A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

ObjectiveAntimicrobial resistance (AMR) has become a global concern and is especially severe in China. To effectively and reliably provide AMR data, we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology, and screened multiple AMR genes in broiler fecal samples.MethodsA high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection. A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.ResultsThe new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction. The sensitivity rate, specificity rate, positive predictive value, negative predictive value and correct indices were 99.30%, 98.08%, 95.31%, 99.79%, and 0.9755, respectively. Utilizing this assay, we demonstrate that AMR genes are widely spread, with positive detection rates ranging from 0 to 97.07% in 273 broiler fecal samples. bla_CTX-M, bla_TEM, mcr-1, fexA, cfr, optr A, and intl1 showed over 80% prevalence. The dissemination of AMR genes was distinct between the two farms.ConclusionsWe successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples. The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.     

稳定表达人α-分泌酶adam10基因启动子荧光素酶报告基因细胞系的构建研究

目的 构建携带adam10基因启动子的荧光素酶报告载体,筛选稳定表达细胞系并分析其活性.方法 提取人神经母细胞瘤细胞(SH-SY5Y细胞)基因组DNA,以其为模板,PCR扩增adam10基因启动子并克隆至荧光素酶报告载体pGL4.17中,构建adam10基因启动子荧光素酶报告载体pGL4.17-adam10,将其转染SH-SY5Y细胞(无启动子的pGL4.17载体作阴性对照,带有CMV启动子的pGL4.51载体作阳性对照),经G418进行稳定表达株的筛选,用1μmol/L维甲酸处理细胞4d后检测其荧光活性.结果 成功扩增到438 bp的adam10基因启动子,pGL4.17-adam10经PCR和双酶切鉴定均正确.SH-SY5Y细胞被该载体转染后经G418筛选得到稳定表达adam10基因启动子的细胞株,经检测具有较强的转录活性;1μmol/L雏甲酸能诱导adam10基因启动子高效表达.结论 成功构建了人adam10基因启动子荧光素酶报告载体,adam10基因启动子在SH-SY5Y细胞中能稳定表达,为深入研究adam10基因的表达调控、多态性分析及其高通量药物筛选提供基础.     

A preliminary study of cytokine gene polymorphism effects on Saudi patients with colorectal cancer

Objectives:To determine the possible associations of polymorphisms in interleukin (IL)-8 (rs4073 T/A), IL-10 (rs1800896 A/G), IL-22 (rs1179251 C/G and rs2227485 C/T), IL-27 (rs17855750 T/G), and transforming growth factor beta 1 (TGFß1) (rs1800469 C/T) with colorectal cancer (CRC) susceptibility in Saudi patients.Methods:The case-control study was carried out between July 2019 and January 2020 in King Khaled University Hospital, Riyadh, Saudi Arabia. A total of 70 patients with CRC and 70 healthy controls were included in the study. Single nucleotide polymorphisms of promoter regions were determined using TaqMan genotyping assays.Results:A statistically significant reduction in CRC risk was identified for carriers of the IL-10 (rs1800896 A/G) AG genotype, IL-22 (rs1179251 C/G) G allele, IL-27 (rs17855750 T/G) G allele and TGFß1 (rs1800469 C/T) CT and TT genotype. While IL-10 (rs1800896 A/G) AA genotype and TGFß1 (rs1800469 C/T) CC genotype were significantly associated with increased susceptibility to CRC. No significant associations were identified between the cytokine polymorphisms of IL-8 (rs4073 T/A) and IL-22 (rs2227485 C/T), and CRC risk.Conclusion:Our findings indicate a significant impact of IL-10 (rs1800896 A/G), IL-22 (rs1179251 C/G), IL-27 (rs17855750 T/G) and TGF-ß1 (rs1800469 C/T) polymorphisms on risk of CRC; while the IL-8 (rs4073 T/A) and IL-22 (rs2227485 C/T) and polymorphisms were not associated with CRC risk.     

