ObjectiveTo investigate the difference in protein expression between hepatocellular carcinoma (HCC) patients with recurrence and those with good prognosis, the differential expression and regulatory mechanism of miR-152-3p target proteins, and the role of miR-152-3p in the recurrence of HCC. MethodsTMT-labeled proteomic sequencing and RT-PCR were used to measure the expression of proteins and the expression of miR-152-3p in the HCC tissue of six patients with recurrence at 2 years after HCC resection and six patients with good prognosis at 5 years. Six databases were used to analyze the target genes of miR-152-3p, and Gene Ontology, DAVID, and REACTOME databases were used to perform target gene screening, enrichment annotation, and signal transduction pathway enrichment analysis. Gene mutation frequency and survival curve analysis were performed for the target genes of miR-152-3p to verify the role of miR-152-3p target genes in patients with HCC recurrence. The independent samples t-test was used for comparison of continuous data between two groups, and a Kaplan-Meier analysis was performed to investigate the survival rates of liver-related genes. ResultsCompared with the patients with HCC recurrence, the patients with good prognosis after HCC resection had a significantly higher transcriptional expression level of miR-152-3p in HCC tissue (P＜0.05). The results of protein sequencing showed that there were 365 differentially expressed proteins in HCC tissue between the patients with good prognosis and the patients with recurrence, and the analysis of HCC recurrence databases showed that 17 proteins were regulated by miR-152-3p. Further analysis of the signaling pathways showed that the function of the 17 target genes regulated by miR-152-3p was enriched in the translation and regulation of mitochondria and ribosome, and multiple enrichment revealed that six target genes were closely associated with mitochondrial respiratory chain complex, i.e., AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1. Gene mutation frequency and survival curve analysis showed that the loss or weakening of the function of mitochondrial respiratory chain-related target proteins seriously affected the prognosis and survival rate of patients. ConclusionThere is a significant difference in the expression of miR-152-3p in HCC tissue between patients with good prognosis and those with recurrence after HCC resection, and miR-152-3p may lead to the recurrence of HCC by regulating the target genes AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1, acting on the mitochondrial respiratory chain, and affecting the oxidative respiratory function of cells.

A rat radiation injury model was constructed, in which myelin basic protein (MBP) expression was detected via western blot. Primary oligodendrocyte precursor cells (OPCs) and neuronal cells were isolated from Sprague-Dawley rats, and expression of neurofilament protein and chondroitin sulphate proteoglycan was detected via immunofluorescence to identify isolated primary cells. Cell counting kit-8 (CCK8) experiments were used to assess the effects of different concentrations of clemastine on OPC proliferation. Five groups of lentiviral shRNA vector targeting muscarinic receptor were constructed, and their transfection efficiencies were quantified using RT-qPCR and western blot. To explore the effect of clemastine on OPC differentiation and myelination, MBP and OLIG2 expression were detected via immunofluorescence and western blot. Clemastine was found to promote MBP formation in rats and inhibit OPC proliferation in a concentration-dependent manner. Compared with the control group, CHRM1, CHRM2, CHRM3, CHRM4, and CHRM5 shRNA expression significantly decreased mRNA and protein expression levels (p<0.01). Among these, CHRM2 displayed the best interference efficiency.CHRM2 interference (M2R) significantly increased MBP and OLIG2 expression in a manner that was further enhanced by clemastine treatment (p<0.05). Clemastine enhances the antimuscarinic effect, promotes the differentiation of OPCs into OLs, and plays an important role in myelination by up-regulating the expression of MBP.

脑卒中是全球第三常见的死亡原因之一,也是致残的主要原因.由于对缺血性脑卒中发生后的细胞和分子变化以及神经元死亡原因的理解尚不完全,目前治疗缺血性脑卒中的有效方法非常少.大量研究表明,急性缺血性脑卒中发生后快速增加的活性氧会迅速压倒抗氧化防御,进而引发一系列病理生理事件,包括炎症反应、血脑屏障破坏、细胞凋亡和自噬等,最终导致神经退行性变和神经细胞死亡.因此,深入了解缺血性脑卒中氧化应激的病理机制及相关的病理事件,对于抑制氧化应激靶向治疗缺血性脑卒中是至关重要的,故本文就氧化应激在缺血性脑卒中中的作用进行综述.

