【摘要】：豬鏈球菌是臨床上重要的人獸共患革蘭氏陽(yáng)性機(jī)會(huì)致病菌,可引起人和動(dòng)物(主要是豬)的腦膜炎、關(guān)節(jié)炎、心內(nèi)膜炎和敗血癥等臨床癥狀,對(duì)養(yǎng)豬業(yè)的健康發(fā)展和公眾健康均造成嚴(yán)重的經(jīng)濟(jì)損失和嚴(yán)重威脅。根據(jù)莢膜抗原可將豬鏈球菌分為35個(gè)血清型,其中以豬鏈球菌2型(Streptococcus suis serotype 2,SS2)流行最廣,也是危害最嚴(yán)重的血清型。正是由于豬鏈球菌包含多個(gè)血清型,應(yīng)用疫苗進(jìn)行豬鏈球菌感染的防治未必能取得較好效果,另外,由于抗生素的濫用和大量使用,使得耐藥性豬鏈球菌日益嚴(yán)重,使得豬鏈球菌的防治情況日益惡化,因此臨床上迫切需要新的抗豬鏈球菌(尤其是SS2)感染策略或者具有新型作用機(jī)制的藥物。隨著近年來(lái)分子生物學(xué)等相關(guān)科學(xué)研究領(lǐng)域的研究日益深入,人們逐步發(fā)現(xiàn)細(xì)菌的致病力與其合成和分泌的各種毒力因子密切相關(guān)。因此對(duì)病原菌感染的控制不僅依賴于抗菌藥物的抗菌活性,同時(shí)也依賴于抗菌藥物對(duì)致病菌感染過(guò)程的干預(yù)作用,因此近年來(lái),以毒力因子為靶標(biāo)進(jìn)行抗感染治療日益受到人們的關(guān)注。在SS2中,其致病力與分選酶A(Sortase A)和豬鏈球菌溶血素(SLY)密切相關(guān),其中Sortase A主要介導(dǎo)SS2表面蛋白錨定;而SLY可裂解宿主細(xì)胞,使得細(xì)菌獲得相應(yīng)的營(yíng)養(yǎng)成分。因此本論文主要以SS2 Sortase A和SLY為靶標(biāo)在中藥中進(jìn)行了抑制劑的篩選,首先通過(guò)構(gòu)建原核表達(dá)載體,實(shí)現(xiàn)了SS2Sortase A和SLY的原核表達(dá)和純化,以Sortase A和SLY的生物學(xué)功能為基礎(chǔ)建立了相關(guān)篩選平臺(tái)進(jìn)行了抑制劑的篩選,結(jié)果發(fā)現(xiàn)銀杏葉提在較低濃度(4-32μg/m L)下即可顯著抑制SLY的成孔活性以及Sortase A的催化活性,但是銀杏葉提取物并不影響SLY的分泌,這一結(jié)果表明銀杏葉提取物是一種潛在的抗SS2感染的抗毒力復(fù)合物。抗菌活性實(shí)驗(yàn)表明銀杏葉提取物對(duì)SS2的最小抑菌濃度(MIC)為256-1024μg/m L,提示銀杏葉提取物對(duì)SS2幾乎沒(méi)有抑菌活性,另外,殺菌活性測(cè)定實(shí)驗(yàn)表明,銀杏葉提取物對(duì)SS2的最小殺菌濃度(MBC)為1×MIC-4×MIC之間,依據(jù)MBC≥32×MIC,則細(xì)菌對(duì)受試藥物已產(chǎn)生耐藥性這一原則,上述結(jié)果表明SS2對(duì)銀杏葉提取物均未產(chǎn)生耐藥性,進(jìn)一步提示銀杏葉提取物代表潛在的抗SS2感染的復(fù)合物。為了進(jìn)一步驗(yàn)證銀杏葉提取物體外對(duì)SS2致病力的影響,本實(shí)驗(yàn)建立了SS2與血管內(nèi)皮細(xì)胞共感染體系,考慮到Sortase A和SLY在細(xì)菌粘附、侵襲、介導(dǎo)細(xì)胞毒性和突破血腦屏障并進(jìn)入腦組織中發(fā)揮著極其重要的作用,因此實(shí)驗(yàn)考察了銀杏葉提取物對(duì)SS2與血管內(nèi)皮細(xì)胞共感染過(guò)程中細(xì)菌粘附和侵襲血管內(nèi)皮細(xì)胞能力、細(xì)菌介導(dǎo)血管內(nèi)皮細(xì)胞毒性作用以及細(xì)菌遷移血管內(nèi)皮細(xì)胞能力的影響。結(jié)果發(fā)現(xiàn),在SS2與血管內(nèi)皮細(xì)胞共感染體系內(nèi),加入銀杏葉提取物可顯著降低SS2對(duì)血管內(nèi)皮細(xì)胞的粘附和侵襲,通過(guò)LDH釋放實(shí)驗(yàn)表明,銀杏葉提取物可明顯抑制SS2介導(dǎo)的細(xì)胞毒性作用;遷移試驗(yàn)表明SS2對(duì)血管內(nèi)皮細(xì)胞的遷移能力受到顯著抑制。上述結(jié)果表明銀杏葉提取物體外顯著抑制SS2的致病力,提示銀杏葉提取物可應(yīng)用于SS2感染的防治。另外,本實(shí)驗(yàn)還應(yīng)用SS2感染小鼠和實(shí)驗(yàn)豬建立了SS2感染實(shí)驗(yàn)動(dòng)物模型,通過(guò)觀察實(shí)驗(yàn)動(dòng)物的死亡率、腦組織菌落定殖數(shù)以及臨床表現(xiàn)進(jìn)一步評(píng)價(jià)了銀杏葉提取物對(duì)SS2體內(nèi)感染的防治作用。結(jié)果表明,銀杏葉提取物可顯著降低SS2感染小鼠的死亡率,經(jīng)藥物治療后,感染小鼠腦組織菌落定殖數(shù)明顯低于無(wú)藥物治療組;另外,銀杏葉提取物治療后顯著改善人工SS2感染實(shí)驗(yàn)豬的臨床癥狀及其感染動(dòng)物的死亡率。上述結(jié)果表明銀杏葉提取物對(duì)SS2感染實(shí)驗(yàn)動(dòng)物(小鼠和實(shí)驗(yàn)豬)均有顯著的保護(hù)作用。綜上所述,抗菌活性較弱或幾乎無(wú)抗菌活性的銀杏葉提取物可直接中和SS2 SLY和Sortase A的生物學(xué)功能,進(jìn)而抑制SS2與血管內(nèi)皮細(xì)胞的粘附和侵襲、SS2介導(dǎo)的細(xì)胞毒性作用以及SS2對(duì)血管內(nèi)皮細(xì)胞的遷移作用,體內(nèi)實(shí)驗(yàn)動(dòng)物(小鼠和實(shí)驗(yàn)豬)感染實(shí)驗(yàn)表明經(jīng)銀杏葉提取物治療后,感染實(shí)驗(yàn)動(dòng)物的死亡率、腦組織菌落定殖數(shù)以及臨床癥狀等指標(biāo)均得到顯著的緩解,表明銀杏葉提取物是一種潛在的抗SS2感染的新型復(fù)合物,且是通過(guò)抑制SS2毒力因子的生物學(xué)功能,干預(yù)細(xì)菌感染過(guò)程而發(fā)揮抗感染作用,是一種具有全新作用機(jī)制的新型抗感染復(fù)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S853.7

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9 雯麗;這些營(yíng)養(yǎng)物質(zhì)能幫助提升腦能量[N];中國(guó)食品報(bào);2011年

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