海分枝桿菌培養(yǎng)及致病性研究
發(fā)布時間：2018-08-28 19:30
【摘要】： 海分枝桿菌(Mycobacterium marinum，M．marinum)為一種腐生性非典型分枝桿菌，其分布遍及世界各國，它存在于海水、淡水中，為接觸感染。隨著對海洋的開發(fā)利用，人、魚共患的疾病存在潛在的危險性，目前已有許多關(guān)于海分枝桿菌對人類感染的病例報道。海水養(yǎng)殖人員、海洋捕撈人員、漁民以及觀賞魚養(yǎng)殖人員等，感染的幾會明顯增加。本文從感染的病變組織中分離到分枝桿菌菌株，通過對其培養(yǎng)和藥物敏感試驗(yàn)、再次感染斑馬魚和實(shí)驗(yàn)鼠以及分離細(xì)菌致病性蛋白的實(shí)驗(yàn)，進(jìn)行致病機(jī)理的研究。 (1)分枝桿菌的分離試驗(yàn)：從人感染的膿液組織中，，分離培養(yǎng)出的分枝桿菌(R1)，以及從活體的蟹體表分離到的分枝桿菌(Y10)，分別與標(biāo)準(zhǔn)海分枝桿菌菌株進(jìn)行對照研究發(fā)現(xiàn)，培養(yǎng)菌落的對光反應(yīng)，即產(chǎn)生黃色反應(yīng)菌落、對9種藥物的敏感性試驗(yàn)結(jié)果、抗酸染色特性、化學(xué)反應(yīng)特性，以及構(gòu)成菌株壁蛋白質(zhì)的分子量大小均表現(xiàn)出一致性，說明這三種分枝桿菌菌株的基因來源有共性。 (2)斑馬魚的感染試驗(yàn)：選擇健康的幼斑馬魚60條，隨機(jī)選取30條，每10條為一組，分別給予腹腔內(nèi)注射0．1 mL液體的Y10、R1和標(biāo)準(zhǔn)海分枝桿菌菌液，分別放置于28℃恒溫的水箱中養(yǎng)殖，觀察斑馬魚的生存情況。腹腔內(nèi)注射0．1 mL海分枝桿菌菌液，30條斑馬魚相繼在7天內(nèi)死亡。從死亡的斑馬魚尸體上可以看到受感染的病變組織，病變組織變成暗棕紅色，打開腹腔可以看到壞死組織，從病變組織中已經(jīng)分離、培養(yǎng)到海分枝桿菌的致病菌株。 (3)小鼠和大鼠的感染模型建立：分別選取12只健康鼠，隨機(jī)分成實(shí)驗(yàn)組和對照組。根據(jù)感染途徑的不同，實(shí)驗(yàn)組又分成實(shí)驗(yàn)組A、B和實(shí)驗(yàn)組C。實(shí)驗(yàn)組A：經(jīng)鼠的尾靜脈注射分枝桿菌菌液；實(shí)驗(yàn)組B：經(jīng)鼠的腹腔注射分枝桿菌菌液；實(shí)驗(yàn)組C經(jīng)消化道感染。對照組鼠給予經(jīng)尾靜脈注射同等劑量的生理鹽水做對照。感染第1周后，重復(fù)感染一次，第8周采取其尾靜脈血分別進(jìn)行細(xì)胞因子(白細(xì)胞介素、干擾素-gamma和腫瘤壞死因子-alpha)、免疫球蛋白(IgA、IgG和IgM)、C-反應(yīng)蛋白和補(bǔ)體(C3和C4)的檢測。細(xì)胞因子檢測采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)；免疫球蛋白、C-反應(yīng)蛋白和補(bǔ)體的檢測方法采用全自動生化分析儀測定。 海分枝桿菌對昆明鼠有致病性，從尸體解剖病理切片標(biāo)本中可以看到明顯的肉芽腫斑塊，肉芽腫包含大量的顆粒狀物質(zhì)，即干酪樣壞死物。感染2周后的小鼠外周靜脈血中白細(xì)胞介素檢測結(jié)果，實(shí)驗(yàn)組A和B小鼠在感染期間血清中白細(xì)胞介素IL-2、IL-4、IL-10、IL-12差異無統(tǒng)計學(xué)意義(p＞0．05)，與實(shí)驗(yàn)組C和對照組小鼠比較有明顯的增加，IL-4和IL-10增加的更加明顯。IL-2和IL-12與對照組差異有統(tǒng)計學(xué)意義(p＜0．05)；IL-4和IL-10與實(shí)驗(yàn)組C和對照組差異有統(tǒng)計學(xué)意義(p＜0．01)。 大鼠感染第8周后外周靜脈血清中，細(xì)胞因子檢測：感染8周后血清中sIL-2R和IFN-γ的分泌明顯的增加，實(shí)驗(yàn)組與對照組之間差異有統(tǒng)計學(xué)意義(p＜0．05)，實(shí)驗(yàn)組A、B和C組之間差異無統(tǒng)計學(xué)意義(p＞0．05)；TNF-α在感染8周后盡管分泌有增加的趨勢，但實(shí)驗(yàn)組和對照組之間差異無統(tǒng)計學(xué)意義(p＞0．05)。補(bǔ)體檢測：實(shí)驗(yàn)組A、B和C組之間，C3、C4、CRP的含量差異無統(tǒng)計學(xué)意義(p＞0．05)，實(shí)驗(yàn)組A、B和C組與對照組之間的差異有統(tǒng)計學(xué)意義(t=3．88-2．58，p＜0．05)。免疫球蛋白檢測：實(shí)驗(yàn)組A、B和C組之間的差異無統(tǒng)計學(xué)意義(p＞0．05)，對照組與實(shí)驗(yàn)組之間的差異有統(tǒng)計學(xué)意義(p＜0．05)。 (4)分離分枝桿菌菌體蛋白，電泳分離，并進(jìn)行分析。分離到分子量65kD左右的蛋白抗原，為主要的致病性蛋白。雙向電泳實(shí)驗(yàn)表明分枝桿菌菌株細(xì)胞壁檢測到490個蛋白質(zhì)斑點(diǎn)。分子質(zhì)量范圍均處于10～100kD。蛋白的pI值主要分布于4．5～6．0，其中在pH4．5～5．5之間蛋白分布比較密集，5．5～6．0之間的蛋白點(diǎn)比較稀散，即偏堿性側(cè)的蛋白幾乎沒有，蛋白主要集中在偏酸性側(cè)。兩株分離株和標(biāo)準(zhǔn)海分枝桿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.
【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：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.
【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378;S855.99
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