不明原因不孕妇女着床期子宫内膜αvβ3、Hox10、MMPs基因的表达

目的明确不明原因不孕妇女子宫内膜中αvβ3、Hox10、MMPs表达和正常生育妇女的差异。方法选取2016年10月—2018年5月收治90例患者作为研究对象,其中正常生育组(对照组)45例,不明原因不孕组(试验组)45例,观察比较两组患者子宫内膜厚度、分型、容积以及内膜下血流的变化,并观察比较患者两侧子宫动脉搏动指数(PI)、阻力指数(RI)、内膜下血管化指数(VI)、血流指数(FI)、血管化血流指数(VFI)的水平及HCG日E 2、LH、P的比较,同时比较两组患者着床期子宫内膜中整合素(αvβ3)mRNA、同源框基因10(Hox10)mRNA、基质金属蛋白酶-9(MMPs-9)mRNA的表达情况。结果试验组患者的子宫内膜厚度、内膜下血流以及PI和RI,VI、FI、VFI与对照组有明显差异,子宫内膜分型、容积及HCG日E 2、LH、P无明显差异,即对照组患者子宫内膜容受性的指标优于试验组,差异有统计学意义(P<0.05);对照组患者在着床期子宫内膜αvβ3mRNA、Hox10mRNA、MMPs-9mRNA表达上亦明显高于试验组,差异有统计学意义(P<0.05)。结论不明原因不孕妇女或许是因为子宫内膜中αvβ3、Hox10、MMPs-9的mRNA表达含量低,从而影响子宫内膜容受性,继而影响临床妊娠,为临床治疗不明原因不孕提供了科学理论依据。     

显性调控相关早反应基因的大规模扫描和分析

目的：动态观察白细胞介素10（IL－10）作用后，淋巴细胞早反应基因表达谱变化，为下一步评价患者免疫状态和诱导治疗提供理论依据。方法：应用DNA微矩阵技术，分离正常人外周血单个核细胞（PBMC），予以抗CD3McAb及CD3McAb IL-10刺激，抽提24h PBMC mRNA，反转录后与芯片杂交，通过生物信息学方法比较分析，结果：培养24h后，抗CD3McAb IL-10组与抗CD3McAb组比较有15个基因发生改变，5个基因表达明显下调，10个基因上调，主要功能在于促进基因编码区连接，影响 细胞骨架和运动，不同部位的磷酸化，去磷酸化，细胞识别，会促进细胞生长作用，细胞凋亡，信号传递的基因则重点涉及钙离子－钙调蛋白，G－蛋白信号通路。结论：在IL－10作用，细胞凋亡，信号传递的基因则重点涉及钙离子－钙调蛋白，G－蛋白信号通路。结论：在IL－10作用下，一系列基因转录活化，其中许多先前与免疫细胞无明确功能的相关基因参与了T细胞活化与调控，是否该群基因即代表IL－10作用下淋巴细胞的基因表达标签，尚须进一步证实；要完全明确T细胞调控网络，还应全面分析T淋巴细胞基因表达谱，从中筛选差异有显著意义的基因，进行功能研究。     

血管活性肠肽对脓毒性休克大鼠的保护作用

目的探讨血管活性肠肽（VIP）对脓毒性休克大鼠的保护作用及其可能机制。方法采用盲肠结扎加穿孔（CLP）法制备脓毒性休克大鼠模型。将30只雄性SD大鼠随机分成3组：假手术组（SO,n=10）、CLP组（n=10）和VIP组（n=10）。VIP组大鼠在行CLP术后即刻给予5nmol VIP,持续监测各组大鼠的平均动脉压（MAP）,统计20h存活率;应用酶联免疫吸附试验（ELISA）检测血清肿瘤坏死因子-α（TNF-α）、白介素-10（IL-10）水平;取大鼠肺、肾和肠等组织行病理检查。结果在8h以后的各时间点,VIP组的MAP显著高于CLP组（P〈0．05）;与CLP组比较,VIP组的TNF-α水平明显降低、IL-10水平明显升高。20h存活率显著提高（P〈0．05）。各脏器病理损伤明显改善。结论VIP通过抑制促炎因子的生成并促进抗炎因子的产生在大鼠脓毒性休克中发挥保护作用。     

