反义寡聚硫代磷酸型、甲基磷酸型核苷酸体外抑制 HSV-Ⅰ增殖与抗原表达

以人单纯疱疹病毒（ＨＳＶ）基因组即刻早期ｍＲＮＡ４和５拼接区１０３４～１０５０密码子序列为靶位点，人工化学修饰、合成特异性８～１４ｍｅｒ反义寡聚硫代磷酸型、甲基磷酸型及未修饰型核苷酸（Ｓ－ＯＤＮ１４、Ｍ－ＯＤＮ８、Ｎ－ＯＤＮ８）体外抑制病毒活性。Ｓ－ＯＤＮ１４５～１０μｍｏｌ／Ｌ浓度即可抑制ＨＳＶ致ＣＰＥ和ＰＦＵ，Ｍ－ＯＤＮ８４０～６０μｍｏｌ／Ｌ表现明显抑制效应，ＥＬＩＳＡ检测Ｓ－ＯＤＮ１４１０μｍｏｌ／Ｌ接近５０％抑制病毒抗原表达。Ｍ－ＯＤＮ８的抑制病毒活性剂量呈现细胞毒性。     

preS2反义寡核苷酸降低端粒酶活性及诱导肝癌细胞凋亡的作用

反义核酸技术从分子水平破坏靶基因 ,已用于多种抗病毒的试验。我们以人工合成的硫代反义寡核苷酸研究针对ＨＢＶ不同区段反义寡核苷酸ＨＢＶ的抑制作用 ,从中筛选了效果最好的针对ＨＢＶｐｒｅＳ2的反义序列 (ａｓ ｐｒｅＳ2 )。为进一步探讨ａｓ ｐｒｅＳ2在肝癌治疗中的应用价值 ,以ＨＢＶＤＮＡ整合的肝母细胞瘤细胞ＨｅｐＧ2 .2 .15为研究对象 ,采用ＦＡＣ ＳＣＡＮ流式仪检测细胞凋亡率 ,ＴＲＡＰ银染测定细胞端粒酶活性 ,同时以ＲＩＡ法检测培养上清中的血清铁蛋白浓度。设计合成针对ｐｒｅ Ｓ2基因 (ｐｅｒ Ｓ2 )翻译起始区 (32 0 3…     

硫代反义寡核苷酸体外对HDV的抑制作用

观察硫代反义寡核苷酸（Ｓ－ＡＳＯＤＮ）体外对ＨＤＶ的抑制作用。方法在ＨＤＶ／ＨＢＶ感染人胎肝细胞中加入不同浓度的针对ＨＤＶ ＳｔｅｍⅠ区６８４－６９８位核苷酸的１５聚Ｓ－ＡＳＯＤＮ,分别采用ＥＬＩＳＡ和斑点杂交法检测上清液中ＨＤＡｇ和细胞中ＨＤＶ ＲＮＡ。结果ＨＢｓＡｇ、ＨＤＡｇ在感染后第２天至第１６天均可测出,以第４天至第１２天达高峰,加入Ｓ－ＡＳＯＮＤ（２、４、６μＭＯＬ／ｌ）ＲＧ ２ＧＤ ,     

LINED CAVAL-ILIAC VENOUS PROSTHESES WITH CULTURED AUTOLOGOUS ENDOTHELIAL CELLS IN NON-HUMAN PRIMATE

ＬＩＮＥＤＣＡＶＡＬ－ＩＬＩＡＣＶＥＮＯＵＳＰＲＯＳＴＨＥＳＥＳＷＩＴＨＣＵＬＴＵＲＥＤＡＵＴＯＬＯＧＯＵＳＥＮＤＯＴＨＥＬＩＡＬＣＥＬＬＳＩＮＮＯＮ－ＨＵＭＡＮＰＲＩＭＡＴＥＬＩＮＥＤＣＡＶＡＬ－ＩＬＩＡＣＶＥＮＯＵＳＰＲＯＳＴＨＥＳＥＳＷＩＴＨＣ...     

