产黑色素普里沃氏菌的超微结构及其外膜蛋白的电泳特性

目的：观察产黑色素普里沃氏菌（Ｐｍ）的超微结构及其外膜蛋白的泳动特性。方法：用电镜和ＳＤＳ－ＰＡＧＥ。结果：３ｄ培养的Ｐｍ具有典型而完整的菌体结构，随培养时间延长（７ｄ），菌体破裂，结构不完整，Ｐｍ的外膜蛋白在ＳＤＳ存在下可出现多条分子量各异的多肽带，但也有不随解离剂和温度影响的成份。结论：Ｐｍ具有典型的革兰氏阴性菌结构，又含有其独特的外膜成份。     

用酶消化法进行鼠原代牙髓细胞的体外培养

目的：试用酶消化法获取鼠原代牙髓细胞，并研究其形态和生长特性。方法：采用０．５％胰酶－０．１％ＥＤＴＡ酶消化法获取ＳＤ鼠原代牙髓细胞，在倒置显微镜下观察其细胞形态和生长情况。结果：细胞形态主要呈梭形和星形，在６ｄ时开始增殖，１３ｄ细胞数达最高，细胞倍增时间为５８．５ｈ（６ｄ～１３ｄ）。结论：用胰酶－ＥＤＴＡ酶消化法可在短期内获得大量的牙髓细胞。     

急性炎症过程中金黄地鼠颊囊SOD活性的变化规律

目的：利用超氧自由基（Ｏ－２）诱导的金黄地鼠颊囊急性炎症损害模型，研究颊囊组织中超氧化物歧化酶（ＳＯＤ）活性的动态变化规律以及外源性ＳＯＤ对其的影响。方法：采用化学发光法测定金黄地鼠颊囊组织中ＳＯＤ活性。结果：（１）模型组早、中期（８ｈ～３ｄ）颊囊ＳＯＤ活性均明显降低（３ｄ：１０．５５±３．５５ｕ／ｍｇ），至６ｄ时才上升到正常范围（１８．８８±２．３８ｕ／ｍｇ）；（２）ＳＯＤ组早期（８ｈ）ＳＯＤ活性显著升高（１１０．８９±２１．２２ｕ／ｍｇ），随后（１ｄ～６ｄ）下降接近正常值（６ｄ：１７．９３±４．７６ｕ／ｍｇ）。结论：外源性ＳＯＤ使内源性ＳＯＤ受到保护而不致于被耗竭或钝化。     

单克隆抗体对变形链球菌生长的影响

用自制的抗远缘链球菌ＯＭＺ１７６２２０ｋＤ表面蛋白抗原的单克隆抗体（ＭＣＡｂ）对ＯＭＺ１７６菌的生长进行研究。结果表明，在一定时间内ＭＣＡｂ（ＷＣ３Ａ６ｄ）与ＯＭＺ１７６菌作用后，其生长率较对照组明显下降（Ｐ＜０．０１）。     

用酶消化进行鼠原代牙髓细胞的体外培养

目的：试用酶消化法获取鼠原代牙髓细胞，并研究其形态和生长特征。方法“采用０．５％胰酶－０．１％ＥＤＴＡ酶消化法获取ＳＤ鼠原代牙髓细胞，在代表团显微镜下观察其细胞形态和生长情况。结果：细胞形态主要呈梭形和星形，在６ｄ时开始增殖，１３ｄ细胞数达最高，细胞倍增时间为５８．５ｈ（６ｄ￣１３ｄ）。结论：用胰酶－ＤＥＴＡ酶消化法可在短期内获得大量的牙髓细胞。     

颌面部枪弹伤软组织内一氧化氮含量与组织细胞损伤

在Ｄ５．８０ｍｍ制式枪弹致伤动物模型上的颌面部，于致伤后２、６、２４、７２ｈ、７ｄ取材，检测组织内ＮＯ的含量，并用ＨＥ和ＮＡＤＰＨ－ｄ染色观察组织形态学变化与一氧化氮合酶（ＮＯＳ）活性的关系。结果发现：伤后组织内ＮＯ含量明显增高，２４ｈ达到高峰，７ｄ维持较低水平；２４～７２ｈ组织呈进行性损伤，组织内可见大量ＮＯＳ阳性细胞，７ｄ组织内修复现象明显，ＮＯＳ阳性细胞明显减少。结果提示：颌面部枪弹伤后组织进行性损伤和修复愈合与Ｌ－精氨酸／ＮＯ途径的激活有关；ＮＯ的大量生成是组织细胞损伤的重要因素之一。     

