group C and control group mice, IL-4 and IL-10 increased significantly. IL-2 and IL-12 were significantly different from control group (p < 0.05); IL-4 and IL-10 were significantly different from experimental group C and control group (p < 0.01).
After 8 weeks of infection, the secretion of sIL-2R and IFN-gamma in the serum of peripheral veins of rats increased significantly, and there was a significant difference between the experimental group and the control group (p < 0.05). There was no significant difference between the experimental group A, B and C (p > 0.05); the secretion of TNF-alpha increased after 8 weeks of infection, although there was an increase in the secretion. There was no significant difference in the contents of C3, C4 and CRP between experimental group A, B and C (p > 0.05). There was significant difference between experimental group A, B and C and control group (t = 3.88-2.58, P < 0.05). There was no significant difference between groups A, B and C (p > 0.05), and there was significant difference between the control group and the experimental group (p < 0.05).
(4) Mycobacterium proteins were isolated, separated by electrophoresis, and analyzed. Protein antigens with molecular weight of about 65 kD were isolated, which were the main pathogenic proteins. The proteins were densely distributed between pH 4.5 and 5.5, and the protein spots between 5.5 and 6.0 were scattered. There were almost no proteins on the alkaline side and the proteins were mainly concentrated on the acidic side.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378;S855.99
[Abstract]:Mycobacterium marinum (M. marinum) is a saprophytic atypical mycobacterium. It exists in seawater and freshwater all over the world. In order to contact with infection, Mycobacterium marinum has been widely used in the world. With the development and utilization of the ocean, there is a potential danger of human and fish co-infected diseases. Mycobacterium strains were isolated from infected pathological tissues, and were cultured and tested for drug sensitivity, re-infected zebrafish, laboratory mice and bacterial pathogenic proteins. Study on pathogenesis.
(1) Mycobacterium isolation test: Mycobacterium (R1) isolated from human infected pus tissue and Mycobacterium (Y10) isolated from the surface of living crabs were compared with the standard Mycobacterium marinum strains. The results showed that the cultured colonies were sensitive to nine drugs, that is, the Yellow reaction colony. The results showed that the characteristics of acid-fast staining, chemical reaction and the molecular weight of the wall proteins of the three strains were consistent, which indicated that the three strains had the same gene sources.
(2) Zebrafish infection test: 60 healthy juvenile zebrafish were randomly selected, 30 of them were randomly selected, and each group was given intraperitoneal injection of 0.1 mL of liquid Y10, R1 and standard Mycobacterium marinum, respectively, and cultured in a constant temperature tank at 28 C to observe the survival of zebrafish. Thirty zebrafish died within seven days. Infected tissues could be seen from dead zebrafish carcasses, which turned dark brown and red. Necrotizing tissues could be seen from the abdominal cavity. Pathogenic strains of Mycobacterium marinum were isolated from the diseased tissues.
(3) Establishment of infection model in mice and rats: 12 healthy mice were randomly divided into experimental group and control group. According to different infection routes, experimental group was divided into experimental group A, B and experimental group C. Experimental group A: Mycobacterium was injected into tail vein of rats; experimental group B: Mycobacterium was injected into abdominal cavity of rats; experimental group C: Mycobacterium was injected into experimental group C. The control group was given the same dose of normal saline via caudal vein as control group. After the first week of infection, the mice were repeatedly infected. At the eighth week, the tail vein blood was taken for cytokines (interleukin, interferon-gamma and tumor necrosis factor-alpha), immunoglobulin (IgA, IgG and IgM), C-reactive protein and complement (C3). Cytokines were detected by enzyme-linked immunosorbent assay (ELISA), immunoglobulin, C-reactive protein and complement by automatic biochemical analyzer.
Mycobacterium marinum is pathogenic to Kunming mice. Obvious granulomatous plaques can be seen in autopsy specimens. Granulomas contain a large number of granular substances, i.e. caseous necrosis. IL-2, IL-4, IL-10 and IL-12 had no significant difference (p > 0.05). Compared with experimental group C and control group mice, IL-4 and IL-10 increased significantly. IL-2 and IL-12 were significantly different from control group (p < 0.05); IL-4 and IL-10 were significantly different from experimental group C and control group (p < 0.01).
After 8 weeks of infection, the secretion of sIL-2R and IFN-gamma in the serum of peripheral veins of rats increased significantly, and there was a significant difference between the experimental group and the control group (p < 0.05). There was no significant difference between the experimental group A, B and C (p > 0.05); the secretion of TNF-alpha increased after 8 weeks of infection, although there was an increase in the secretion. There was no significant difference in the contents of C3, C4 and CRP between experimental group A, B and C (p > 0.05). There was significant difference between experimental group A, B and C and control group (t = 3.88-2.58, P < 0.05). There was no significant difference between groups A, B and C (p > 0.05), and there was significant difference between the control group and the experimental group (p < 0.05).
(4) Mycobacterium proteins were isolated, separated by electrophoresis, and analyzed. Protein antigens with molecular weight of about 65 kD were isolated, which were the main pathogenic proteins. The proteins were densely distributed between pH 4.5 and 5.5, and the protein spots between 5.5 and 6.0 were scattered. There were almost no proteins on the alkaline side and the proteins were mainly concentrated on the acidic side.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378;S855.99
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