目的:探讨CD4+/CD8+比值降钙素原(Procallcitonin,PCT)、C反应蛋白(C-reactiveprotein,CRP)水平变化与感染性休克(Septicshock,SS)患者疾病转归的相关性.方法:选取我院2020年4月-2022年2月收治的135例SS患者为研究对象,根据患者28d疾病转归分为生存组(91例)和病死组(44例).比较两组一般临床资料,比较两组治疗前、治疗3d、7d后CD4+/CD8+比值、PCT、CRP水平,分析治疗3d、7d后CD4+/CD8+比值、PCT、CRP水平与急性生理与慢性健康评估(AcutephysiologyandchronichealthevaluationⅡ,APCHEⅡ)评分、序贯器官衰竭(Sepsis-relatedorganfailureassessment,SOFA)评分及疾病转归的相关性,偏回归分析影响SS患者疾病转归的危险因素.结果:病死组年龄、治疗3d后、7d后APACHEⅡ评分、SOFA评分较生存组高(P<0.05);病死组治疗3d、7d后CD4+/CD8+比值低于生存组,PCT、CRP水平高于生存组(P<0.05);治疗3d、7d后,CD4+/CD8+比值与SOFA评分、APCHEⅡ评分、疾病转归呈负相关,PCT、CRP水平与SOFA评分、APCHEⅡ评分、疾病转归呈正相关(P<0.05);Logistic回归分析显示,治疗3d后CD4+/CD8+比值≤1.10、PCT>19.18μg/L、CRP>122.98mg/L,治疗7d后CD4+/CD8+比值≤1.18、PCT>18.03μg/L、CRP>111.65mg/mL是SS患者病死的危险因素(P<0.05).结论:CD4+/CD8+比值、PCT、CRP水平与SS患者疾病转归密切相关,检测其水平对临床预测患者疾病转归具有指导意义.

Objective:To establish a candidate reference method for the determination of angiotensin Ⅱ in human plasma by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and to evaluate its performance.Methods:Using [ 13C 6- 15N]-angiotensin Ⅱ as the internal standard, the plasma was accurately weighed by gravimetric method and mixed with a certain amount of internal standard. At the same time, enzyme inhibitor was added. After zinc sulfate solution protein precipitation and reversed-phase solid-phase extraction plate treatment, it was analyzed by liquid chromatography tandem mass spectrometry. The multi reaction ion monitoring mode(MRM)was selected by mass spectrometry to detect specific ion fragments of angiotensin Ⅱ and internal standard. The linearity, sensitivity, precision, recovery rate and uncertainty of the performance of the established method were evaluated according to ISO15193. Results:Angiotensin Ⅱ had good linearity in the range of 10-1 000 pg/g ( r=0.999 5), the lower limit of quantification was 7.68 pg/g, the analytical recoveries were 97.14% to 102.85%, intra-batch imprecision≤3.21%, inter-batch imprecision≤2.96%, and total imprecision≤3.67%. Conclusion:A method for the determination of plasma angiotensin Ⅱ was established by ID-LC-MS/MS. The method is accurate and reliable, and is expected to be a reference method for the determination of plasma angiotensin Ⅱ.

目的:观察1例圆头精子症患者的精子形态,并进行全外显子测序,探索其发病的遗传学基础.方法:取1例圆头精子症患者和1名健康对照精液各2μL,滴于GoldCyto Spermblue精子形态染色分析玻片上,用光学显微镜观察精子形态.抽取患者静脉血3 mL,提取基因组DNA,进行全外显子测序,并采用实时荧光定量PCR和Sanger测序方法对全外显子测序检测到的基因变异进行验证.结果:患者所有精子均为圆形头,仅见深染的细胞核,无顶体;该患者DPY19L2基因纯合缺失,同时携带DNAH1基因c.871+3G>A杂合突变以及ZMYND10基因c.833G>C杂合突变.结论:DPY19L2基因纯合缺失可能是本例圆头精子症的发病原因.

