病毒性心肌炎与支原体肺炎患儿心肌损伤标志物水平的检验意义

目的:探讨病毒性心肌炎与支原体肺炎患者心肌损伤标志物水平检测意义。方法:回顾性分析医院收治的病毒性心肌炎患儿53例和肺炎支原体肺炎患儿49例分别作为病毒性心肌炎组和支原体肺炎组,选取同期体检正常儿童50例作为对照组,分别检测心肌酶指标和心肌蛋白指标。结果:病毒性心肌炎组心肌肌钙蛋白I(c Tnl)、肌红蛋白(MYO)显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组和对照组组间差异显著,具有统计学意义(P0.05)。病毒性心肌炎组肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、门冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)均显著高于支原体肺炎组、对照组,差异显著(P0.05);支原体肺炎组、对照组组间对比差异显著,具有统计学意义(P0.05)。2组入院10 d c Tnl、MYO均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d c Tnl、MYO显著高于支原体肺炎组,具有统计学意义(P0.05)。2组入院10 d CK、CK-MB、AST、LDH均低于入院第1 d,具有统计学意义(P0.05);病毒性心肌炎组入院第10 d CK、CK-MB、AST显著高于支原体肺炎组,差异具有统计学意义(P0.05)。根据ROC曲线分析临床性能,c Tnl、MYO、CK、CK-MB、AST、LDH的临界值分别为0.38μg/L、56.2μg/L、236.58 U/L、32.8 U/L、71.6 U/L、232.8 U/L,灵敏度分别为82.7%、85.4%、84.8%、89.6%、90.2、79.8%。结论:心肌损伤标志物可作为诊断病毒性心肌炎和支原体肺炎的重要指标,应用ROC回归曲线确定各指标的临界值,还可对两种疾病进行鉴别诊断。     

PCR技术在附红细胞体感染模型中的应用

目的以猪附红细胞体(E.suis)为例,建立套式PCR方法,定性测定E.suis感染大鼠血液中DNA,检验套式PCR方法在其动物模型应用中的实用性和可操作性。方法用阳性血液样品分别经腹腔、皮下及肌肉感染大鼠,定期采血,进行常规血涂片染色,并从血液中分离纯化E.suis,提取DNA,进行套式PCR法扩增,凝胶电泳定性。结果套式PCR经扩增后得到了一长度为498bp的特异条带,测序鉴定确为目的片段;在整个实验过程中未见感染大鼠明显的临床症状。结论可以用大鼠建立猪附红细胞体动物感染动物模型;附红细胞体的感染方式有以下几种:腹腔、皮下及肌肉感染。在隐性感染的整个阶段,PCR方法依靠其高敏感性,作为猪附红细胞体感染大鼠模型检测指标之一,有其必要性和重要性。     

Polydatin suppresses the development of lung inflammation and fibrosis by inhibiting activation of the NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 inflammasome and the nuclear factor-κB pathway after Mycoplasma pneumoniae infection

Mycoplasma pneumoniae (MP) can infect both the upper and lower respiratory tracts. Polydatin (PD), a traditional Chinese medicine, is known to have anti-inflammation and antifibrosis properties. However, the protective effects of PD against MP pneumonia (MPP) remain unclear. So, the aim of this study was to describe the therapeutic effects and underlying mechanisms of PD against MPP. BALB/c mice were assigned to three groups: a normal control group, MP infection group, or PD-treated MP infection group. BEAS-2B cells transfected with or without NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 (NLRP3) were used to confirm the protective mechanisms of PD. Immunohistochemical analysis, Western blot analysis, enzyme-linked immunosorbent assay, and flow cytometry were used in this study. The results showed that PD treatment suppressed MP-induced lung injury in mice by suppressing the expression of inflammatory factors and inhibiting the development of pulmonary fibrosis. Meanwhile, PD treatment inhibited activation of the NLRP3 inflammasome and nuclear factor κB (NF-κB) pathway. Overexpression of NLRP3 reversed the protective effect of PD against MP-induced injury of BEAS-2B cells. Taken together, these results indicate that PD treatment suppressed the inflammatory response and the development of pulmonary fibrosis by inhibiting the NLRP3 inflammasome and NF-κB pathway after MP infection.     