单克隆抗体对变链球菌粘附的影响

分别用抗ＯＭＺ１７６菌（ｄ血清型）和抗ＭＴ６Ｒ菌（ｃ血清型）表面蛋白抗原２２０ＫＤ和１８５ＫＯ分子量的单克隆抗体（ＭｃＡｂ）对血清型变链球菌ｃ、ｄ作用后，通后３Ｈ核素标记测试细菌的粘附性。结果显示：抗２２０ＫＤ、抗１８５ＫＤ表面蛋白抗原的ＭｃＡｂｓ对相应血清型的变链球菌的粘附均呈现明显的抑制作用，两种ＭｃＡｂｓ对不相应血清型变链球茵的粘附也有一定的抑制作用。因而认为ＭｃＡｂ通过局部被动免疫抗粘附原理对变链球菌粘附具有抑制作用。     

单克隆抗体对变形链球菌生长的影响

用自制的抗远缘链球菌ＯＭＺ１７６ ２２０ＫＤ表面蛋白抗原的单克隆抗体对ＯＭＺ１７６菌的生长进行研究。结果表明，在一定时间内ＭｃＡｂ与ＯＭＺ１７６菌作用后，其生长率较对照组明显下降。     

颌面部伧弹伤软组织内一氧化氮含量与组织细胞损伤

在Ｄ５．８０ｍｍ制成枪弹致伤动物模型上的颌面部，于致伤后２，６，２４，７２ｈ，７ｄ取材，检测组织内ＮＯ的含量，并用ＨＥ和ＮＡＤＰＨ－ｄ染色观察组织形态学变化与一氧化氮合理活性的关系。结果发现：伤后组织内ＮＯ含量明显增高，２４ｈ达到高峰，７ｄ维持较低水平；２４－７２ｈ组织呈进行性损伤，组织内可见大量ＮＯＳ阳性细胞，７ｄ组织内修复现象明显，ＮＯＳ阳性细胞明显减少。     

金黄地鼠颊囊癌生成过程中过氧化脂质和超氧化物歧化酶活性的动态研究

实验选用４８只金黄地鼠做实验对象，随机分为６组，其中４组用０．５％ＤＭＢＡ丙酮溶液涂布颊囊粘膜诱癌，第９周时粘膜癌变。分别于６、９、１２、１８周日才进行Ｐ—ＬＰＯ和Ｅ—ＳＯＤ测定，动态地分析肿瘤发生发展过程中血浆过氧化脂质和红细胞超氧化物歧化酶活性含量的变化情况，探讨Ｐ—ＬＰＯ和Ｅ—ＳＯＤ含量变化与肿瘤间的关系。结果发现，诱癌早期，肿瘤未出现时，Ｐ—ＬＰＯ呈逐步上升趋势，Ｅ－ＳＯＤ则逐步下降；肿瘤生成后，前期Ｐ—ＬＰＯ开始下降，但未低于正常水平，后期则急剧升高并超过癌变前最高水平，而Ｅ—ＳＯＤ在肿瘤早期便急剧上升并持续到后期与Ｐ—ＬＰＯ呈高含量水平平衡．实验结果表明，自由基和其清除物含量的测定对预测肿瘤的发生发展和提供诊疗依据有一定帮助。     

Extracellular vesicle-associated and soluble trypsin-like enzyme fractions of Porphyromonas gingivalis W50

Soluble vesicle-associated trypsin-like enzyme fractions (VSF) were prepared by sonication from extracellular vesicles (ECV) from strains W50 and W50/BE1. High-(H), intermediate-(I) and low-(L) molecular-weight VSF enzyme subfractions were identified by non-dissociative gel filtration chromatography with Mr 160, 95 and 60 kDa respectively. The chromatographic profiles of W50 VSF from 48-h and 72-h cultures were identical. W50/BE1 VSF displayed a higher ratio of the 160 to 60 kDa components. This ratio was reduced in VSF from 72-h cultures. Extracellular soluble protein (EP) trypsin profiles were similar to their respective VSF, but the 60 kDa component predominated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a loss of soluble extracellular polypeptides with culture age. A polyclonal antiserum to EP subfraction L reacted in immunoblots with a 50 kDa peptide of subfraction L of W50. Whole EP and its subfraction H displayed a 50 kDa immunoreactive peptide but no peptides of higher molecular weight. This antiserum reacted with a similar sized peptide, and with lower-molecular-weight components in whole ECV. Gelatin substrate zymography of whole EP following non-reducing SDS-PAGE revealed a major 80 kDa protease that increased with culture age. Minor protease bands of 70 and 50 kDa were also detected.     