Objective To construct a double luciferase reporter gene vector with PRDM1 gene promoter as target,and establish drug screening cell model in vitro,hope to find small molecule compounds in Chinese herbal medicine library by this model.Methods Total DNA was extracted from 293T cells and it was used for amplifying the fragment contained PRDM1 gene promoter (267 bp/1 257 bp) by PCR.The amplified product was inserted into pGL3-Basic vector.The PCR product and pGL3-PRDM1 vector were verified by sequencing and alignment.The pGL3-PRDM1 and pRL-TK vector were co-transfected into engineer cells.The activity of PRDM1 gene promoter could be assayed by measuring luciferase.The method was optimized by changing ratio of two vectors.Results The highest transfection efficiency and luciferase activity were found in ratio of n(pGL-PRDM1) ∶ n(pRL-TK)=2 ∶1,and with the recombinant luciferase report gene vector contained the length of 1 257 bp amplified fragment transfecting into U266 cells.Moreover,the inductor (5-Aza-CdR) of PRDM1 gene was used for authenticating the method(P＜0.01),the fluoresscence in tensity and promoter activity of PRDM1 gene were enhanced in a dose dependent manner with 5-Aza-CdR.The activity of the promoter of PRDM1 gene was significantly decreased from the concentration of 5 μmol/L of Artemisinid(P＜0.05).The total glucosides of paeoniflorin promoted the promoter activity of PRDM1 gene at a low concentration,and inhibited the promoter activity of PRDM1 gene at a high concentration.Artesunate has no effect on cell proliferation.The effect of total glucosides of paeony on cell proliferation was more complicated.Conclusion A drug selection model targeting PRDM1 gene promoter has been successfully established,and artesunate has been screened to inhibit the promoter activity of PRDM1 gene.

目的:探讨坎地沙坦对左室射血分数保留的心力衰竭(HFpEF)心室重构及心功能的影响.方法:将24只新西兰白兔随机分成对照组、HFpEF组和给药组,每组各8只,HFpEF组给予缩窄腹主动脉,给药组在缩窄腹主动脉后给予坎地沙坦酯片1 mg/(kg,d)灌胃,经心脏彩超及血流动力学测定对比其心功能的不同,经常规病理染色比较各组心肌标本的病理及间质重构的差异.结果:术后90 d,HFpEF组较对照组室壁显著增厚,左房内径(LAD)明显扩大,二尖瓣舒张早期血流峰值与舒张晚期血流峰值之比(E/A)显著升高、等容舒张时间(IVRT)明显增加(P＜0.01),而射血分数(EF)正常;左室舒张末压(LVEDP)明显升高,左室松弛时间常数(tau)也显著延长(P＜0.01);心肌间质增生及重构明显(P＜0.01).而给药组的室壁厚度、间质增生及重构程度都较HFpEF组轻,但较对照组重(P＜0.05,P＜0.01).结论:坎地沙坦能显著改善压力负荷诱导的心室重构及心功能的改变,从而延缓整个病理过程发展到HFpEF.

目的 探讨抗核小体抗体(AnuA)和抗dsDNA抗体在狼疮肾炎(LN)诊断中的价值.方法 对98例LN患者(LN组)和79例非LN的系统性红斑狼疮(SLE)患者(非LN组)采用酶联免疫吸附(ELISA)法检测AnuA和ds-DNA抗体,并进行比较.两组标本行抗Sm抗体、抗nRNP抗体、抗核糖体P蛋白和抗中性粒细胞胞浆抗体(ANCA)检测,并进行相关性分析.结果 LN组患者血清中AnuA和抗dsDNA抗体的阳性率分别为63.69％和67.86％,非LN组阳性率分别为44.21％和51.39％,LN组阳性率明显高于非LN组(P＜0.05).LN组与非LN组和ANCA比较,差异有统计学意义(P＜0.05),抗SM抗体抗nRNP抗体和抗核糖体P蛋白比较,差异无统计学意义(P＞0.05).结论 AnuA和抗dsDNA抗体可作为诊断SLE的指标,其与SLE患者发生LN密切相关.同时检测多种自身抗体有助于诊断SLE和LN.

目的 研究抗环瓜氨酸肽(CCP)抗体在风湿性疾病中的临床意义,探讨抗CCP抗体在类风湿关节炎(RA)和系统性红斑狼疮(SLE)及其他风湿性疾病诊断中的应用价值.方法 采用酶联免疫吸附实验(ELISA)方法检测150例RA患者、120例SLE患者及100例健康人血清中的抗CCP抗体,并对3组检测结果进行相关性分析.结果 抗CCP抗体在RA组中的敏感性为77.3％,特异性为95.9％,抗CCP抗体在SLE患者组中的敏感性为6.7％,特异性为44.8％.RA组的抗CCP抗体的敏感性及特异性均明显高于SLE组及健康对照组,差异有统计学意义(P ＜0.05);SLE患者中关节炎组的抗CCP抗体阳性率为9.5％,较非关节炎组(3.5％)高,差异有统计学意义(P＜0.05).结论 抗CCP抗体可作为RA诊断的良好指标,抗CCP抗体与SLE患者关节炎的发生及骨侵蚀密切相关.

