【作者】 钟世顺；

【导师】 张振书；

【作者基本信息】 第一军医大学 ， 内科学， 2004， 博士

【摘要】 双歧杆菌是人体肠道中一种最重要的生理菌，对人体健康具有重要意义，已有大量有关双歧杆菌抗肠道致病菌感染的报道。但双歧杆菌活菌制剂在应用于肠屏障功能障碍时也存在几个问题：一是该活菌制剂在制造与保存方面尚有一定难度，二是活菌在肠屏障功能障碍病人肠道可能由于局部气体和生长底物的不足而难以足量繁殖。近年来，有国外学者报道：双歧杆菌对肠上皮细胞的粘附可能有蛋白质样物质参与，还有人从长双歧杆菌SBT2928培养上清中分离出一种蛋白质成分，该种蛋白质成分能抑制ETEC对其糖脂结合受体(GA1)的粘附，同时也能抑制其对肠上皮细胞HCT-8的粘附且呈剂量依赖效应。由此提示双歧杆菌可能存在分泌型粘附素成分。1999年，我室也从青春型双歧杆菌1027耗尽培养上清液中提取纯化出一种分子量为16KDa的蛋白质，但未对其生理作用进行进一步研究。本研究的目的是：(1)通过化学层析等方法较大量地制备双歧杆菌分泌型粘附素成分，体外观察其对双歧杆菌粘附肠上皮细胞的介导作用；(2)评价其体外对ETEC、EPEC和艰难梭菌等致病菌粘附肠上皮细胞的竞争性抑制作用；(3)评价其对肠上皮细胞系增殖／凋亡活性及上皮细胞形态学变化的影响；(4)探讨其对上皮细胞内转录因子NF-kappaB活性的影响，以评价其生物调节活性，并从分子水平初步探讨其作用机理。 一、双歧杆菌分泌型粘附素的分离纯化及其对双歧杆菌粘附肠上皮细胞的介导作用 采用硫酸铵沉淀、Superdex 75凝胶过滤和Q-Sepharose FF离子交换色谱法对双歧杆菌分泌型粘附素进行分离纯化，并经SDS一PAGE鉴定，获得一分子量大约为1 6Kda的蛋白质;为研究其对双歧杆菌粘附肠上皮细胞的介导作用，采用粘附试验进行观察。结果表明:粘附素介导的双歧杆菌对体外培养肠上皮细胞的粘附具有浓度和时间依赖性，当其浓度为30 p g/ml、时间为3小时时，粘附达到饱和状态;粘附素悬浮的双歧杆菌对Lovo细胞的粘附能力与耗尽培养上清悬浮的双歧杆菌的粘附能力无显著性差别(分别为18.28士6.55和19.90土6.07，P>0.05)，但明显强于用PBS悬浮的双歧杆菌(8.50士3.30，尸<0.01)，细菌呈线性粘附于细胞周围。上述结果表明，本实验成功分离纯化了双歧杆菌分泌型粘附素，其分子量大约为16KDa;粘附素能特异性地介导双歧杆菌粘附于肠上皮细胞周围，并且具有浓度和时间依赖性。 二、双歧杆菌分泌型粘附素对致病菌粘附肠上皮细胞的竟争抑制作用Lovo细胞预先与不同浓度的双歧杆菌分泌型粘附素孵育30分钟后，再与ETEC、EPEC和艰难梭菌进行粘附试验，结果发现，除粘附素浓度为1林留ml和5林g/m1时不能明显抑制病原菌对Lov。细胞的粘附外，10林留ml，20林g/ml，30林g/m1浓度组均能明显抑制病原菌对Lovo细胞的粘附，且随浓度的逐渐增加，这种抑制作用也逐渐增强。粘附素浓度为30“留ml时，对ETEC、EPEC或艰难梭菌粘附Lovo细胞的抑制作用与双歧杆菌+ETEC、双歧杆菌+EPEC或双歧杆菌+艰难梭菌组相比无显著差异(P>0.05)。ETEC和EPEC呈线状粘附于细胞周围，而艰难梭菌呈簇状粘附于细胞周围。粘附素浓度为1“g/ml和5林g/m1时，ETEC、EPEC和艰难梭菌粘附肠上皮细胞的平均荧光强度与无粘附素时相比无显著差别;而10林留ml，20林g/m1，30林留ml浓度组的平均荧光强度均显著低于ETEC、EPEC或艰难梭菌单独作用组(尸<0.01)，且随浓度的逐渐增加，荧光强度也逐渐减弱。其中30林g/ml浓度组的平均荧光强度与双歧杆菌+ETEC、EPEC或艰难梭菌组相比无显著差别(P>O.05)。上述结果提示，在体外实验中，青春型双歧杆菌1027的纯化粘附素能有效地抑制ETEC、EPEC和艰难梭菌对肠上皮Lovo细胞的粘附且呈剂量依赖效应。 三、双歧杆菌分泌型粘附素对脂多糖和H202调节肠上皮细胞增殖和凋亡的影响采用3H一TdR掺入法测定细胞增殖的结果表明，100雌/LLPS能显著促进肠上皮细胞的增殖，而双歧杆菌分泌型粘附素则能抑制LPS对肠上皮细胞的促增殖作用，且随粘附素浓度的升高，抑制作用有 4逐渐增强的趋势(P<0.01一0.05)。而200林mol几玩O:对肠上皮细胞有明显的抑制增殖作用(P<O.01)，双歧杆菌分泌型粘附素可对抗玩02的这种抑制增殖作用(尸<0.01一0.05)。流式细胞仪检测细胞凋亡发现，对照组细胞凋亡率为(4.40士0.52)%，LPS处理组细胞凋亡率为(14.63士0.57)%，加入双歧杆菌分泌型粘附素后，细胞凋亡率逐渐下降，随着粘附素浓度的逐渐升高，这种作用更为明显(P<0.01一0.05)。而HZo:处理组，其细胞凋亡率达(23.28士0.68)%，双歧杆菌分泌型粘附素处理后，细胞凋亡率也逐渐下降，而且随着粘附素浓度的逐渐升高，这种抑制作用也更为明显(p<0 .01一0.05)。丫淀橙染色、电镜下观察细胞形态学变化发现，LPS和玩02处理组可见较多凋亡细胞，核浓缩，聚集于核周边或裂解成碎片;而先经粘附素处理后，凋亡细胞数则明显减少。光镜下观察见LPS或H20:处理组细胞变小、变圆，胞浆颗粒明显增多，部分细胞脱落漂浮于培养基中;细胞先经粘附素处理后，变小、变圆，胞浆颗粒增多的细胞较单独LPS或HZ更多还原