反义Gαq／11亚基ODNs对平滑肌细胞DNA合成和PC蛋白表达的 …

目的：探讨Ｇｑ蛋白介导血管活性多肽对ＶＳＭＣＤＮＡ合成及增殖细胞核抗原（ＰＣＮＡ）蛋白表达的影响。方法：用硫代的Ｇαｑ／１１亚基反义寡聚脱氧核苷酸，以６μｍｏｌ／Ｌ的浓度加入含１０＾－７ｍｏｌ／Ｌ刺激无血清ＤＭＥＭ培养基中体外培养ＶＳＭＣ。用＾５Ｈ－ＴｄＲ掺入法测定ＶＳＭＣＤＮＡ合成，免疫细胞化学技术检测ＶＳＭＣＰＣＮＡ蛋白表达。结果：Ｇαｑ／１１ＡＳ－ＯＤＮｓ可明显抑制ＶＳＭＣＤＮＡＲ的合成，在     

反义硫代寡核苷酸体外抑制柯萨奇病毒B3增殖的研究

目的 在ＣＶＢ３易感的Ｖｅｒｏ细胞系中观察与ＣＶＢ３ＲＮＡ５’ＮＣＲ的ｎｔ５８１－６０１区域区补的２１聚硫代ＡＯＤＮ抗病毒活性。方法 采用ＭＴＴ法测定细胞活性,直接观察ＣＰＥ,测定培养上清ＴＣＩＤ５０,并分别用ＥＬＩＳＡ和往点杂交法检测ＣＶＢ３抗原及其ＲＮＡ。结果 １．ＡＯＤＮ可推迟和减轻ＣＰＥ,且随ＡＯＤＮ浓度增加,对ＣＰＥ的抑制作用增强,感染细胞存活率也随ＡＯＤＮ浓度升高而升高;２．ＡＯＤＮ可     

STUDY OF NOVEL MEMBRANOUS MATERIAL FOR CHARCOAL KIDNEY

ＳＴＵＤＹＯＦＮＯＶＥＬＭＥＭＢＲＡＮＯＵＳＭＡＴＥＲＩＡＬＦＯＲＣＨＡＲＣＯＡＬＫＩＤＮＥＹＳＴＵＤＹＯＦＮＯＶＥＬＭＥＭＢＲＡＮＯＵＳＭＡＴＥＲＩＡＬＦＯＲＣＨＡＲＣＯＡＬＫＩＤＮＥＹＧｕＨａｎｑｉｎｇ；ＬｕＭｏｚｕ（Ｔｉａｎｊｉｎｉｎｓｔｉｔｕ...     

A FREQUENCY DOMAIN QUANTITATIVE MEASUREMENT METHOD FOR BLOOD FLOW VELOCITY WITH BIDIRECTIONAL DOPPLERULTRASOUND AVOIDING DOWN-BAND CHANNEL

ＡＦＲＥＱＵＥＮＣＹＤＯＭＡＩＮＱＵＡＮＴＩＴＡＴＩＶＥＭＥＡＳＵＲＥＭＥＮＴＭＥＴＨＯＤＦＯＲＢＬＯＯＤＦＬＯＷＶＥＬＯＣＩＴＹＷＩＴＨＢＩＤＩＲＥＣＴＩＯＮＡＬＤＯＰＰＬＥＲＵＬＴＲＡＳＯＵＮＤＡＶＯＩＤＩＮＧＤＯＷＮ－ＢＡＮＤＣＨＡＮＮＥＬＷａ...     

ELECTRON MICROSCOPE ANALYSIS OF MITOCHONDRIA ON RENAL TUBULE CELL SUFFERED WARM ISCHEMIC AND REPER FUSIVE DAMAGE IN RABBITS

ＥＬＥＣＴＲＯＮＭＩＣＲＯＳＣＯＰＥＡＮＡＬＹＳＩＳＯＦＭＩＴＯＣＨＯＮＤＲＩＡＯＮＲＥＮＡＬＴＵＢＵＬＥＣＥＬＬＳＵＦＦＥＲＥＤＷＡＲＭＩＳＣＨＥＭＩＣＡＮＤＲＥＰＥＲＦＵＳＩＶＥＤＡＭＡＧＥＩＮＲＡＢＢＩＴＳＥＬＥＣＴＲＯＮＭＩＣＲＯＳＣＯＰＥＡ...     