Objective To investigate the positive rate of HIV antibody in inpatients of a general hospital.Methods The plasma HIV antibodies of 244 636 inpatients were detected from June 2010 to May 2012,then the positive cases were analyzed.Results Totally 291 plasma samples were positive by the antibody screening test for HIV and the positive rate was 0.119%.The plasma HIV antibodies of 113 514 inpatients were detected from June 2010 to May 2011,89 plasma samples were positive with the positive rate of 0.078%.The plasma HIV antibodies of 131 122 inpatients were detected from June 2011 to May 2012,202 plasma samples were positive with the positive rate of 0.154%.The 291 positive cases were distributed in 22 clinical departments including Pneumology Department,Infectious Disease Department,Neurology Department,Hematology Department,Dermatological Department,Vasculocardiology Department,Otolaryngological Department,Internal Emergency Department,Oncology Department and Department of Nephropathy and Rheumatism,accounted for 81.44%of the total cases.Conclusion The positive rate of HIV antibody among inpatients is high and shows an increasing trend.The test of HIV antibodies is an important way for finding the HIV/AIDS infector in general hospital.The HIV antibody test should be strengthened in inpatients for early detection of infections and control the transmission of AIDS.

目的 观察人肝脏胶原样凝集素1(CL-L1)在肝癌细胞株HepG2内的定位及其功能.方法 利用pcDNA3.1/myc-his和pEGFP-N1载体将CL-L1基因转染HepG2细胞,通过免疫荧光标记技术及激光共聚焦显微镜观察CL-L1在HepG2细胞内的定位及HepG2细胞的克隆形成能力.结果 CL-L1主要定位于HepG2细胞的细胞质基质中,高尔基体、内质网和细胞核中不存在CL-L1.CL-L1基因过表达可引起HepG2细胞克隆形成能力的明显下降.结论 CL-L1定位于HepG2细胞的细胞质基质,可能是肝癌相关抑癌基因.

我们通过建立裸鼠食管鳞癌细胞EC9706的移植瘤模型,观察TPX2干扰前后对移植瘤的影响和肿瘤组织中TPX2的表达水平. 一、材料与方法 1.材料:人食管癌细胞株EC9706(中国医学科学院肿瘤医院惠赠).裸鼠:BALB/c裸小鼠15只,雄性,4周龄,体质量16～20 g[中国科学院动物所,SCXK(京)2009-0004].TPX2兔抗人多克隆抗体(由德国 Ulrike Bauer惠赠).免疫组织化学试剂盒(北京中山生物技术有限公司).TPX2与β-肌动蛋白(β-actin)引物(北京奥科生物技术有限责任公司),逆转录-聚合酶链反应(RT-PCR)试剂盒(大连TaKaRa公司).

OBJECTIVE:To construct small interfering RNA (siRNA) expression vectors targeting human MAGE3 gene and to observe the effects of gene silencing of MAGE3 by RNA interference on location and metastasis of lung carcinoma cells.METHODS:MAGE3 mRNA targeted hairpin siRNA was devised and the oligonucleotide strands of DNA fragments encoding the above siRNA were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into pSUPERneoGFP, followed by amplification and DNA sequencing, then transfected into human lung carcinoma NCI-H446. The expression of MAGE3 gene mRNA and protein were examined by RT-PCR and Western blotting. Colony formation assay and Boyden chamber assay were performed to detect the effects of MAGE3 on colony formation and metastasis.RESULTS:The DNA fragments encoding MAGE3-targeted siRNA were cloned into the pSUPERneoGFP and confirmed by restrictive enzyme digestion and DNA sequencing. RT-PCR and Western blotting revealed a strongly decreased expression level of MAGE3. The lung carcinoma cells transfected by siRNA group was significantly lower than others, an effect on its colony formation and invasiveness. The colony formation of lung carcinoma cells transfected by siRNA in soft agar and the number of cells penetrating matrigel both reduced, there is significant difference compared with untransfected group and transfected empty vector.CONCLUSION:An siRNA vector targeting human MAGE3 gene has been successfully constructed. Expression silencing of MAGE3 by RNA interference could reduce location and metastasis of lung carcinoma cells effectively.

