不同连翘制剂对甲型流感病毒NP基因转染后表达的影响

目的：观察连翘苷、连翘醇提液、连翘水煎剂等3种连翘制剂对甲型流感病毒甲1型流感病毒核蛋白（ nucleoprotein,NP）基因转染后表达的影响。方法：将甲型流感病毒NP基因转染Hela细胞,用甲型流感病毒胶体金检测3种连翘制剂对转染后细胞内和上清核蛋白表达情况,用RT_PCR检测转染后Hela细胞内NP基因的拷贝数。结果：NP重组质粒组上清胶体金检测为阳性,连翘苷组上清为阴性、细胞内为弱阳性;连翘水煎剂及连翘醇提物组上清均为弱阳性。连翘苷组NP基因表达量较NP重组质粒组减少（ P<0.05）。结论：连翘苷抑制甲型流感病毒NP基因转染后表达作用最强。     

A pathogenic trace of Tannerella forsythia – shedding of soluble fully active tumor necrosis factor α from the macrophage surface by karilysin

Tannerella forsythia is implicated as a pathogen causing chronic and aggressive periodontitis. However, its virulence factors, including numerous putative proteases, are mostly uncharacterized. Karilysin is a newly described matrix metalloprotease‐like enzyme of T. forsythia. Since pathogen‐derived proteases may affect the host defense system via modulation of the cytokine network, the aim of this study was to determine the influence of karilysin on tumor necrosis factor‐α (TNF‐α). The results showed that karilysin cleaved the membrane form of TNF‐α on the surface of macrophages, and that this led to an increased concentration of soluble TNF‐α in the conditioned medium. Importantly, despite partial degradation of soluble TNF‐α by karilysin, the released cytokine retained its biological activity, inducing apoptosis and stimulating autocrine pathway of pro‐inflammatory gene expression. Notably, the observed effect required proteolytic activity by karilysin, since a catalytically inactive mutant of the enzyme did not affect TNF‐α secretion. The shedding was independent of the activity of ADAM17, a major endogenous TNF‐α converting enzyme. Karilysin‐dependent TNF‐α release from the cell surface is likely to occur in vivo because human plasma, the main constituent of gingival crevicular fluid, only slightly affected the sheddase activity of karilysin. Taken together, these results indicate that karilysin modulates the host immune response through regulation of TNF‐α secretion, and should therefore be considered as a new virulence factor of T. forsythia.     

慢性牙周炎患者龈下菌斑中福赛斯坦纳菌与临床指标的关系

目的分析慢性牙周炎患者龈下菌斑中福赛斯坦纳菌（T.forsythensis）的分布情况,及其与牙周临床指标的关系,探讨T.forsythensis与慢性牙周炎发生发展的关系。方法采集108例慢性牙周炎患者的216个病变位点和108个健康位点的龈下菌斑,共324个样本,采用16S rRNA聚合酶链反应检测T.forsythensis的分布情况。对324个取样位点进行临床检查,记录探诊深度、临床附着丧失和探诊出血情况,分析T.forsythensis与临床指标的相互关系。结果慢性牙周炎患者病变位点与健康位点T.forsythensis的检出率分别为59.72%和3.70%,二者间有统计学差异（P<0.001）。随着探诊深度和临床附着丧失的增加,T.forsythensis的检出率也升高,Spearman相关系数分别为0.48和0.51。探诊出血阳性位点T.forsythensis的检出率为61.31%,明显高于探诊出血阴性位点的检出率8.80%（P<0.001）。结论T.forsythensis与慢性牙周炎发生、发展关系密切。     

慢性牙周炎龈下菌斑中福赛斯坦纳菌的检测

目的检测慢性牙周炎患者和牙周健康者龈下菌斑中福赛斯坦纳菌的数量和所占比例,探讨福赛斯坦纳菌与牙周炎发生发展的关系。方法采集经常规聚合酶链反应（PCR）法检测福赛斯坦纳菌为阳性的61例慢性牙周炎患者和12例牙周健康者的龈下菌斑,应用TaqMan实时荧光定量PCR法对菌斑的细菌总量和福赛斯坦纳菌的数量进行定量检测,构建含有福赛斯坦纳菌和真细菌靶基因的重组质粒,建立定量标准。结果本研究设计的引物和探针具有良好的特异性和敏感性;慢性牙周炎患者病变位点的福赛斯坦纳菌数量和细菌总量均高于牙周健康者的健康位点,且福赛斯坦纳菌在龈下菌斑中的比例也比健康位点高（P<0.05）;龈下菌斑的细菌数量与探诊深度呈正相关（P<0.001）;龈下菌斑中福赛斯坦纳菌所占比例在不同的探诊深度间无统计学差异（P>0.05）。结论龈下菌斑中福赛斯坦纳菌的数量与牙周状况有密切关系,实时荧光定量PCR法是研究牙周病病因及治疗方法的有效手段。     

连翘对重症急性胰腺炎大鼠肝组织中NF-κB和Foxp3表达的影响

目的探讨核因子NF-κB和Foxp3在重症急性胰腺炎（SAP）肝损伤中的作用及连翘对其表达活性的影响。方法雄性Wistar大鼠80只,随机分成假手术组（SO组）、SAP组和干预组,其中干预组分连翘高、中、低剂量组和阳性对照组（PDTC）。牛磺胆酸钠溶液在胰胆管远端注射造模,SO和SAP组于术后3、6、12 h,干预组于术后12 h处死大鼠,分别留取标本。测各组血淀粉酶（AMY）、ALT及TNFα水平,鲎试剂法测血浆内毒素水平,流式细胞术测外周血Treg百分数,对胰腺及肝脏进行病理学检查及评分,RT-PCR法检测肝脏组织中NF-κBmRNA和Foxp3mRNA表达量。组间比较采用单因素方差分析,进一步进行多重比较,采用LSD法进行统计学处理,各指标间相关性分析采用直线相关分析。结果与SO组比较,SAP组中各项指标均随时间升高,于12 h达高峰。与SAP12 h组相比,干预组（大鼠死亡率为0）肝脏组织中的NF-κBmRNA和Foxp3mRNA表达明显降低（P〈0.01）,与Treg呈正相关（r=0.738,P〈0.01）。随连翘剂量增加,AMY、ALT及TNFα水平均明显降低,肝脏和胰腺组织炎症明显减轻,高剂量组和阳性对照组相比较无明显差异（P〉0.05）。结论 NF-κB的激活参与SAP肝损伤的发生,连翘能显著降低NF-κB的活性及肝脏组织中NF-κBmRNA和Foxp3mRNA的表达,减轻SAP肝损伤的严重程度。     

连翘中连翘苷含量测定方法的试验研究

目的改进高效液相色谱法测定连翘中连翘苷的含量测定方法。方法采用甲醇索氏提取,中性氧化铝柱层析分离纯化、高效液相色谱法测定连翘中连翘苷含量。结果改进了高效液相色谱法测定连翘中连翘苷的含量测定方法。结论该连翘苷含量测定方法准确、重现性好、回收率为97.09%、含量限度制定合理、可行,可较好地控制连翘质量。     

金翘感冒口服液的质量标准研究

目的研究金翘感冒口服液的质量标准。方法对金翘感冒口服液中主要药物金银花、连翘、甘草进行薄层色谱鉴别;采用RP-HPLC法测定绿原酸的含量。结果薄层色谱检出绿原酸、连翘药材、甘草次酸特征斑点,绿原酸进样量在0.224~1.120μg线性关系良好,平均加样回收率99.81%,RSD为0.89%。结论方法简便易行,灵敏,结果准确,重现性好,可有效控制金翘感冒口服液的质量。