反义寡脱氧核苷酸在鸭体内抑制鸭乙型肝炎病毒复制与 …

目的 研究反义核酸的抗病毒作用。方法 设计合成了针对鸭乙型肝炎病毒（ＤＨＢＶ）前Ｓ（ＰｒｅＳ）基因区第９５１－９６８位核苷酸的硫代反义寡脱氧核苷酸（ＡＳ－ＯＤＮ），以２０μｇ／ｇ体重／日剂量对３只腹腔感染ＤＨＢＶ５．２毒株后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性鸭连续静脉注射１０天，同时以等体积生理盐水注射另３只感染鸭作为对照。结果 对照鸭注射生理盐水后，血清ＤＨＢｓＡｇ及ＤＨＢＶ ＤＮＡ阳性未     

Diagnostic Value of Monitoring Human Cytomegalovirus Late pp67 mRNA Expression in Renal-Allograft Recipients by Nucleic Acid Sequence-Based Amplification

The diagnostic value of monitoring human cytomegalovirus (HCMV) late pp67 mRNA expression by nucleic acid sequence-based amplification (NASBA) after renal-allograft transplantation was evaluated. RNAs were isolated from 489 whole-blood specimens of 42 patients for the specific amplification of the late pp67 (UL65) mRNA. NASBA results were compared to results from the pp65 antigenemia assay, virus isolation by cell culture, and serology. The sensitivity value for NASBA proved to be higher than that for the antigenemia assay (50 versus 35%) for the detection of HCMV infection, while the sensitivity values of cell culture and NASBA were comparable (54 and 50%, respectively). NASBA detected the onset of HCMV infection simultaneously with cell culture and the antigenemia assay. Both the antigenemia assay and NASBA are very specific (100%) and highly predictive (100%) for the onset of HCMV infection. Antiviral therapy with ganciclovir resulted in negative results for cell culture, the antigenemia assay, and NASBA. In conclusion, monitoring HCMV pp67 mRNA expression by NASBA is a highly specific method for the detection of HCMV infection in renal-allograft recipients and is more sensitive than the antigenemia assay. Furthermore, NASBA can be used to monitor the progression of HCMV infections and the effect of antiviral therapy on viral activity.     

Antiviral drug resistance of human cytomegalovirus

The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.     

Human cytomegalovirus immediate early gene expression in the osteosarcoma line U2OS is repressed by the cell protein ATRX

The control of human cytomegalovirus (HCMV) immediate early (IE) gene expression in infected human fibroblasts was compared with that in the U2OS human osteosarcoma cells. Viral IE expression was stimulated by the virion protein pp71 and repressed by the cell protein hDaxx in fibroblasts, as expected from published data. Neither of these events occurred in infected U2OS cells, suggesting that this cell line lacks one or more factors that repress HCMV IE expression. The chromatin remodeling factor ATRX is absent from U2OS cells, therefore the effect of introducing this protein by electroporation of plasmid DNA was investigated. Provision of ATRX inhibited HCMV IE expression, and the presence of the HCMV-specified virion phosphoprotein pp71 overcame the repression. The experiments demonstrate that ATRX can act as a cellular intrinsic antiviral defense in U2OS cells by blocking gene expression from incoming HCMV genomes. In contrast, ATRX did not affect the replication of herpes simplex virus type 1, showing that there are differences in the way U2OS cells respond to the presence of the herpesviral genomes.     

Rapid antiviral DNA-DNA hybridization assay for human cytomegalovirus

A rapid DNA-DNA hybridization technique that can be accomplished in 4 to 5 days was compared with plaque reduction assay to determine its reliability in performing antiviral assays for human cytomegalovirus (HCMV). The assay involves lysing infected cells, direct wicking of denatured DNA onto membranes and hybridization using a 125I-labeled HCMV DNA probe. Using ten ganciclovir sensitive clinical HCMV strains for comparison, the DNA hybridization technique correlated well with the plaque assay. Clinical HCMV strains previously identified as resistant to ganciclovir were also readily identified. The DNA-DNA hybridization assay is less tedious and more rapid than plaque reduction assays, and thus, provides an excellent alternative for evaluation of the antiviral activity of drugs against HCMV.     

Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.     

