孕早期人巨细胞病毒宫内活动性感染的诊断方法学研究

目的 探讨诊断孕早期HCMV宫内活动性感染的实验方法及其价值。方法 留取 68例有异常妊娠史的早孕妇女绒毛组织 ,分为两份 ,一份提取总RNA ,采用RT PCR检测其中的HCMVmRNA ,一部分制成冰冻切片 ,分别采用原位杂交技术 (ISH)检测HCMVmRNA ,免疫组织化学法检测HCMV早期抗原。结果 采用RT PCR检测到的孕早期绒毛组织HCMVmRNA阳性率为 39 7% ;ISH阳性率为 2 3 5 % ;免疫组化检测HCMV早期抗原阳性率2 2 1 %。RT PCR检出率明显高于ISH(P <0 0 5) ,而ISH与免疫组化的阳性率无显著性差异 (P >0 0 5)。结论 测定绒毛组织中的HCMVmRNA、早期抗原是HCMV宫内活动性感染的客观指标。RT PCR的敏感性最高 ,但ISH与免疫组化可同时作定位、定量观察 ,可操作性强 。

海洛因依赖者活动性巨细胞病毒感染和红细胞免疫功能检测

目的:了解海洛因依赖、红细胞免疫及巨细胞病毒感染之间的关系。方法:采用红细胞酵母菌花环实验检测43例海洛因依赖者及11例伴有活动性巨细胞病毒感染的海洛因依赖者红细胞免疫功能。结果:与正常对照组相比海洛因依赖者和伴有活动性巨细胞病毒感染的海洛因依赖者红细胞C_(3b)受体花环率(RC_(3b)R)、红细胞免疫粘附促进因子活性(RFER)、红细胞促中性粒细胞吞噬率(ERPN)显著下降(P<0.01),红细胞免疫复合物花环率(RICR)、红细胞免疫粘附抑制因子活性(RFIR)明显升高(P<0.01)。海洛因依赖者活动性巨细胞病毒感染率明显高于正常对照组(P<0.01),伴有活动性巨细胞病毒感染的海洛因依赖者与单纯海洛因依赖者相比,其红细胞C_(3b)受体花环率进一步降低(P<0.05),而红细胞免疫复合物花环率(RICR)则进一步增高(P<0.01)。结论:长期吸食海洛因可以导致红细胞免疫功能的明显损害,巨细胞病毒是海洛因依赖者的常见机会性感染因子,巨细胞病毒的活动性感染可以进一步损伤红细胞免疫功能。

肾移植受者巨细胞病毒RNA检测及其临床意义

目的建立一种早期快速准确的诊断移植术后人巨细胞病毒感染的新方法。方法采用核酸基础序列扩增法(nucleic acid sequence-based amplification,NASBA)检测肾移植术后患者外周血标本的巨细胞病毒即刻早期(immediate-early,IE)mRNA及晚期结构抗原pp67的mRNA,同时行抗原血症(pp65)检测,并将结果相比较。结果在接受肾移植的55例患者中,IE-mRNA阳性20例;有症状的活动性巨细胞病毒感染13例。IE-mRNA的敏感度、特异度、阳性预测值和阴性预测值分别为92.3%,83.3%,60.0%和97.1%。结论移植术后IE-mRNA序列扩增法检测能够早期、快速、客观的反映人巨细胞病毒活动性感染的状况,对临床抗病毒药物治疗具有指导意义。

核酸基础序列扩增技术在诊断移植后巨细胞病毒感染中的应用

人类巨细胞病毒感染是引起器官移植受者致病和死亡的重要原因,早期、敏感、特异地诊断活动性巨细胞病毒感染,已成为选择抗病毒治疗时机的关键。本文就巨细胞病毒感染的诊断现状、核酸基础序列扩增技术原理、特点及应用前景作了综述。