OBJECTIVE:To examine the infectivity of human adenovirus type 55 (HAdV-55) in human intestinal cells.METHODS:Caco-2 cells were cultured in vitro, and infected with HAdV-3, 7, 14 and 55. The expression of viral proteins in infected cells was detected with immunofluorescence method. The intracellular and supernatant viral DNA levels were determined with fluorescent quantitative PCR at different points of time. The level of infectious virus particles in the supernatant of Caco-2 cells was determined with adenovirus sensitive HEp-2 infection assay.RESULTS:Immunofluorescence assay showed positive result for the expression of HAdV-55 virus protein in Caco-2 cells 48 h post infection. HAdV-3, 7, 14, and 55 showed sustained replication and proliferation in Caco-2 cells. The level of viral DNA in infected cells and the supernatant increased with the infection time, and the viral DNA level of HAdV-55 was significantly higher than those of HAdV-3, 7 and 14. The infectious virus particles of HAdV-55 in Caco-2 supernatant were more than those of HAdV-3, 7 and 14, showing statistically significant difference ( P<0.05). Caco-2 cells were infected with low doses of virus (1×TCID 50), and the cytopathic effect (CPE) of HAdV-55 infection wells was more significant than that of HAdV-3, 7 and 14 infection wells.CONCLUSION:This study found that human intestinal cells were susceptible to HAdV-55, and the infection level was higher than that of other common respiratory infections caused by adenovirus types 3, 7 and 14.

Objective To analyze the genetic and evolutionary properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF 1ab/S/M proteins and select antigen epitope sequences of mRNA vaccines. Methods We analyzed the worldwide SARS-CoV-2 genome sequences in this study and have focused on the protein and nucleic acid sequences of the ORF 1ab/S/M. The neighbor-joining tree was employed to map the global distribution of genetic differences. Based on current research on SARS-CoV-2 and SARS-CoV-2 genetic differences, we predicted candidate mRNA vaccines for SARS-CoV-2. Results The SARS-CoV-2 ORF 1ab nucleic acid sequence similarity is 100.0%, while the homology is 99.3% in the global hot region; the S-protein nucleic acid sequence similarity is 100.0%, while the homology is 97.5%; the M-protein nucleic acid sequence similarity is 100.0%, while the homology is 99.9%. Global distribution of ORF 1ab/S/M proteins indicates that there is a significant genetic difference between the Americas and Eurasia. Potential vaccine antigen epitope mRNA sequences (11 B cell responses and 13 T cell responses) were selected for SARS-CoV-2 ORF 1ab protein; 6 B cell responses and 4 T cell responses antigen epitope mRNA sequences were selected for the Spike protein; 3 B cell responses and 7 T cell responses antigen epitope mRNA sequences were selected for the membrane protein. Conclusion There are significant genetic differences in the global hot spot of SARS-CoV-2 in the Americas and Eurasia. Through our new antigen design strategy to screen linear epitopes, we predicted many sequences in ORF 1ab/S/M coding region that potentially raising an immune response. Our study will benefit the discovery of the mRNA vaccine (tandem antigen epitope sequence), antibody discovery, and potentially understanding related immune mechanisms. DOI: 10.11855/j.issn.0577-7402.2020.11.04

实习是医学生完成理论课程后,进入医院实际工作环境,将所学理论进行实践应用的过程.良好的实习能够锻炼实习生的技术水平,启发医学生理论联系实际的思考,促进医学生后续更快适应工作岗位.实习质量管理是实习生管理的重要内容,然而由于实习生一旦离开学校抵达实习单位,学校对实习生管理的责任自然而然地交给了实习单位,学校对实习生一般只是下发各类通知、填报各类信息以及少量的检查督导.实习单位一般对实习生管理比较规范,但同时也存在注重结果忽视过程、个别管理不到位、人员管控不彻底、实习质量监督不严格等,实习质量监督中存在诸多问题.解春宝等[1]探讨了医学检验专业实习生中存在的问题,指出实习生的实习态度、带教老师的师资力量以及实习生管理方面参次不齐,存在各种各样的问题.