Human mannose-binding lectin inhibits human cytomegalovirus infection in human embryonic pulmonary fibroblast

A limited number of drugs have been used for treatment of human cytomegalovirus (HCMV), all sharing the similar antiviral mechanism of inhibiting virus replication. This study investigates the anti-HCMV activities of mannose-binding lectin (MBL) from blocking virus entry and inhibiting virus spread. Recombinant human MBL was produced in CHO cells and native human MBL was isolated from human serum. A HCMV neutralization test was performed by pre-treating HCMV with each diluted MBL solution. Then the treated HCMV was inoculated onto the human embryonic pulmonary fibroblasts (HELF), which was followed by HCMV-DNA detection, PP65 positivity examination and confocal imaging of the infected cells. To test the activity of MBL in inhibiting viral spreading after viral invasion, HCMV growth inhibition test was performed. The infected cells were incubated with each diluted MBL, every 24 h, the supernatant was tested for HCMV-DNA. After 72 h, cells were collected for HCMV-DNA and PP65 examination. Then the cytopathic effect was observed and cell viability was measured at the 5 days after infection. HCMV neutralization test revealed 10 μg/mL MBL significantly decreased the HCMV invasion in HELF and the anti-HCMV activity can be blocked by 20 mg/mL mannan. HCMV growth inhibition test indicated that at 48 h after HCMV invasion, the HCMV-DNA level in the culture supernatant with 10 μg/mL MBL was lower than the control. After 72 h, both the HCMV-DNA levels and PP65 positivity in cells incubated with MBL were reduced. This is the first to report on the anti-HCMV activities of MBL by in vitro studies.     

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. The significant clinical impact of HCMV infection and progression to HCMV disease among allogeneic hematopoietic stem cell transplant recipients has been reduced by prophylactic, preemptive, and curative treatments using ganciclovir, valganciclovir, foscarnet, and cidofovir. Resistance to (val)ganciclovir results from mutations localized in HCMV UL97 gene (encoding the pUL97 phosphotransferase), UL54 gene (encoding the pUL54 DNA polymerase), or both genes, whereas foscarnet and cidofovir resistance results from mutations localized within UL54 gene only. This review is focused on HCMV antiviral drug resistance, including the functions of target genes of antivirals, the mechanisms of antiviral resistance, the different mutations in pUL97 and pUL54 that have been identified in either clinical isolates or laboratory strains, and their impact on HCMV susceptibility to antiviral drugs. It emphasizes the importance of proving that observed genetic changes confer resistance so they can be distinguished from polymorphisms. Because of the emergence of HCMV resistance to currently available drugs, novel drugs are urgently needed for the therapeutic management of HCMV‐resistant infections in hematopoietic stem cell transplant patients. Copyright © 2016 John Wiley & Sons, Ltd.     

Human antibody response to human cytomegalovirus-specific DNA-binding proteins

Human antibody responses to human cytomegalovirus (HCMV) specific DNA-binding proteins were studied in serum samples by the Western blot technique. The molecular weights of six DNA-binding proteins found in HCMV-infected cells, ranged from 52kD to 18kD. The sera obtained from patients with acute HCMV infections reacted well with the six HCMV specific DNA-binding proteins. The strongest reactivity was observed with the 52kD and 35kD proteins. The sera from healthy HCMV seropositive donors reacted only with the 52kD DNA-binding protein as visualized in Western blots, but 2 out of 8 sera failed to react with any HCMV specific DNA-binding proteins.     

Inflammatory cells in transplanted kidneys are infected by human cytomegalovirus.

To determine which cells in kidney grafts are infected with human cytomegalovirus (HCMV) before and after transplantation, kidney specimens were studied by in situ hybridization with 35S-labeled DNA probes representing HCMV immediate-early and late genes. Pretransplantation biopsies and serial posttransplantation biopsies were obtained from 7 renal grafts. All of the transplant recipients were HCMV-seronegative at the time of transplantation and all developed primary HCMV infections. HCMV nucleic acids were not detected in biopsies taken from the healthy donor kidneys before transplantation. However, biopsies taken at various intervals after transplantation showed abundant hybridization with HCMV immediate-early and late gene probes. Virtually all of the hybridizing cells were mononuclear inflammatory cells in the interstitial spaces of the kidney. Occasional hybridization was seen with renal tubular or glomerular cells. No cytomegalic cells were seen. Biopsy specimens taken after systemic anti-HCMV chemotherapy with phosphonoformate showed no uniform reduction in HCMV gene expression. These studies demonstrate that the principal HCMV-infected cells in kidneys of renal transplant patients with primary HCMV infections are infiltrating inflammatory cells.