SAT法检测人巨细胞病毒pp67 mRNA在人类免疫缺陷病毒合并人巨细胞病毒感染患者临床诊疗中的价值探讨

目的探讨实时荧光核酸恒温扩增检测技术(simultaneous amplification and testing,SAT)检测人巨细胞病毒(human cytomegalovirus,HCMV)基质表层蛋白pp67(phosphoprotein67,pp67)mRNA在人类免疫缺陷病毒(human immunodeficiency virus,HIV)合并HCMV感染患者临床诊疗中的价值。方法利用SAT技术构建检测HCMV pp67 mRNA的方法,评估SAT法检测HCMV pp67 mRNA重复性、特异性以及检测下限;收集HIV阳性HCMV活动性感染患者69例、HIV阳性HCMV非活动性/潜伏感染154例、HIV阴性HCMV非活动性/潜伏感染79例患者的血清、肺泡灌洗液、脑脊液等样本进行HCMV pp67 mRNA检测,评估特异度、灵敏度、正确率、阴性预测值、阳性预测值以及约登指数,评估HCMV pp67 mRNA在辨别HCMV活动性感染中的临床价值。结果SAT法检测HCMV pp67 mRNA的精密度(变异系数<10%)可满足临床检测要求,与其他病原体无交叉反应,特异性良好,试剂检测下限为400 copies/mL;特异度87.55%,灵敏度60.87%,正确率81.46%,阴性预测值0.88,阳性预测值0.59以及约登指数0.48;SAT法检测HCMV pp67 mRNA结果阳性率与临床诊断结果差异无统计学意义(P=0.894)。结论SAT法检测HCMV pp67 mRNA可用于HCMV活动性感染的辅助诊断。

小儿巨细胞病毒活动性感染的两种诊断方法比较

目的 通过血清人巨细胞病毒 (HCMV)IgM和尿快速培养HCMV检测临床标本的比较 ,进一步评价血清特异性IgM诊断活动性HCMV感染的价值。 方法 ①ELISA法检测患儿血抗HCMV IgG、IgM。②通过检测尿培养物中HCMV即刻早期抗原 (IEA)快速诊断活动性HCMV感染。结果 ① 119例尿培养阳性者 ,血抗HCMV IgG均为阳性。②与尿快速培养法相比 ,血IgM的特异性和敏感性分别达到 97 7%和 6 9 7% ;血IgM阳性数与尿快速培养阳性数比率在≤ 1个月、～ 1岁和～ 3岁组分别为 4 5 8%、75 75和 82 4 % ,表明IgM检测结果在各年龄组均有假阴性 ,其中尤以 1个月以下婴儿多见。③ 2例 3个月左右患儿血IgM阳性而尿培养阴性。 11例随访患儿中 ,9例在结束更昔洛韦治疗后 1个月复查 ,3例IgM迟于尿培养转阴 ,2例尿培养阴性而血IgM仍为阳性 ,表明IgM检测结果可有假阳性。 结论 血清特异性IgM检测可用于诊断活动性CMV感染 ,但其意义有一定局限性 。

肾移植患者术前活动性巨细胞病毒感染对术后感染的影响

移植后早期活动性巨细胞病毒(CMV)感染高达60%～80%[1],被认为不仅是肾移植术后近期死亡的重要原因,而且可能与急性排斥反应和慢性移植物肾病有关.肾移植后能否有效、及时地防治CMV感染是影响移植效果的关键[2].鉴于肾移植前活动性CMV感染的发生状况、移植前活动性CMV感染对移植后的影响目前尚不清楚,本研究旨在通过检测CMV-pp65抗原血症,探讨移植前活动性CMV感染对移植后活动性CMV感染的严重程度及发生CMV病的影响,为临床防治CMV感染提供依据.

重症患者巨细胞病毒活动性感染与Th1/Th2型细胞因子的研究进展

重症患者巨细胞病毒(CMV)感染率极高,并且CMV感染后多呈潜伏性感染,同时感染CMV的重症患者往往缺乏典型的临床表现。但是当CMV再激活,即CMV活动性感染,此时可严重影响重症患者病情转归,导致多种不良预后的发生。究其机制为,CMV可通过影响辅助性T淋巴细胞1型和2型细胞(Th1/Th2)的功能,即通过调控Th1/Th2产生的细胞因子的数量及比例,来改变机体免疫状态,使CMV难以清除及易于再激活。因此Th1/Th2细胞因子的表达对CMV的再激活、复制和散播有着极其重要的作用及意义。本文就重症患者CMV活动性感染与Th1/Th2型细胞因子相互作用机制的研究进展作一综述。