目的 评价灭活人55型腺病毒(human adenovirus-55,HAdV-55)在小鼠体内免疫原性.方法 将HAdV-55(SF04/SC/2016)毒株经0.1％甲醛60℃热灭活12h.将灭活HAdV-55与弗氏完全佐剂等体积混合,充分乳化,皮下注射BALB/c小鼠,200 μL/只;2周后,将灭活HAdV-55与弗氏不完全佐剂等体积混合,充分乳化,加强免疫2次,间隔2周,同时设PBS对照组.每次免疫1周后经小鼠静脉采血,分离血清,ELISA法检测血清抗HAdV-55特异性抗体水平;通过免疫荧光法、ELISA法和体外中和试验检测末次免疫的血清抗体水平.结果 灭活HAdV-55免疫小鼠产生了特异性抗HAdV-55抗体,随着免疫次数的增加,抗体滴度增大.灭活HAdV-55免疫小鼠血清可与HEp-2细胞内HAdV-55蛋白反应,产生荧光;总IgG水平均高于PBS对照组小鼠;可在HEp-2细胞上中和HAdV-55的感染,中和效价为1∶640～1∶1280.结论 灭活HAdV-55可作为免疫原诱导小鼠产生中和抗体,具有良好的免疫原性.

目的 了解新药头孢他啶-阿维巴坦(CZA)在耐碳青霉烯类肺炎克雷伯菌(CRKP)中的药物敏感性,为临床合理用药提供依据.方法 选取2015-2017年从临床各科标本中分离获得的96株CRKP,用改良碳青霉烯灭活试验(mCIM试验)和乙二胺四乙酸(EDTA)协同碳青霉烯灭活试验(eCIM试验)检测碳青霉烯酶,用K-B法检测CZA的药物敏感性.结果 96株CRKP中,mCIM阳性44株(45.83％)、eCIM阳性20株(20.83％)、产丝氨酸酶24株(25.00％)、产金属酶20株(20.83％)、CZA敏感78株(81.25％).其中,疑为假敏感2株.结论 CZA对CRKP存在着较高的敏感性,检测CRKP是否产碳青霉烯酶,以及产酶类型的检测更有利于指导临床对该药物的合理使用.

目的:探讨吉西他滨对HepG2.2.15细胞中乙肝病毒前基因组RNA (pregenomic RNA,pgRNA)转录以及乙肝病毒复制的影响及其可能的作用机制.方法:使用不同浓度吉西他滨处理HepG2.2.15细胞,分别使用CCK-8法、流式细胞术、实时定量聚合酶链式反应(real-time polymerase chain reaction,RT-PCR)、酶联免疫吸附测定(enzyme-linked immuno sorbent assay,EHSA)法检测细胞活性、细胞凋亡、细胞周期、细胞内乙肝病毒pgRNA、细胞培养上清液中乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B e antigen,HBeAg)、乙肝病毒DNA(hepatitis B virus DNA,HBV DNA);实时定量PCR法检测吉西他滨作用下HepG2.2.15细胞中与pgRNA转录有关的调控基因肝细胞核因子4α (hepatocyte nuclear factor 4,HNF4α)、P38蛋白激酶(P38 mitogen-activated protein kinase,P38MAPK)、环磷酸腺苷效应元件结合蛋白1(CAMP responsive element binding protein 1,CREB1)、核转录因子p65 (nuclear factor-kappa B p65,NF-κb p65)、组蛋白去乙酰化酶1(histone deacetylase 1,HDAC1)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(peroxisome proliferator-activated receptor gamma coactivator 1-alpha,PGC-1α)、叉头转录因子O1 (forkhead box protein O1,FOXO1)、相关调控基因沉默信息调节因子2相关酶1(sirtuin 1,SIRT1)、P53 mRNA水平以及pgRNA的表达量,分析这些基因随吉西他滨浓度变化与pgRNA随浓度变化的相关性,找出与pgRNA变化相关性最高的基因,并使用Western blot在蛋白质水平验证吉西他滨对此基因蛋白质表达量的影响.结果:吉西他滨对HepG2.2.15细胞的生长显示出浓度以及剂量依赖性的抑制作用;吉西他滨使细胞凋亡率增高;吉西他滨使细胞更多的细胞停滞在G0G1期,而处于S期细胞比例降低;吉西他滨明显增加HepG2.2.15细胞内乙肝病毒pgRNA,72 h之内呈现明显的时间与剂量依赖性;吉西他滨作用细胞24 h会使细胞培养上清液中的HBeAg增加;吉西他滨促进HNF4α mRNA、HNF4α蛋白质表达量增加,且在所检测的与乙肝病毒转录调节有关的基因中,HNF4α mRNA变化趋势与HBV pgRNA变化趋势相关性最高.结论:吉西他滨可在细胞水平促进乙肝病毒pgRNA转录以及乙肝病毒的复制,其机制为通过促进乙肝病毒转录因子HNF4α的表达实现的.

