银杏叶提取物抗猪链球菌2型感染作用及应用

发布时间：2019-04-07 20:10

【摘要】：猪链球菌是临床上重要的人兽共患革兰氏阳性机会致病菌,可引起人和动物(主要是猪)的脑膜炎、关节炎、心内膜炎和败血症等临床症状,对养猪业的健康发展和公众健康均造成严重的经济损失和严重威胁。根据荚膜抗原可将猪链球菌分为35个血清型,其中以猪链球菌2型(Streptococcus suis serotype 2,SS2)流行最广,也是危害最严重的血清型。正是由于猪链球菌包含多个血清型,应用疫苗进行猪链球菌感染的防治未必能取得较好效果,另外,由于抗生素的滥用和大量使用,使得耐药性猪链球菌日益严重,使得猪链球菌的防治情况日益恶化,因此临床上迫切需要新的抗猪链球菌(尤其是SS2)感染策略或者具有新型作用机制的药物。随着近年来分子生物学等相关科学研究领域的研究日益深入,人们逐步发现细菌的致病力与其合成和分泌的各种毒力因子密切相关。因此对病原菌感染的控制不仅依赖于抗菌药物的抗菌活性,同时也依赖于抗菌药物对致病菌感染过程的干预作用,因此近年来,以毒力因子为靶标进行抗感染治疗日益受到人们的关注。在SS2中,其致病力与分选酶A(Sortase A)和猪链球菌溶血素(SLY)密切相关,其中Sortase A主要介导SS2表面蛋白锚定;而SLY可裂解宿主细胞,使得细菌获得相应的营养成分。因此本论文主要以SS2 Sortase A和SLY为靶标在中药中进行了抑制剂的筛选,首先通过构建原核表达载体,实现了SS2Sortase A和SLY的原核表达和纯化,以Sortase A和SLY的生物学功能为基础建立了相关筛选平台进行了抑制剂的筛选,结果发现银杏叶提在较低浓度(4-32μg/m L)下即可显著抑制SLY的成孔活性以及Sortase A的催化活性,但是银杏叶提取物并不影响SLY的分泌,这一结果表明银杏叶提取物是一种潜在的抗SS2感染的抗毒力复合物。抗菌活性实验表明银杏叶提取物对SS2的最小抑菌浓度(MIC)为256-1024μg/m L,提示银杏叶提取物对SS2几乎没有抑菌活性,另外,杀菌活性测定实验表明,银杏叶提取物对SS2的最小杀菌浓度(MBC)为1×MIC-4×MIC之间,依据MBC≥32×MIC,则细菌对受试药物已产生耐药性这一原则,上述结果表明SS2对银杏叶提取物均未产生耐药性,进一步提示银杏叶提取物代表潜在的抗SS2感染的复合物。为了进一步验证银杏叶提取物体外对SS2致病力的影响,本实验建立了SS2与血管内皮细胞共感染体系,考虑到Sortase A和SLY在细菌粘附、侵袭、介导细胞毒性和突破血脑屏障并进入脑组织中发挥着极其重要的作用,因此实验考察了银杏叶提取物对SS2与血管内皮细胞共感染过程中细菌粘附和侵袭血管内皮细胞能力、细菌介导血管内皮细胞毒性作用以及细菌迁移血管内皮细胞能力的影响。结果发现,在SS2与血管内皮细胞共感染体系内,加入银杏叶提取物可显著降低SS2对血管内皮细胞的粘附和侵袭,通过LDH释放实验表明,银杏叶提取物可明显抑制SS2介导的细胞毒性作用;迁移试验表明SS2对血管内皮细胞的迁移能力受到显著抑制。上述结果表明银杏叶提取物体外显著抑制SS2的致病力,提示银杏叶提取物可应用于SS2感染的防治。另外,本实验还应用SS2感染小鼠和实验猪建立了SS2感染实验动物模型,通过观察实验动物的死亡率、脑组织菌落定殖数以及临床表现进一步评价了银杏叶提取物对SS2体内感染的防治作用。结果表明,银杏叶提取物可显著降低SS2感染小鼠的死亡率,经药物治疗后,感染小鼠脑组织菌落定殖数明显低于无药物治疗组;另外,银杏叶提取物治疗后显著改善人工SS2感染实验猪的临床症状及其感染动物的死亡率。上述结果表明银杏叶提取物对SS2感染实验动物(小鼠和实验猪)均有显著的保护作用。综上所述,抗菌活性较弱或几乎无抗菌活性的银杏叶提取物可直接中和SS2 SLY和Sortase A的生物学功能,进而抑制SS2与血管内皮细胞的粘附和侵袭、SS2介导的细胞毒性作用以及SS2对血管内皮细胞的迁移作用,体内实验动物(小鼠和实验猪)感染实验表明经银杏叶提取物治疗后,感染实验动物的死亡率、脑组织菌落定殖数以及临床症状等指标均得到显著的缓解,表明银杏叶提取物是一种潜在的抗SS2感染的新型复合物,且是通过抑制SS2毒力因子的生物学功能,干预细菌感染过程而发挥抗感染作用,是一种具有全新作用机制的新型抗感染复合物。
[Abstract]:Streptococcus suis is a clinically important human animal with Gram-positive opportunistic pathogens and can cause clinical symptoms such as meningitis, arthritis, endocarditis, and septicemia in humans and animals (mainly pigs), The health development and public health of the pig industry have serious economic losses and serious threats. Streptococcus suis can be divided into 35 serotypes according to the membrane antigen, in which Streptococcus suis serotype 2 (SS2) is the most popular, and is also the most serious serotype. Due to the fact that the streptococcus suis contains a plurality of serotypes, the prevention and treatment of the streptococcus suis infection by the application of the vaccine do not necessarily achieve a good effect, and the drug resistance of the streptococcus suis is increasingly serious due to the abuse and the large amount of use of the antibiotics, so that the prevention and treatment of the streptococcus suis is getting worse, Therefore, there is an urgent need for new anti-swine streptococci (especially SS2) infection strategies or drugs with a new mechanism of action. As the research in the fields of molecular biology and other related scientific research has been in depth, it is found that the pathogenic force of bacteria is closely related to the various virulence factors of its synthesis and secretion. Therefore, the control of the infection of the pathogenic bacteria not only depends on the antibacterial activity of the antibacterial agent, but also depends on the intervention effect of the antibacterial agent on the pathogenic bacteria infection process. In SS2, the pathogenic force of SS2 is closely related to Sortase A and Slysin (SLY). Sportase A mainly mediates the anchoring of SS2 surface protein, and SLY can lyse the host cell, so that the bacteria can obtain the corresponding nutrient components. Therefore, the expression and purification of SS2Sportase A and SLY were carried out by using SS2 Sportase A and SLY as targets. On the basis of the biological function of Sportase A and SLY, the screening of the inhibitor was established based on the biological function of Sportase A and SLY. The results showed that the activity of SLY and the catalytic activity of