强直性脊柱炎患者外周血中TNF-α、IL-17、IL-23、IL-33水平检测及其与病情的相关性分析

目的分析强直性脊柱炎患者外周血中肿瘤坏死因子-α(TNF-α)、白介素-17(IL-17)、白介素-23(IL-23)、白介素-33(IL-33)水平及其与病情的相关性。方法选择2012年11月至2014年11月接受住院治疗的强直性脊柱炎患者78例作为研究对象,另取67例同期在本院进行健康体检的正常人作为正常对照组,对比两组血清TNF-α、IL-17、IL-23、IL-33含量,疾病活动性因子红细胞沉降率(ESR)、C反应蛋白(CRP)水平,进一步分析TNF-α、IL-17、IL-23、IL-33含量与ESR、CRP水平的相关性。结果(1)研究组患者的血清TNF-α、IL-17、IL-23、IL-33含量明显高于对照组(P<0.05);(2)研究组患者的血清ESR及CRP水平显著高于正常对照组(P<0.05);(3)强直性脊柱炎患者中,外周血TNF-α、IL-17、IL-23、IL-33含量与疾病活动性因子ESR、CRP水平呈正相关(P<0.05)。结论强直性脊柱炎患者外周血中TNF-α、IL-17、IL-23、IL-33水平显著上升,且其幅度与疾病活动性呈正相关,可以对疾病的严重度及治疗效果做出客观评估。

Magnolol attenuates sepsis-induced gastrointestinal dysmotility in rats by modulating inflammatory mediators

AIM： To investigate the protective effects of magnolol on sepsis-induced inflammation and intestinal dysmotility. METHODS： Sepsis was induced by a single intraperitoneal injection of lipopolysaccharide （LPS）. Male Wistar rats were randomly assigned to one of three treatment groups： magnolol prior to LPS injection （LPS/Mag group）; vehicle prior to LPS injection （LPS/Veh group）; vehicle prior to injection of saline （Control/Veh）. Intestinal transit and circular muscle mechanical activity were assessed 12 h after LPS injection. Tumor necrosis factor-α （TNF-α）, interleukin-10 （IL-10）, monocyte chemoattractant protein-1 （MCP-1） and inducible nitric oxide synthase （iNOS） mRNA in rat ileum were studied by RT-PCR 2 h after LPS injection. Nuclear factor-κB （NF-κB） activity in the intestine was also investigated at this time using electrophoretic mobility shift assay. In addition, antioxidant activity was determined by measuring malondialdehyde （MDA） concentration and superoxide dismutase （SOD） activity in the intestine 2 h after LPS iniection.RESULTS： Magnolol significantly increased intestinal transit and circular muscle mechanical activity in LPS- treated animals. TNF-α, MCP-1 and iNOS mRNA expression in the small intestine were significantly reduced after magnolol treatment in LPS-induced septic animals, compared with untreated septic animals. Additionally,magnolol significantly increased IL-10 mRNA expression in septic rat ileum. Magnolol also significantly suppressed NF-κB activity in septic rat intestine. In addition, magnolol significantly decreased MDA concentration and increased SOD activity in rat ileum. CONCLUSION： Magnolol prevents sepsis-induced suppression of intestinal motility in rats. The potential mechanism of this benefit of magnolol appears to be modulation of self-amplified inflammatory events and block of oxidative stress in the intestine.

Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

AIM： To investigate the anti-neoplastic effect of inositol hexaphosphate （InsP6 or phytic acid） on dimethylhydrazine （DMH）-induced colon tumor in rats and its effect on blood natural killer （NK） cell activity. METHODS： Healthy Wistar rats, 4 wk old, were divided into control group （fed with common food） and InsP6 group （fed with common food＋2% sodium inositol hexaphosphate in the drinking water）, 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously （20 mg/kg body weight） once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenase-release assay. RESULTS： Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups. CONCLUSION： InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats.