Objective:To construct the lentiviral expression vector of murine Mash1 for further study on neural development and neural differentiation. Methods:The entire Mash1 cDNA was amplified by RT-PCR from a mouse embryo and then was ligated with pGEM-T vector. The ligation product was transformed into the JM109 competent cells. The positive recombinant clones were selected and identified by α-complementation, PCR, restriction endonuclease digestion and DNA sequencing. The cloning vector and the Lentivirus were cut by BamHⅠand XholⅠ. Then they were ligated and transformed. The enzyme analysis was used to confirm the recombinant vector. Results:The results of PCR, enzyme analysis and DNA sequencing analysis have confirmed that the right Mash1 gene was cloned. Conclusion:The cDNA clone of murine Mash1 and its lentiviral expression vector Lentivirus-Mash1 are successfully constructed.

Aim: To construct efficient expression vector of HBsAg and B19-VP2 gene. Methods: HBsAg gene and B19 VP2 gene were amplified using PCR, and cloned with pGEM-T vector and sequencing. The HBsAg gene was inserted into the prokaryotic expression vector pGEX-5X-1 and the recombinant pGEX-5X-1-HBs was obtained. Then VP2 gene was inserted into pGEX-5X-1-HBs in order to form pGEX-5X-1-HBs-VP2, which was transformed into E.Coli. BL21(DE3) and induced to express the fusion protein by IPTG. Finally it were confirmed by Western-blot. Results: pGEX-5X-1-HBs-VP2 recombinant vector could express efficiently in BL21. By using Western-blot, it was confirmed that the fusion protein had both the antigen epitopes of HBsAg and B19 VP2 protein partially. Conclusion: The pGEX-5X-1-HBs-VP2 expression vector has been constructed successfully. And its expression product has antigenic characterization of HBsAg and B19 VP2 protein. It provides information for developing B19 and HBsAg recombinant polyvalent vaccine.

Aim: To explore the immunogenicity of pcDNA3/VP2 as gene vaccine. Methods: B19 VP2 gene was amplified using PCR. B19 VP2 was recombined with pcDNA3 to form pcDNA3/VP2. pcDNA3/VP2 packed with liposome was inoculated into laboratory rabbit. B19 VP2 antibody was detected in rabbit's serum using ELISA.Results: Eukaryotic vector pcDNA3/VP2 was successfully constructed, which had better immunogenicity as gene vaccine and could stimulate rabbit to generate higher titre antibody. Conclusion: B19 VP2 gene can be used as target gene of B19 virus gene vaccine.

Aim: To clone the complete genome of B19 virus(Zhengzhou strain) and analyze its sequence homology by comparing with the known sequence in GenBank. Methods: PCR was used for genome cloning of B19 virus which was isolated from the serum of an abortion patient in Zhengzhou. It would get 7 gene segments which overlaped each other. The length of each gene segment was 814 bp(1~814), 849 bp(753~1 601), 663 bp(1 523~2 185), 738 bp(2 124~ 2 861), 1 011 bp(2 790~3 800), 1 014 bp (3 747~4 760),914 bp(4 681~5 594). Then each gene segment was inserted into T vector and sequenced. The sequence data were compared with the the known sequence in GenBank. Results: The complete genome of B19 virus (Zhengzhou strain) which had 5 594 nucleotides was well matched with the known sequence(NC_000883). There were 10 basic group differences between them. Furthermore, only one caused amino acid difference(C→G at the 3 752th necleotide caused Ser→Cys). Among the 5 594 nucleotides, A accounted for 29.58%( 1 655/5 594), T, 26.52%(1 484/5 594), G, 22.88%(1 280/5 594), C, 21.02% (1 175/5 594). There were 3 ORFs in the genome. The 1st one began from the 615th to the 2 630th nucleotide,which coded 672 amino acids; the 2nd one began from the 2 623rd to the 4 968th nucleotide, which coded 782 amino acids; the 3rd one began from the 3 304th to the 4 968th nucleotide, which coded 555 amino acids. Conclusion: The B19 virus of Zhengzhou strain shares the same genotype with the known sequence.