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。     

颊纤毛菌对变异链球菌产酸及黏附作用的影响

目的:研究分别使用颊纤毛菌(L.buccalis)及其上清液与变异链球菌(S.m)共培养,观察S.m的产酸、黏附变化及乳酸脱氢酶(ldh)、葡糖基转移酶(gtfb、gtfc、gtfd)基因表达变化.方法:首先从龈上菌斑中分离口腔L.buccalis,采用生化鉴定和测序鉴定;将L.buccalis单独培养组、S.m单独培养组、L.buccalis与S.m共培养组和S.m加入L.buccalis上清液培养组均培养9 h,比较每小时各组细菌产酸能力的变化;再将L.buccalis单独培养组、S.m单独培养组、分别加入1、2、3 mL L.buccalis菌液的S.m共培养组和分别加入1、2、3 mL L.buccalis上清液的S.m菌液培养组,用实时定量RT?PCR法测定各组ldh、gtfb、gtfc和gtfd的基因表达;在S.m中分别加入50、100、150μL L.buccalis上清液培养,在激光共聚焦显微镜下观察S.m黏附数量变化.结果:本实验成功分离L.buccalis,S.m中加入L.buccalis或L.buccalis上清液培养后pH下降速率增快,细菌的黏附数量增多,且随L.buccalis或L.buccalis上清液体积的增加ldh、gtfb、gtfc和gtfd的基因表达增高.结论:L.buccalis及其上清液对S.m的产酸及黏附能力具有促进作用.     

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。     

嗜酸乳杆菌对人舌癌细胞增殖的影响

目的研究嗜酸乳杆菌对人舌癌细胞Tca8113增殖及细胞周期的影响。方法体外培养Tca8113细胞,分别将不同稀释度（原液和4、16倍稀释）的嗜酸乳杆菌上清液、灭活菌液和无细胞提取物与Tca8113细胞共培养,采用倒置显微镜观察细胞形态并行细胞计数,磺酰罗丹明B（SRB）法测定细胞增殖率,流式细胞术分析嗜酸乳杆菌各组分对Tca8113细胞增殖及细胞周期的影响,激光扫描共聚焦显微镜（CLSM）检测细胞内自由基和Ca2+含量。结果嗜酸乳杆菌各组分作用于Tca8113细胞48 h后,在倒置显微镜下观察,细胞由菱形、多角形、铺路石状变为细长形。细胞计数与SRB实验分析：在不同稀释度同一培养时间与不同培养时间同一稀释度培养条件下,嗜酸乳杆菌各组分均可明显抑制Tca8113细胞增殖,抑制力随稀释度增加而降低,随培养时间延长而增强。流式细胞术分析：嗜酸乳杆菌各组分作用Tca8113细胞48 h后,细胞增殖指数降低（P<0.01）。CLSM检测：嗜酸乳杆菌各组分作用Tca8113细胞48 h后细胞内自由基和Ca2+含量均升高（P<0.01）。结论嗜酸乳杆菌代谢产物、灭活菌液、无细胞提取物均可抑制Tca8113细胞增殖,可能与菌体及其代谢产物引起细胞内自由基含量增多、Ca2+超载有关。     

成纤维细胞上清提取物对表皮细胞增殖迁移的影响

目的:在人皮肤成纤维细胞培养上清液中寻找有效的生长因子样活性组分.方法:收集超滤成纤维细胞的培养上清液,采用MTT法、划痕试验检测对人皮肤表皮细胞增殖和诱导迁移的影响.结果:上清液中分子量5～10 KD组和5～30 KD组的试验组对表皮细胞有明显促增殖趋势,而5～30 KD组有较强的促进单层细胞伤口愈合的能力,二者与对照组比较有显著性差异(P＜0.01).结论:人成纤维细胞培养上清液的提取物中含有促进表皮细胞增殖及迁移能力的活性物质,可以尝试用其替代部分条件培养基进行对组织工程皮肤种子细胞的实验研究.     

变异链球菌对人唾液腺细胞系免疫球蛋白受体影响的初步研究

目的:观察变异链球菌(S.mutans)对人唾液腺细胞系(HSG)中多聚免疫球蛋白受体(Pigr)表达的影响.方法:用免疫荧光染色验证细胞系来源与Pigr在HSG中的表达.将对数生长中期的S.mutans水热灭活后与HSG细胞共培养,CCK-8法观察细胞增殖,qPCR与Western blot检测S.mutans对Pi...     