Farnesoid X receptor expression is reduced in human hepatocellular carcinoma

Farnesoid X receptor (FXR, NR1H4) is a member of nuclear hormone receptor superfamily. Previously studies showed that FXR-/- mice spontaneously developed liver tumors when they aged, however, the relevance of which to human hepatocellular carcinoma (HCC) is unclear. The aim of this study is to observe whether FXR expression is also downregulated in HCC and discuss the mechanism of the reduced FXR expression in HCC. Expression of FXR and small heterodimer partner (SHP) was measured by real-time PCR and immunohistochemical technique. Effect of pro-inflammatory cytokines on expression of FXR and its promoter activity were determined in primary hepatocytes or HepG2 and Huh7 cell lines. Our results showed that expression of FXR and its target gene SHP in human HCC was strongly downregulated compared to the normal liver tissues. In addition, pro-inflammatory cytokines were able to decrease FXR expression by inhibiting the FXR promoter activity. In conclusion this work demonstrates FXR expression is strongly downregulated in human HCC, which may be caused by decreased FXR promoter activity, suggesting a potential role of FXR in human HCC development.

Effect of Haimiding on the functioning of red cell membrane of FC and H_(22) tumor-bearing mice

AIM: To study the effect of Haimiding on the functioning of red cell membrane of FC and H22 tumor-bearing mice. METHODS: The membrane fluidity of red cells is measured with DPH fluorescence probe as a marker; the amount of red cell membrane proteins is measured using polyacrylamide gel electrophoresis; the amount of sialic acid (SA) on the surface of red cell membrane and the scalability of these cells are measured using colorimetric analysis. RESULTS: Haimiding can lower the membrane fluidity of red cells in tumor-bearing mice and the amount of their membrane proteins, while increasing the amount of sialic acid in the membrane of red cells in these mice and enhancing the ability of the membrane of their red cells to reseal. CONCLUSION: The anti-tumor effect of Haimiding on tumor-bearing mice is due to its ability to improve and restore the functions of the membrane of their red cell and to enhance the immune effect of the organisms.

Up-regulation of Fas Ligand Expression by Sirtuin 1 in both Flow-restricted Vessels and Serum-stimulated Vascular Smooth Muscle Cells

Objective To study the role of sirtuin 1 （SIRT1） in Fas ligand （FasL） expression regulation during vascular lesion formation and to elucidate the potential mechanisms. Methods SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell （VSMC） lysate. Smooth muscle cell （SMC）-specific human SIRT1 transgenic （Tg） C57BL/6 mice and their littermate wild-type （WT） controls underwent complete carotid artery ligation （ligation groups） or the ligation-excluded operation （sham groups）. The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 （SIRT1H363Y）, and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis. Results SIRTI was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation （P〈0.001） and VSMCs treated with serum （P〈0.05 at the transcriptional level, P〈0.001 at the protein level）. No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL （P〈0.001）. The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 （P〈0.001）, while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 （P〈0.001）. Conclusions Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT 1.

Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.

ANTITUMOR ACTIVITY OF IMMUNOCONJUGATES COMPOSED OF BOANMYCIN AND MONOCLONAL ANTIBODY

Boanmycin (bleomycin A6 . BM) . an antitumor antibiotic, was conjugated to monoclonal antibodies including R19, H 111 and CCT2. The immunoconjugates exhibited selective cytotoxicity to related target cells including cecum cancer Hce-8693 cells, liver cancer BEL-7402 cells and leukemia CEM cells. They were highly effective against related human tumor xenografts in nude mice, and the inhibition rates by the conjugates were much higher than those by free BM. The inhibition rate by R19-BM conjugate against human cecum cancer xenografts reached 90%. BY immunoelectron microscopy, CCT2-BM conjugate showed specific binding and internalization in leukemia CEM cells. The results indicate that boanmycin-monoclonal antibody immunoconjugates are highly active both in vitro and in vivo.

Effects of methylation status of caspase-8 promoter on antitumor activity of TRAIL to human gastric cancer cells

Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2′-deoxycytidine (5-Aza-CdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus line （BIVL） was established by transfecting baby hamster kidney （BIV） cells infection in vitro, a BIV indicator cell with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.