Haemagglutinating and haemolytic activity of the extracellular vesicles of Bacteroides gingivalis W50

The extracellular vesicles (ECV) and extracellular protein (EP) fractions of Bacteroides gingivalis W50 showed haemagglutinating (HA) activity towards sheep erythrocytes. Similar fractions from the nonpathogenic strain W50/BE1 did not haemagglutinate. W50 ECV HA activity was not inhibited by various glycosidase, phospholipase or protease pretreatments, sugars or amino acids, including arginine or lysine. The haemagglutinating activity of ECV was associated only with the extracellular vesicle membrane. The EP and ECV of both strains displayed haemolytic activity. This activity was apparently depressed in the presence of 10 mM dithiothreitol (DTT). All EP and ECV fractions degraded certain structural sheep erythrocyte membrane proteins. The greatest activity was displayed by W50 ECV and W50/BE1 EP and was enhanced by DTT. In the presence of DTT, the ECV of both strains degraded purified human haemoglobin but this activity was greatly reduced in its absence.     

几株口腔厌氧菌胞外膜泡,外膜蛋白复合物的提取

采用盐析、超滤、超速离心等方法提取并比较了５种口腔常见厌氧菌（ＰｇＷ５０，Ｐｇ３８１，ＰｅＡＴＣＣ３５４０６，ＰｉＡＴＣＣ２５２６１，ＰｍＡＴＣＣ２５８４５）的胞外膜泡（ＥＣＶ）、外膜蛋白复合物（ＯＭＰＣ），结果显示：二种提取物外观性状均较好，呈淡灰色，其中ＰｇＷ５００ＯＭＰＣ的湿沉淀有一定粘度。ＥＣＶ的提取率由高至低分别为：ＰｇＷ５０（１９．８４ｍｇ／Ｌ）、Ｐｇ３８１（１８．７５ｍｇ／Ｌ）、Ｐｅ（１５．６５ｍｇ／Ｌ）、Ｐｍ（９．６０ｍｇ／Ｌ）、Ｐｉ（７．４０ｍｇ／Ｌ）；ＯＭＰＣ提取率分别为：ｐｇＷ５０（８７．３６ｍｇ／Ｌ）、Ｐｇ３８１（７２．５６ｍｇ／Ｌ）、Ｐｉ（５７．４０ｍｇ／Ｌ）Ｐｍ（４１．２８ｍｇ／Ｌ）、Ｐｅ（３２．７６ｍｇ／Ｌ）．在ＥＣＶ的提取中，采用超滤和超离心法提取ＥＣＶ的量要高于盐析法，表明营养条件不是细菌ＥＣＶ形成的决定因素。     

非白色假丝酵母菌代谢产物对人脐静脉内皮细胞株ECV304细胞增殖的诱导作用

目的 研究3种常见的非白色假丝酵母菌对人脐静脉内皮细胞株ECV304细胞增殖及细胞周期的影响。方法 制备热带假丝酵母菌、克鲁斯假丝酵母菌和光滑假丝酵母菌上清液并行倍比稀释,设1、4、16倍3个稀释度以及对照组。体外培养ECV304细胞,分别将3种非白色假丝酵母菌不同稀释度的上清液与ECV304细胞共培养,采用 MTT法测定培养24、48、72 h后的细胞增殖率;倒置显微镜观察培养48 h后细胞密度的变化;流式细胞术测定培养 48h后对ECV304细胞周期的影响。结果 培养24 h后,3种假丝酵母菌1倍稀释的上清液均可促进细胞增殖,4倍稀释的克鲁斯假丝酵母菌也可促进细胞增殖（P<0.05）;培养48、72 h后,克鲁斯假丝酵母菌和光滑假丝酵母菌上清液仍可促进ECV304细胞增殖,而热带假丝酵母菌上清液无论稀释度高低均不能促进ECV304细胞增殖。培养48 h后,克鲁斯假丝酵母菌和光滑假丝酵母菌上清液组的细胞密度明显增高,同时其G/G1期细胞所占比例降低,细胞增殖指数（P）升高（P<0.05）;而热带假丝酵母菌组的细胞密度和PI均无明显变化（P>0.05）。结论 克鲁斯假丝酵母菌和光滑假丝酵母菌的代谢产物对ECV304细胞的增殖有诱导作用,临床上应加强非白色假丝酵母菌感染的检测和治疗。     

牙龈卟啉菌（Pg）的胰酶样蛋白酶（TLP）检测

对Ｐｇ的ＥＣＶ和ＯＭＰＣ的ＴＬＰ检测结果显示：ＥＣＶ中的酶（ＴＬＰ）和蛋白含量均高于ＯＭＰＣ，ＥＣＶ中的ＴＬＰ活性在细菌生长早期随细菌的快速增长而升高，与细菌的生长基本同步，说明：（１）ＴＬＰ的酶合成是发生在细菌生长早期；（２）ＥＣＶ的形成是生长期细菌的一个特征，而不是老化的细菌或者非生长期细菌的代谢产物。     