Sportase A could be significantly inhibited by the extraction of ginkgo biloba at a lower concentration (4-32 & mu; g/ m L). But the ginkgo leaf extract does not affect the secretion of the SLY, which results in that the ginkgo leaf extract is a potential anti-SS2-infected anti-toxic compound. The results of the antibacterial activity showed that the minimum inhibitory concentration (MIC) of the ginkgo biloba extract on the SS2 was 256-1024ug/ mL, suggesting that the ginkgo leaf extract has little antibacterial activity on the SS2, The minimum bactericidal concentration (MBC) of the extract of Ginkgo biloba to SS2 was 1-MIC-4-MIC, and according to the MICC-32-MIC, the drug-resistance of the bacteria to the tested drug was the principle. The results showed that the SS2 had no drug resistance to the ginkgo biloba extract. It is further suggested that the ginkgo leaf extract is representative of the potential anti-SS2-infected complex. In order to further verify the effect of ginkgo biloba extract on the pathogenic force of SS2 in vitro, this experiment established a system of co-infection of SS2 and vascular endothelial cells, taking into account that Sportase A and SLY were in bacterial adhesion and invasion, The effect of ginkgo biloba extract on the adhesion and invasion of vascular endothelial cells during the co-infection of SS2 and vascular endothelial cells was studied. The effect of bacteria on the cytotoxicity of vascular endothelial cells and the ability of bacteria to migrate vascular endothelial cells. The results showed that in SS2 and vascular endothelial cell co-infection system, the addition of ginkgo biloba extract could significantly reduce the adhesion and invasion of SS2 on vascular endothelial cells. The migration test showed that the migration ability of SS2 to vascular endothelial cells was significantly inhibited. The results show that the extract of Ginkgo biloba extract can inhibit the pathogenic force of SS2 in vitro, and it is suggested that the extract of Ginkgo biloba leaf can be used in the prevention and treatment of SS2 infection. In addition, SS2-infected mice and experimental pigs were also used to establish the animal model of SS2 infection. By observing the mortality of the experimental animals, the number of colonization of the brain tissue and the clinical manifestations, the prevention and treatment effect of the extract of Ginkgo biloba on the infection of SS2 was further evaluated. The results showed that the ginkgo biloba extract can significantly lower the mortality of the mice infected with SS2, and after the treatment, the number of colonization of the infected mice is significantly lower than that of the non-drug-free treatment group; in addition, The clinical symptoms and the mortality of the infected animals of the artificial ss2-infected experimental pigs are significantly improved after the ginkgo leaf extract is treated. The results showed that the ginkgo biloba extract had a significant protective effect on the SS2 infected animals (mice and experimental pigs). In conclusion, the ginkgo leaf extract with weak antibacterial activity or little antibacterial activity can directly neutralize the biological functions of SS2 SLY and Sportase A, and further inhibit the adhesion and invasion of SS2 and vascular endothelial cells, SS2-mediated cytotoxicity, and the migration effect of SS2 on vascular endothelial cells, the experimental animals (mice and experimental pigs) of the in-vivo experiment showed that after the treatment of the ginkgo leaf extract, the mortality of the infected experimental animals, the number of colonization of the brain tissue and the clinical symptoms were significantly relieved, It is suggested that the extract of Ginkgo biloba is a new type of complex of anti-SS2 infection, and it is a new anti-infection complex with a new mechanism by inhibiting the biological function of the virulence factor of SS2 and intervening in the process of bacterial infection.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S853.7

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