配色颜料及唾液对硅橡胶表面白色念珠菌粘附性的影响

目的:探讨赝复配色颜料及口腔唾液对MDX44210硅橡胶表面白色念珠菌(Candidaalbicans)粘附性的影响。方法:对MDX44210硅橡胶试样表面的C.albicans粘附数量进行计数,采用χ2检验和方差分析。结果:C.albicans的粘附数量在着色和未着色MDX44210硅橡胶材料组间无显著差异(P>0.05)。有唾液获得膜存在时,MDX44210硅橡胶表面的C.albicans粘附数量低于无唾液获得膜存在时(P<0.01)。结论:配色颜料对MDX44210硅橡胶的C.albicans表面粘附性能无影响,而口腔唾液有降低C.albicans粘附的作用。     

脂质体鱼肝油酸钠促ECV-304系细胞凋亡作用的实验研究

目的探讨鱼肝油酸钠的脂质体包裹剂型对于人脐静脉内皮细胞（humanumbilicalveinendothelialcell，ECV）-304细胞系的作用。方法采用甲基噻唑四唑法（methylthiazolyltetrazolium，MTT）、透射电镜、DNA凝胶电泳和流式细胞仪染色等方法观察脂质体鱼肝油酸钠对ECV-304细胞的作用。结果细胞毒理学显示，脂质体鱼肝油酸钠组活细胞数目下降速度较慢。电镜显示脂质体鱼肝油酸钠组细胞凋亡，凝胶电泳出现典型DNA梯度，PI染色后流式细胞仪检测出现典型亚二倍体峰，凋亡率为22．23％。结论与普通鱼肝油酸钠制剂不同，脂质体鱼肝油酸钠可以促进ECV-304细胞系凋亡。     

平阳霉素白蛋白微球抑制血管内皮细胞生长的实验研究

目的:观察平阳霉素白蛋白微球(PYM AMS)对体外培养的人脐静脉血管内皮细胞株(ECV304)生长的抑制作用。方法:采用MTT法、流式细胞仪检测不同浓度的PYM AMS、PYM注射剂和空白AMS作用24、36、48、72h后,血管内皮细胞的生长抑制率、细胞凋亡率。结果:空白AMS对ECV304生长没有抑制作用,反而有轻度促进生长的作用;PYM AMS和PYM注射剂对ECV304生长有抑制作用。统计分析表明,相同剂量的PYM AMS与PYM注射剂抑制作用基本一致,而抑制作用具有剂量依赖性和时间依赖性。而且PYM AMS和PYM诱导血管内皮细胞凋亡的作用与细胞生长抑制的作用有良好的相关性,有浓度依赖性和时间依赖性,同时PYM AMS有缓释作用。结论: 本研究的PYM AMS能够对血管内皮细胞造成损伤,其损伤程度与浓度、时间成正相关,与单用PYM基本一致,并且有缓释作用效果;而空白AMS不具有损伤血管内皮细胞的作用。     

成纤维细胞上清提取物对成纤维细胞增殖迁移的影响

目的：在人皮肤成纤维细胞培养上清液中寻求出有效的生长因子样活性组分。方法：收集超滤成纤维细胞的培养上清液，采用MTT法、划痕试验等检测其对人皮肤成纤维细胞增殖和诱导迁移的影响。结果：上清液组中分子量10～30kD的试验组对成纤维细胞有明显促增殖趋势，而大于30kD组有较强的促进单层细胞伤口愈合的能力，二者与对照组比较均差异显著（P〈0．01）。结论：人成纤维细胞培养上清液的提取物中含有促进成纤维细胞增殖和迁移能力的活性物质。     

Cytotoxicity of MTA and Portland cement on human ECV 304 endothelial cells

AIM: To evaluate the cytotoxic effects of two brands of mineral trioxide aggregate (MTA) (Pro-Root MTA and MTA Angelus) and Portland cement (PC) on the human ECV 304 endothelial cell line. METHODOLOGY: Endothelial ECV 304 cells were incubated at 37 degrees C in an atmosphere of 95% air, 5% carbon dioxide and 100% humidity for 7 days and grown in F12 medium supplemented with 10% fetal bovine serum with 50 microg mL(-1) of gentamicin sulphate. Effects of the materials on mitochondrial functions were measured by a colorimetric assay. At each experimental time interval (24, 48 and 72 h), a dimethyl-thiazol-diphenyl tetrazolium bromid assay was conducted to measure cell viability. All assays were repeated three times to ensure reproducibility. Results were expressed as average absorbance (A(570/nm)) +/- SD and the data were analysed statistically by one-way analysis of variance and the Bonferroni post-test. A P-value < 0.05 was considered statistically significant. RESULTS: No statistically significant difference was shown between any of the experimental materials (P > 0.05). CONCLUSIONS: The two brands of MTA analysed, as well as the PC, initially showed a similar elevated cytotoxic effect that decreased gradually with time allowing the cell culture to become reestablished.     

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.     

Growth interaction between Candida albicans and Streptococcus salivarius: in vitro and in vivo studies.