The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50

A soluble trypsin-like enzyme (STE) was purified from a cell- and particle-free culture supernatant of this bacterium by a combination of ultra-centrifugation, ammonium-sulphate precipitation and gel-filtration chromatography on Sephacryl S-200. Trypsin-like activity in the culture supernatant was associated with a 58 kDa peptide and also with a higher molecular-weight complex. The STE and extracellular vesicle (ECV) fraction of B. gingivalis W50 rapidly degraded human plasma fibronectin in the presence and the absence of 10 mM dithiothreitol (DTT). The STE yielded a range of lower molecular-weight fibronectin digestion products. Under conditions where little activity was expressed by mammalian trypsin, both STE and ECV depolymerized a denatured and a native type I collagen substrate. Quantitative and qualitative differences were observed in the patterns of digestion products generated by both STE and ECV fraction following incubation with and without 10mM DTT. Inclusion of DTT appeared to reduce the degradative effect of both ECV and STE towards the type I collagen and plasma fibronectin substrates.     

细菌膜泡（ECV）,外膜蛋白复合物（OMPC）的氨基酸组成分析

采用酸水解法比较分析了ＰｇＷ５０、Ｐｇ３８１的ＥＣＶ、ＯＭＰＣ中１７种氨基酸的不同含量，结果显示：ＥＣＶ的蛋白结构基本上与同菌的ＯＭＰＣ相近，而在某些氨基酸浓度（ｎｍｏｌ／Ｌ）存在差异，说明在ＥＣＶ形成过程中其外膜可能发生某些生化改变；不同菌的ＯＭＰＣ之间、ＥＣＶ之间在氨基酸含量上也存在着差异，表明不同种的细菌，其ＯＭＰＣ在结构上各有差异，相应形成的ＥＣＶ都会发生某些特殊的变化；另外，天冬氨酸、甘氨酸可能也是组成Ｐｇ、Ｐｅ细菌细胞壁的重要的氨基酸成分。     

The distribution of trypsin-like enzyme activity in cultures of a virulent and an a virulent strain of Bacteroides gingivalis W50

The distribution of trypsin-like enzyme activity was studied in 48- and 72-h batch cultures of Bacteroides gingivalis W50 and an avirulent variant (W50/BE1) of the parent strain. Activity was measured at pH 7.4 in cells, the extracellular vesicle (ECV) and soluble extracellular protein (EP) fractions recovered by ammonium sulphate precipitation from spent growth medium. Both organisms produced cell surface and extracellular vesicles, but whilst strain W50 produced more ECV, W50/BE1 yielded more of the EP fraction by weight. Whole cultures of W50 displayed a three-fold greater trypsin activity than those of W50/BE1. However, 90% of the total enzyme activity of W50 cultures was associated with the particulate fraction (cells and ECV totalled), whereas this fraction accounted for only 10-30% of the total for W50/BE1. Unlike W50/BE1, the specific activities of W50 cells and ECV rose in 72-h cultures. Conversely, cultures of W50/BE1 displayed an increase in the yield and specific activity of the EP fraction.     

白假丝酵母菌对人脐静脉内皮细胞株ECV304细胞增殖的诱导作用

目的 研究白假丝酵母菌(S.albicans)对人脐静脉内皮细胞株ECV304细胞增殖及细胞周期的影响.方法 体外培养ECV304,实验分为S.albicans上清液组、S.albicans灭活菌液组、上清液和灭活菌液混合组、对照组.采用MTT法、细胞计数法、倒置显微镜及流式细胞术分别观察各组对ECV304细胞增殖及细...     

平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其机制的实验研究

目的：探讨平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其相关机制。方法：用不同浓度的平阳霉素作用ECV304细胞不同时间,以MTT法比较平阳霉素作用24、48及72h后的细胞抑制率,应用DNA电泳实验、流式细胞术分析细胞凋亡、坏死情况、细胞周期及Fas、Bcl-2蛋白的表达。结果：MTT法显示随平阳霉素浓度升高和作用时间的延长,细胞生长抑制越明显DNA电泳证实合适浓度平阳霉素作用下细胞发生凋亡,过高浓度细胞则发生坏死;流式细胞术显示细胞凋亡率和坏死率随药物浓度和作用时间增加而增高,过高浓度细胞坏死占主导;平阳霉素各浓度作用24h后,G2—M期百分率增高,S期百分率降低,G0—G1期百分率无明显变化;Fas表达随药物浓度增加而递减,Bcl-2表达随药物浓度增加而升高。结论：平阳霉素能明显抑制ECV304细胞生长,并具有浓度和时间依赖性;平阳霉素可能通过诱导凋亡和坏死作用以抑制ECV304细胞的体外生长和增殖;细胞凋亡发生在G2—M期,是通过上调Fas蛋白并下调Bcl-2蛋白的表达来实现的。