山羊传染性胸膜肺炎病原体4株国内分离株的重新分类

山羊传染性胸膜肺炎(Contagious Caprine Pleuropneumonia,CCPP)是由山羊支原体山羊肺炎亚种(M.capricolum subsp.capripneumoniae,Mccp)引起的高度接触性传染病,对4株CCPP中国分离株进行分子特征研究,确定其分类地位。针对3段基因(A、B、C),对扩增产物进行酶切鉴定和测序,将结果与丝状支原体簇的6个成员进行遗传衍化分析。在A片段,4株中国分离株的扩增产物经PstI酶切后的结果与Mccp代表株F38相同,为548、420、128等3条带,其他5个丝状支原体簇成员只有420、128bp两条带。在B片段,序列分析结果显示4株中国分离株与F38同源性为99.5%,与山羊支原体山羊亚种Mcc代表株kid的同源性为98.9%,与丝状支原体山羊亚种MmcZZ株同源性仅为95.4%。在C片段研究发现,4株中国分离株的序列与Mmc模式株PG3株同源性为67.4%~67.6%,与2株Mcc8601-50和California Kid同源性为95.1%~98.4%,与3株Mccp97097ET、Gabes和F38的同源性为99.6%~99.8%。通过对中国分离的87001、87002、367、1653等4株CCPP病原体的分子特征研究,首次提出其与山羊支原体山羊肺炎亚种(Mccp)亲缘关系最近,应归属为Mccp,并将国内流行的山羊传染性胸膜肺炎的病原定名为Mccp。     

Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity

Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.     

Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases

The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.     

阿奇霉素序贯治疗联合麻杏石甘汤加味对痰热闭肺型肺炎支原体肺炎患儿Th17/Treg细胞因子和NLRP3炎症小体通路的影响

摘要 目的：观察阿奇霉素序贯治疗联合麻杏石甘汤加味对痰热闭肺型肺炎支原体肺炎（MPP）患儿辅助性T细胞17（Th17）/调节性T细胞（Treg）细胞因子和NOD样受体蛋白3（NLRP3）炎症小体通路的影响。方法：选择山东省中医院2019年4月~2021年5月期间收治的痰热闭肺型MPP患儿106例,根据随机数字表法分为对照组和研究组,各为53例,对照组患儿接受阿奇霉素序贯治疗,研究组患儿接受阿奇霉素序贯治疗联合麻杏石甘汤加味治疗,对比两组疗效、中医证候积分、Th17/Treg细胞因子和NLRP3炎症小体通路相关指标,记录两组不良反应发生情况。结果：与对照组相比,研究组的临床总有效率明显更高（P<0.05）。两组治疗后次证积分、主证积分、总积分均较治疗前下降,且研究组低于对照组（P<0.05）。两组治疗后白介素-17A（IL-17A）、白介素-25（IL-25）水平均较治疗前下降,白介素-10（IL-10）、白介素-35（IL-35）水平均较治疗前升高,且研究组的变化程度大于对照组（P<0.05）。两组治疗后NLRP3 mRNA、接头蛋白凋亡相关斑点样蛋白（ASC）mRNA、半胱天冬酶1（caspase-1） mRNA均较治疗前下降,且研究组的下降程度大于对照组（P<0.05）。两组在治疗期间均未曾出现明显不良反应。结论：阿奇霉素序贯治疗联合麻杏石甘汤加味治疗痰热闭肺型MPP患儿疗效显著,可促进症状改善,作用机制可能与调节Th17/Treg细胞因子和NLRP3炎症小体通路有关。     

小儿消积止咳口服液辅助治疗对肺炎支原体肺炎患儿呼吸肺功能和Th1/Th2免疫应答的影响

摘要 目的：观察小儿消积止咳口服液辅助治疗儿童肺炎支原体肺炎（MPP）的疗效及对呼吸肺功能和Th1/Th2免疫应答的影响。方法：选择我院2019年9月到2021年9月期间收治的MPP患儿80例。根据随机数字表法分为对照组（n=40）和研究组（n=40）,对照组患儿接受大环内酯类药物治疗,研究组患儿在对照组的基础上辅助小儿消积止咳口服液治疗,对比两组疗效、呼吸肺功能、Th1/Th2免疫应答和不良反应。结果：与对照组的临床总有效率相比,研究组的明显升高（P<0.05）。两组治疗7 d后用力肺活量（FVC）、第1秒用力呼气容积（FEV1）、最大呼气流量（PEF）均较治疗前升高,且研究组的变化程度大于对照组（P<0.05）。两组治疗7 d后Th1细胞因子干扰素γ ( IFN-γ) 的水平、Th2细胞因子白介素-6（IL-6）均较治疗前下降,IFN-γ/IL-6较治疗前升高,且研究组的变化程度大于对照组（P<0.05）。两组均有不良反应发生,但组间对比未见明显统计学差异（P>0.05）。研究组的肺部啰音消失时间、咳嗽消失时间、X线阴影消失时间、退热时间短于对照组（P<0.05）。结论：小儿消积止咳口服液辅助治疗儿童MPP,可提高治疗效果,改善呼吸肺功能和Th1/Th2免疫应答,且未见明显不良反应增加,效果显著。