不明原因复发性流产患者子宫内膜/早孕蜕膜组织HOXA-1O基因的表达

目的探讨不明原因复发性流产（URSA）与子宫内膜／早孕蜕膜组织HOXA-10基因表达的关系。方法采用原位杂交技术，以HOXA-10反义核苷酸为探针检测51例URSA患者及38例正常生育（NF）组的分泌期子宫内膜／早孕蜕膜组织HOXA-10mRNA的表达水平，并以灰度阳性单位（PU值）表示。结果NF组子宫内膜分泌早期HOXA-10mRNA表达的PU值腺体为（6．66±0．11），间质为（6．76±0．15）；分泌中期分别为（10．95±0．90）及（11．46±1．08）；分泌晚期分别为（11．05±1．12）及（11．54±1．10）；蜕膜组织分别为（11．55±1．14）及（11．93±1．92）；分泌中晚期及蜕膜组织的Pu值显著高于分泌早期。URSA组不同时期子宫内膜及早孕蜕膜组织中HOXA-10mRNA的表达水平基本一致，无明显的分泌中晚期及早孕期峰，并且分泌中晚期及早孕蜕膜组织的PU值明显低于NF同期组。两组各期腺体和间质的PU值无显著差异。结论子宫内膜HOXA-10基因在分泌中晚期及早孕蜕膜组织中的高表达可能和孕卵着床及妊娠维持密切相关，其表达缺陷可能是导致URSA发生的重要原因之一。     

消炎痛对阻塞性黄疸患者外周血淋巴细胞白细胞介素-10、白细胞介素-2水平的影响

目的研究阻塞性黄疸患者外周血淋巴细胞白细胞介素-10(IL-10)、白细胞介素-2(IL-2)水平和血浆中前列腺素E2(PGE2)水平的变化,同时观察消炎痛对上述指标的影响。方法给阻塞性黄疸患者口服消炎痛(25mg,1次/8小时),用药前和用药后7d取外周血,分别用放射免疫和ELISA法测血浆中PGE2水平及外周血淋巴细胞IL-10、IL-2水平。结果用药前阻塞性黄疸患者血浆中PGE2水平及外周血淋巴细胞IL-10水平较正常人明显升高,而IL-2水平明显下降,且IL-10、IL-2的水平与PGE2水平显著相关;用药后,患者血淋巴细胞IL-2水平明显升高,而IL-10水平及血浆中PGE2水平明显下降,与用药前相比差异有统计学意义。结论阻塞性黄疸患者淋巴细胞IL-10、IL-2水平的变化与其血浆中PGE2水平升高有关,消炎痛能够明显缓解这种作用。     

清炎颗粒对肾盂肾炎小鼠外周血IL-8与IL-10的影响

目的：探讨清炎颗粒治疗肾盂肾炎的免疫学机制。方法：采用孙氏方法改进造模,治疗组分别按剂量3.12、6.24、12.48g·kg^-1·d^-1予清炎颗粒,模型组予生理盐水,对照组予至灵胶囊灌胃,喂养至3d、7d、15d、30d、60d取血清,用ELISA法检测其IL-8、IL-10的水平。结果：清炎颗粒能显著降低不同时段血清IL-8的水平、提高不同时段血清IL-10的水平。结论：免疫调控可能是清炎颗粒治疗肾盂肾炎的作用机制之一。     

Testikuläre Ischämie

Background

Ischemia and reperfusion (I/R) lead to cellular damage. A disturbance of testicular perfusion occurs during the therapy of cryptorchidism and in cases of testicular torsion. This results in the activation of mediator cells with an increasing synthesis of mediators of infection like TNF-α and the expression of cell adhesion molecules like ICAM (intercellular adhesion molecule) and VCAM (vascular cell adhesion molecule) at the cellular surface.

Methods

The expression of the cytokines IL-10 and TNF-α and the adhesion molecules ICAM and VCAM after defined testicular I/R injury in nine male transsexuals was evaluated with rt-PCR. Furthermore we examined lactate and the diameter of the testicular tubulus under ischemic conditions.

Results

During ischemia ICAM, IL-10, and VCAM do not show significant changes on the side of testicular ischemia and the contralateral side; the same was seen for the tubulus diameter. TNF-α and the testicular lactate values showed a significant change of the expression pattern.

Discussion

The statistical changes of TNF-α and testicular lactate are the expression of leukocyte migration, infectious reaction, and immune response. To what extent the TNF-α expression represents a severe immunological reaction remains undefined. This human study shows primary results for the immunological understanding of and cellular response to testicular ischemia.