目的 建立一种简易可行的质控方法代替室内质量控制,对移动式车载血细胞分析仪实施质量监控,确保在长距离机动后血细胞分析仪检测结果在控.方法 选择Sysmex XE 2100血细胞分析仪为基准仪器,选取门诊患者血常规结果正常、异常(高水平)2个浓度的静脉新鲜血标本,在基准仪器上测定定值为靶值;以移动式车载迈瑞BC 5800血细胞分析仪为比对仪器,比较两种设备检测白细胞计数(WBC)、红细胞计数(RBC)、血红蛋白浓度(HGB)、红细胞压积(HCT)、血小板计数(PLT)等项目的相对偏倚.以美国临床实验室改进修正案CLIA'88规定的允许总误差的1/2作为判断标准,对比对结果进行分析,判断标准为:2个浓度的比对结果偏倚均在设定范围之内,即认定比对合格.结果 比对的偏倚平均值为:WBC 3.36％,RBC 1.89％,HGB 1.71％,HCT 1.34％,PLT 3.49％,均在设定范围之内,判定为比对合格.结论 在车载迈瑞BC 5800血细胞分析仪长途机动后,利用新鲜血进行比对操作,能够快速监控仪器质量,确保检测结果的准确性,提供准确的医疗保障服务.

目的 探讨LPS对丙型肝炎病毒(HCV)复制以及I型干扰素(IFN-Ⅰ)信号通路的影响.方法 CCK8法检测LPS对肝癌细胞株Huh7细胞增殖的影响,100 mg/L LPS处理Huh7,并加入0.5感染复数(MOI) HCVcc感染细胞,荧光定量PCR以及免疫荧光法检测细胞内病毒水平;流式细胞仪检测LPS处理Huh7细胞后细胞凋亡与周期的改变;West-ern blot检测LPS对IFN-α、IFN-β蛋白水平表达的影响.结果 各浓度LPS对Huh7细胞的增殖无明显影响;100 mg/LLPS可以抑制HCV的感染及复制水平(P＜0.05);LPS处理能够减轻Huh7细胞凋亡,并使S期细胞明显增多.LPS刺激细胞后,IFN-α、IFN-β表达显著上调,HCV感染可以促进IFN-α、IFN-β的表达,LPS抑制HCV感染引起的IFN-β表达上调,但对IFN-α表达无明显影响.结论 LPS可以在细胞水平抑制HCV的复制及感染,可能与其促进IFN-α、IFN-β的表达有关,而与细胞毒性作用和细胞凋亡无关.

目的 对成都某部人感染甲型流感病毒进行分离鉴定和基因突变分析. 方法 采集甲型流感患者咽拭子标本,通过MDCK细胞分离病毒毒株;采用免疫荧光法鉴定其感染细胞能力,采用基因分型特异性引物鉴定病毒亚型,PCR扩增血凝素基因(HA)和神经氨酸酶基因(NA)后测序,与NCBI数据库在线比对并利用MEGA软件构建系统发育进化树,分析突变位点. 结果 从甲型流感患者咽拭子标本中分离出1株流感病毒,经型特异性引物PCR鉴定为H1N1 (09pdm)亚型,该毒株在37℃时对细胞致病力较强.免疫荧光检测到分离毒株感染细胞内甲型流感病毒核蛋白(NP)高表达,甲型流感病毒NP蛋白在细胞核和细胞质中均有大量分布.利用反转录PCR和测序获得该毒株HA和NA全长基因序列.在线比对及系统发育树分析显示,该毒株HA和NA序列与2017-2018流感季其他国家流行株同源性均＞99％.对HA氨基酸突变位点进行分析,其序列的155位点存在组氨酸-酪氨酸(H-Y)点突变,该点突变也发生Influenza A/Hawaii/24/2018(H1N1) (MH245873)、Influenza A/North Carolina/19/2018(H1N1) (MH245873)和Influenza A/Missouri/51/2017(H1 N1) (MH083792)毒株上.NA氨基酸序列与近期流行的毒株均相同. 结论 本起流感病毒株H1N1(09pdm)的HA、NA基因序列与同期其他国家流行株高度相似,但在HA氨基酸序列的155位点存在一个组氨酸-酪氨酸的点突变,其意义尚不清楚.