【Abstract】 Bifidobacteria, the predominant bacteria in the human intestinal microflora, are considered to be microorganisms with a great influence on human health. There have been many studies demonstrating that strains of bifidobacteria have anti-infectious properties against enteropathogenic bacteria. However, there existed a few flaws when live bifidobacteria agents was used in gut barrier dysfunction: live bifidobacteria agents were difficult to produce and preserve, and also difficult to breed sufficiently due to lack of local gas and growth substrate. In recent years, it was reported the occurrence of bifidobacteria adhering to the human intestinal cells by a mechanism of adhesion which involves a proteinaceous component. It was observed that SBT2928 produced a proteinaceous factor, binding inhibitory factor (BIF), which prevented the binding of the ETEC to GAl in vitro, and the binding of ETEC to the human intestinal epithelial cell line HCT-8 was reduced by BIF treatment in a dose-dependent manner. These results showed there was adhesin component in bifidobacteria. In 1999, we extracted and purified a protein with a molecular weight of 16 KDa from spent culture supernatant of bifidobacterium adolescentis 1027 (B. ado 1027), but it’s physiological functions was not studied. In the present study, our aims were: (1) To produce mass bifidobacterial secretion adhesin by chemistry chromatography, and observe the role of adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells mediated by it in vitro. (2) To evaluate7competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cells by adhesin in vitro. (3) To study the effect of adhesin on proliferation, apoptosis and morphology of intestinal epithelial cells. (4) To detect the effect of adhesin on expression of NF- K B activity, which could regulate expression of cytokines, and hope to investigate the mechanism of adhesin in molecular levels.Isolation and purification of bifidobacterial secretion adhesin and its role on adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells The adhesin of bifidobacterium was isolated and purified by ammonium sulfate deposition, Superdex 75 gel filtration and Q-Sepharose FF ion exchange chromatography, and the adhesin was analyzed by SDS-PAGE. A protein with a molecular weight of 16 KDa was obtained. Adhesion test was used to study the adhesion of human bifidobacterial strain to cultured human intestinal epithelial cells. Results showed that the adhesion was time and dose-dependent, and adhesiveness achieved saturation when adhesin in the concentration of 30ug/ml, and lasting 3 hours. The adhesion of bifidobacteria to Lovo cell was no significantly difference between pretreatment of adhesin and spent culture supernatant (18.28 + 6.55 and 19.90 ?.07, respectively. P>0.05), but the adhesion was significantly stronger than that of by the pretreatment of PBS(8.50?.30, P.01). The adhesion of bifidobacteria to intestinal epithelial cell was linear. These results suggested a 16KDa protein was purified and identified as the adhesin of bifidobacterium. The binding of human bifidobacterial strains to human intestinal epithelial cells mediated by the adhesin appeared to be specific, time and dose-dependent.Competitive inhibition of adherence of enteropathogens to intestinal epithelial cells by bifidobacterial secretion adhesin The number of adhesion of ETEC, EPEC and Clostridium difficile to Lovo cells was counted and the results were analyzed by comparison after 30 min of incubation indifferent concentration of adhesin. Results showed the adhesin at thegconcentration of 10 ug/ml, 20 ug/ml and 30 ug/ml except at 1 ug/ml and 5 ug/ml could significantly inhibit the adhesion of ETEC, EPEC and clostridium difficile to intestinal epithelial cell line Lovo. Moreover, we observed that the ability of inhibition was enhanced with increase in the concentration of adhesin. Inhibition of更多还原