Suppression of Candida albicans in the mouth by oral flora has been proposed as one of the mechanisms preventing candidal overgrowth. According to Liljemark and Gibbons (1973), Streptococcus salivarius plays a significant role in this process. The aim of this investigation was to study the growth interaction between C. albicans and S. salivarius in vitro and in vivo. An aerobic continuous-flow system was used for the in vitro study. Pure and mixed cultures of C. albicans (NCPF 3118) and S. salivarius (NCTC 8618) were inoculated into a buffered medium containing either 0.1 per cent or 0.001 per cent glucose concentrations and incubated at 37 degrees C for 55 hours. Two in vivo investigations were undertaken using inbred germfree C3H mice. In the first, mice were exposed to a mixed suspension of S. salivarius and C. albicans for 48 hours. In the second the mice were exposed to S. salivarius for 48 hours. Fourteen days later they were contaminated with C. albicans. A comparison of growth curves showed no growth inhibition between the species. The in vivo studies showed that oral lesions from candidal infestation occurred in all mice. We were therefore unable to demonstrate in vitro or in vivo suppression of C. albicans in the presence of S. salivarius.     

Antimicrobial effects of glass ionomer cements containing bioactive glass (S53P4) on oral micro-organisms in vitro

The antimicrobial effects of two glass-ionomer cements (GICs), GC Fuji II and Fuji II LC, mixed with different amounts (0 wt%, 10 wt% and 30 wt% of the total powder weight) of bioactive glass (BAG), S53P4, on Streptococcus mutans and Candida albicans were studied in vitro. The growth inhibition was tested using agar diffusion. The materials were also studied in a liquid media. The effect of the material extracts on acid production was studied using cell suspensions. The antimicrobial activity of the materials was examined by incubating the cell suspensions with the material powder. In the agar diffusion test, only the GICs containing 30 wt% BAG inhibited the growth of S. mutans. When the materials were tested in culture medium, no inhibitory effects on S. mutans were detected. The only materials to inhibit acid production of S. mutans were the GIC extracts without added BAG. Furthermore, they also had antibacterial activity against S. mutans when tested as powders. We found very few effects of the tested materials on C. albicans. The only material with an antimicrobial effect on C. albicans was BAG when incubated in a suspension with C. albicans. This is the first time that this effect has been demonstrated for C. albicans. By adding BAG to GICs the structure of the material becomes more brittle than the structure of GICs without BAG. Thus, in addition to the composition of the tested materials, their structure may also have influenced the results. In summary, commercially available GICs and GIC disks containing 30 wt% of BAG exerted antibacterial effects on S. mutans. BAG exerted antimicrobial effects on both S. mutans and C. albicans.     

纳米载银树脂基托的体外抗菌效果

目的研究纳米载银树脂基托在体外对变形链球菌和白色念珠菌的抗菌效果。方法先测定纳米载银抗菌剂对变形链球菌和白色念珠菌的最小抑菌浓度,并以此为依据在聚甲基丙烯酸甲酯基托树脂粉中添加不同比例的抗菌剂,制成抗菌树脂基托,用贴膜法测定该树脂基托对变形链球菌和白色念珠菌的抗菌率。结果纳米载银抗菌剂对变形链球菌的最小抑菌浓度为0.1mg/ml,对白色念珠菌的最小抑菌浓度为1mg/ml。当添加抗菌剂的浓度分别为1、2、5、10mg/ml时,树脂基托对变形链球菌的抗菌率分别为67.4%、71.3%、99.0%、99.5%;对白色念珠菌的抗菌率分别为25.8%、54.8%、90.3%、93.0%。结论载银抗菌树脂基托在体外表现了一定的抗变形链球菌和抗白色念珠菌的效果,当树脂基托中抗菌剂的浓度达到5mg/ml时,抗菌效果满意。     

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。     

口腔白色假丝酵母菌毒力因子与随机扩增多态性DNA条带的多元回归分析

目的探讨白色假丝酵母菌毒力因子与随机扩增多态性DNA（RAPD）电泳条带之间的关系,构建多元回归模型。方法采用体外法对92株白色假丝酵母菌的细胞外磷脂酶活性、分泌性蛋白酶活性、芽管生成率、对口腔黏膜细胞的黏附力进行检测;并通过RAPD方法进行扩增,电泳后分析扩增条带。对上述毒力因子和电泳条带进行多元回归分析。结果白色假丝酵母菌的细胞外磷脂酶活性与RAPD扩增后350、450、650和1 300 bp 4个条带明显相关（P<0.05）;分泌性蛋白酶活性与350、1 200 bp 2个条带明显相关（P<0.05）;芽管生成率与400、550 bp 2个条带明显相关（P<0.05）。结论白色假丝酵母菌RAPD部分电泳条带与部分毒力因子的强弱有关,其所含基因信息可能参与该菌毒力因子的调节。