腺病毒(ADV)为无包膜双链DNA病毒,基因全长约3.6 kb,目前已知的腺病毒种类超过80个型别(http://hadvwg.gmu.edu),分为A～G共7个血清组,每个血清组下面包含多个型别,其中大多数型别对人类有致病性,人腺病毒(HAdV)感染是急性呼吸道感染的重要病原之一[1].腺病毒感染全年均可出现,高发于夏季和冬季,儿童和免疫力较差的成人易感.2018年9～11月,美国新泽西州的一家儿童护理机构爆发HAdV-7感染疫情,造成36例儿童感染,11例死亡的严重后果,是近年来腺病毒感染人最严重的一起公共卫生事件(https://www.nj.gov/health/cd/topics/adenovirus.shtml).

目的 探讨联合检测血清及脑脊液脂蛋白a[(Lp(a)]含量对脑出血和脑梗死患者的临床意义,为疾病诊断和鉴别诊断提供参考.方法 选取脑出血患者59例(脑出血组)及脑梗死患者102例(脑梗死组),另选取同期做疾病排查诊断无脑出血和脑梗死的患者52例(对照组),在入院72 h内采集脑脊液和血标本,采用乳胶免疫比浊法测定各组血清及脑脊液Lp(a)含量.结果 与对照组相比,脑出血组和脑梗死组血清Lp(a)含量均显著增高(P＜0.05);脑出血组脑脊液Lp(a)含量无显著变化(P＞0.05),而脑梗死组脑脊液Lp(a)含量显著升高(P＜0.05).将脑梗死患者按出院结局分为存活组与死亡组,死亡组血清与脑脊液Lp(a)含量均高于存活组(P＜0.05).结论 联合检测血清和脑脊液Lp(a)含量能辅助鉴别诊断脑梗死与脑出血,并对疾病预后具有一定的提示作用.

Objective To map the immunodominant linear B-cell epitopes of Campylobacter jejuni (C.jejuni) PEB1 antigen and evaluate the protective immune responses elicited by these epitopes in a mouse model of oral infection with C.jejuni. Methods The B-cell immunodominant PEB1 epitopes were identified using synthetic overlapping peptide ELISA. BALB/c mice were immunized with the immunodominant PEB1 peptides conjugated with KLH plus CFA/IFA, and the IgG titers against these peptides were detected using ELISA. Seven days after the last immunization, the mice were orally infected with C.jejuni 11168, and the bacterial burden and expression of tumor necrosis factor-α (TNF-α) mRNA in the jejunum were analyzed using qRT-PCR in 28 d after the challenge. We assessed the effect of the antibodies against the immunodominant PEB1 epitopes in mediating the opsonophagocytic killing of C.jejuni by HL-60 cells. We also assessed the bacterial burden in the jejunum following C.jejuni challenge in a B cell-knockout mice immunized with the peptides. Results The immunodominant peptides PEB155-72aa, PEB197-114aa, and PEB1211-228aa all induced strong IgG responses to PEB1 antiserum, and the antisera of these immunodominant peptides also showed strong IgG responses to recombinant PEB1. Compared with the antiserum of CFA/IFA, the antisera of these immunodominant peptides induced significantly enhanced opsonophagocytic activity of HL-60 cells (P < 0.01). Both the bacterial burdens and TNF-α mRNA expression level in the jejunum were significantly lowered in the mice immunized with the 3 immunodominant peptides in comparison with the control mice immunized with CFA/IFA (P < 0.01). In B cell-knockout mice, immunization with the 3 peptides did not provide immune protection against C.jejuni, and the bacterial burden in the jejunum after C.jejuni challenge was significantly greater than that in immunized wild-type mice (P < 0.01). Conclusion We successfully identified 3 linear B-cell epitopes of C.jejuni PEB1 (PEB155-72aa, PEB197-114aa, and PEB1211-228aa), which exhibit good immunogenicity and immunoprotective activities and may facilitate the future development of vaccines against C.jejuni infection.

目的 探讨采用阿德勒个体心理学理论培养检验实习生人文素养的效果.方法 以2015年1月～2016年1月在本院检验科的实习生(n=38)为对照组,采用传统的检验科实习教学方式,即师徒式带教;以2017年1月～2018年1月在本院检验科的实习生(n=42)为试验组,专业带教实习的方法同对照组,同时,根据阿德勒个体心理学理论,对实习生进行人文素质培养.结果 试验组操作和理论考试成绩、自我评定各项评分和教师对实习生满意度评价分值都远远高于对照组(P＜0.05).结论 采用阿德勒个体心理学理论培养检验实习生人文素养,不但能够有效的提升实习生的整体素质,还能够提高他们对专业理论的掌握和实际操作技能,获得更好的实习效果,此教学理论和方法值得进一步探索和推广.

目的 探讨红景天苷(Sal)对HepG2.2.15细胞的乙型肝炎病毒DNA(HBV DNA)、HBV前基因组RNA(pgRNA)的影响以及其可能的作用机制.方法 使用0、50、100 μmol/L的Sal处理HepG2.2.15细胞,分别使用CCK8、流式细胞仪、实时定量PCR检测细胞活性、细胞周期和凋亡、上清液HBV DNA、细胞内pgRNA以及相关调控基因沉默信息调节因子2相关酶1(SIRT1)、过氧化物酶体增殖物激活受体γ辅助激活因子-1α(PGC-1α)、AMP依赖蛋白激酶α2(AMPKα2)、P38蛋白激酶(P38 MAPK)和Forkhead转录因子1(FOXO1) mRNA表达水平;分析pgRNA和各基因的相关性.结果 Sal对HepG2.2.15的细胞活性、细胞凋亡率、细胞周期没有较大影响;50、100 μmol/L Sal处理细胞6d后,细胞上清液中的HBV DNA较对照组明显增加(F=42.337,P=0.006);pgRNA的变化趋势与SIRT1 mRNA变化趋势的相关性明显(r =0.891),pgRNA变化趋势与其它基因PGC-1α、AMPKα2、P38MAPK、FOXO1的mRNA变化趋势相关性系数r分别为0.553、0.135、-0.943、-0.809.结论 Sal可以通过影响SIRT1途径促进HBV的复制,慢性乙型肝炎患者和HBV携带者使用Sal时应关注乙肝病毒复制情况.

BACKGROUND:Hepatitis C virus (HCV) has been classified as a strictly hepatotropic pathogen for a long time, and hepatocytes are target cells for HCV infection. More and more studies showed non-liver cells supported HCV entry and replication, such as macrophages. The mechanisms of HCV entry into macrophages are still not clear.AIMS:This study aims to determine the way of HCV entry into macrophages.METHODS:Cell culture-derived infectious HCV particles (HCVcc) were prepared using Huh7 cells transfected with HCV RNA. CD81-knockdown cells were obtained through siRNA transfection. HCV RNA levels were determined by RT-qPCR. Flow cytometry analyses were used to determine cell surface levels of CD11b, CD68, and CD81. ELISA and western blotting were performed to quantify the protein levels of IL-1β, IL-6, and TNF-α. Phagocytic ability was determined by neutral red uptake assay.RESULTS:CD81 knockdown could not inhibit HCVcc entry into macrophages. The entry of HCV into macrophages could not be blocked by pooled IgG from chronic hepatitis C patient's sera. Macrophages derived from THP-1 cells displayed stronger phagocytic capacity, which also swallowed more HCV RNA. Treatment of macrophages with endocytic inhibitor, methyl-β-cyclodextrin, decreased the internalization of HCV. HCV uptake by macrophages was related to the reorganization of F-actin cytoskeleton and PI3Ks activation. HCV infection significantly increased the expression of IL1β and IL6 in macrophages and promoted apoptosis of macrophages.CONCLUSIONS:HCV entry into macrophages mainly depends on phagocytosis of macrophages.

目的 血管平滑肌细胞为动脉粥样硬化(AS)病变组织的主要细胞之一.流感病毒对人主动脉平滑肌细胞(HASMCs)的感染及对其细胞因子表达的影响报道较少.文中旨在探讨甲型流感病毒(IAV)和乙型流感病毒(IBV)感染HASMCs及对其细胞因子表达的影响.方法 采用IAV和IBV刺激HASMCs,分别为甲型组、乙型组,另设对照组(不加病毒).免疫荧光法检测细胞内流感病毒核蛋白表达,CCK8法检测细胞增殖,甲型组、乙型组取上清液分别加入正常培养基和病毒生长液,RT-PCR检测流感病毒RNA水平的变化,并用收集的上清液感染MDCK细胞,检测流感病毒核蛋白.RT-PCR检测感染流感病毒24 h后细胞因子的表达变化.结果 CCK8实验结果显示,HASMCs感染流感病毒后第3、4天,甲型组、乙型组细胞增殖较对照组明显减慢(P＜0.05).第3天,乙型组细胞上清RNA表达量较第2天明显增加(P＜0.05),第4天细胞上清RNA表达量较第3天明显下降(P＜0.01).甲型组正常培养基、病毒生长液IAV RNA水平分别为0.842±0.148、15.182±1.932,差异有统计学意义(P＜0.01).乙型组正常培养基、病毒生长液IBV RNA水平分别为0.962±0.033、4.029±0.681,差异有统计学意义(P＜0.01).甲型组、乙型组MCP-1的相对表达量[(4.364±0.193)、(3.348±0.507)]较对照组(1.001±0.001)均明显增高(P＜0.05);IL-6、TNF-α 亦明显增高(P＜0.05).结论 IAV、IBV均可感染HASMCs,并引起AS相关细胞因子(IL-6、TNF-α、MCP-1)表达的增加,为流感病毒感染导致AS的机制研究提供了理论依据.

目的 分析成都某部感染乙型流感病毒遗传进化与血凝素(HA)基因突变位点.方法 通过犬肾上皮细胞(MDCK细胞)体外分离患者咽拭子标本流感病毒毒株,用PCR获取乙型流感病毒HA基因并测序,与NCBI数据库在线比对并利用MEGA 6.06软件构建系统发育进化树,分析突变位点.结果 通过MDCK细胞接种,分离出1株乙型流感病毒株,以感染病例咽拭子核酸及分离毒株的核酸为模板进行PCR扩增得到1 755 bp全长HA基因,获得的序列提交至GenBank数据库,获得基因登录号为MH236281.通过在线比对及系统发育树构建,该病例感染的病毒为Yamagata系乙型流感病毒.与乙型流感病毒Yamagata系的代表毒株Influenza B/Yamagata/16/88(GenBank No.M36105)相比,HA基因点突变碱基为57个;与世界卫生组织(WHO)推荐的疫苗株InfluenzaB/Utah/08/2014 (GenBank No.KU592766)相比,突变碱基数为20个.进一步对HA1氨基酸突变位点进行分析,与四川地区往年流行株相比均发生了不同程度的突变,其中与2010年四川温江的分离株(GenBank No.KP461138)相比突变位点较少,仅有4处点突变;与WHO推荐的疫苗株Influenza B/Utah/08/2014相比,有2个氨基酸位点发生了变异,分别为L176Q和M255V,但突变没有处于HA1上的抗原决定簇区域.其中176位点是一个全新的突变,以往四川流行毒株、Yamagata系的代表毒株Influenza B/Yamagata/16/88及WHO推荐的疫苗株Influenza B/Utah/08/2014的HA1 176位点均为亮氨酸(L),而本研究病例感染的乙型流感毒株HA1 176位点突变为谷氨酰胺(Q).结论 成都地区驻地部队2017-2018流行季感染的乙型流感病毒HA基因已发生一些突变,但突变尚未造成抗原性的改变.