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1.
从一名国内感染的艾滋病人采血,分离其外周血单核细胞(PMCs)。首先与正常的PMCs共培养,4周后检测其HIV-1p24抗原(ELISA)达到峰值。用此时的细胞及其上清分别感染Jurkat-tat、CEM、MT4细胞,可很快地在这三株细胞中检测到HIV生长,HIV在Jurkat-tat细胞中生长情况最好,同时用病人血清直接感染Jurkat-tat和MT4细胞,4周后检测其细胞上清HIV-1p24抗原(ELISA法)为阳性,但OD值很低(约为PMC共培养组的一半)。用病人的少量全血与正常PMCs共培养,得到的结果与分离病人PMCs法相近。应用间接免疫荧光法(IFA)、免疫酶法(IEA)、蛋白印迹法及HIV-1POl基因和Env基因特异引物的聚合酶链反应(PCR)等证实为HIV-1病毒。分离的HIV在Jurkat-tat细胞中连续传代,细胞被感染后2-3天即出现以大量融合细胞为主的细胞病变,感染后7-10天细胞几乎全部死亡。病毒在连续传代过程中的生长特征及致细胞病变特征不变。此病毒命名为CA-2毒株。  相似文献   

2.
从一名国内感染的艾滋病人分离人免疫缺陷病毒   总被引:3,自引:0,他引:3  
赵永森  徐克沂 《病毒学报》1994,10(3):216-220
从一名国内感染的艾滋病人采血,分离其外周血单核细胞。首先与正常的PMCs共培养,4周后检测其HIV-1p24抗原达到峰值。用此时的细胞及其上清分别感染Jurkat-tat,CEM,MT4细胞,可很快地在这三株细胞中检测到HIV生长。HIV在Jurkat-tat细胞中生长情况最好。同时用病人血清直接感染Jurkat-tat和MT4细胞,4周后检测其细胞上清HIV-1P24抗原为阳性,但OD值很低。用  相似文献   

3.
广东两株人免疫缺陷病毒的分离和鉴定   总被引:1,自引:0,他引:1  
从我省两名经性途径感染艾滋病的病人中采血,分离外周血单核细胞(PBMCs),将其与正常人PBMCs共培养分离人免疫缺陷病毒(HIV),3周后检测上清HIV-1p24抗原(ELISA法)超过阈值。将共培养第四周细胞和上清分别感染H9细胞(T细胞淋巴瘤传代细胞),一周后检测HIV-1 p24怕,证明有病毒生长。用HIV-1 ENV基因引物的套式聚合酶链反应(Nested PCR)证实,两株新分离的病毒  相似文献   

4.
用单克隆抗体LCA,UCHL1,MT1,MB1,MB2,LN1和LN2对65例非何杰金氏淋巴瘤常规福尔马林固定石蜡包埋组织切片作免疫表型研究。证实B细胞性淋巴瘤50例(77%)。T细胞性淋巴瘤11例(17%)(4例染色不良,未能分型)。根据国际工作分类,本组T细胞淋巴瘤所占比例分别是11%,27%和13%。全部病例中LCA阳性率97%。T细胞抗体UCHL;阳性率(91%)明显高于MT1(73%),且与B淋巴细胞无交叉反应。4种B细胞抗体阳性率分别为LN。(96%),MB2(94%),LN1(86%),MB1(84%)。LN2和LN1对源于滤泡中心细胞淋巴瘤有更强的反应。研究结果表明上述单克隆抗体可有效用于非何杰金氏淋巴瘤石蜡切片的免疫组织化学标记,合理选用单克隆抗体有助于进行免疫学分型。  相似文献   

5.
用电泳迁移分析方法研究了21nt脱氧寡核苷酸G3TG2TGT2G5TG2TGT(CP1)与129bp的乙肝病毒(HBV)核衣壳启动子(Cp)片段内一位点结合形成的三链DNA的特异性及稳定性.在克隆有HBV基因组的质粒pCP10的酶切产物中,CP1仅与含Cp的129bp片段结合.在20mmol/LMg2+溶液中其解离常数(Kd)为1.4×10-7mol/L.不同离子稳定三链DNA的效果依次为sp4+(精胺)>Mg2+>Zn2+>Na+>K+,离子之间存在相互竞争作用.比CP1多一误配碱基的脱氧寡核苷酸G2TG2TGTG3TG2TG2TG2T(CP2)在20mmol/LMg2+溶液中与Cp结合的Kd值约为CP1的1/7,而在60mmol/LK+或5mmol/LZn2+溶液中检测不到它与Cp的结合,这进一步显示了三链DNA形成的特异性.细胞的生理离子浓度被认为是:Sp4+1mmol/L,Mg2+10mmol/L,K+140mmol/L,因此,CP1在细胞内将能特异地与Cp结合并具有较好的稳定性.  相似文献   

6.
将编码丙型肝炎病毒(HCV)E2蛋白417~750位氨基酸的DNA片段 克隆到真核表达载体pcDNA 3.1(-)中的CMV IE启动子下游,构建成HCV E2重组真核表达质粒 pcE2。ELISA法检测pcE2 DNA免疫兔血清中的E2抗体变化和维持规律,结果显示免疫20d已有 抗体产生,30d后开始进入高峰,40d时达到最高值,至第90d抗体水平保持平稳,抗体滴度 达到1∶1600左右。流式细胞计数仪(FACS)检测pcE2 DNA免疫鼠CD4+、CD8+T淋巴细胞变 化情况,与注射空载体pCDNA3.1(-)的阴性鼠相比,CD4+淋巴细胞水平略有上升,CD8+ 细胞水平有较大升高,增幅达35.46%。免疫组化检测结果显示注射pcE2的小鼠组织中有明显 的阳性着色,而注射pcDNA3.1(-)的对照组小鼠免疫组化结果为阴性。以上结果表明:pcE2 在实验动物内表达出的HCV E2蛋白可以引起免疫动物的体液免疫应答和细胞免疫应答,尤其 是MHC-1限制性杀伤性CD8+T淋巴细胞水平的提高对清除 病毒是十分有利的,因此HCV E2 DNA免疫有可能成为预防和治疗HCV感染的一条新途径。  相似文献   

7.
李田昌  佟利家 《生理学报》1996,48(4):337-342
内皮素(endothelin,ET)是已知的体内活性最强的缩血管物质,其缩血管作用由G蛋白偶联受体所介导。但ET强大的促血管平滑肌细胞(VSMC)增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉VSMC,探讨丝裂素活化蛋白激酶(MAPK)在ET促细胞增生中的作用。结果表明:ET-1呈时间和浓度依赖性地促进细胞摄取 ̄3H-TdR和激活MAPK,此作用可被蛋白激酶C(proteinkinaseC,PKC)抑制剂Staurosporine(STP),H-7和ET_A受体拮抗剂BQ123所抑制,但不被酪氨酸激酶抑制剂HerbimycinA(Herb)所抑制,用PKC激动剂PMA(Phorbolmyristateacetate)预处理VSMC,使其PKC活性下调,可显著减弱ET-1对MAPK的激活能力。本结果提示:(1)MAPK参与ET-1所致的VSMC增生;(2)ET-1促细胞增生与激活MAPK的作用是由ET_A受体和PKC介导的。  相似文献   

8.
目的和方法:将红细胞生成素(EPO)3'-增强子野生片断及点突变片断借脂质体主人脐静脉内皮细胞株ECV-304,用半定量RT-PCR测定正常秘缺氧诱导因子-1(HIF-1)诱导剂氧化钴(CoCl2)作用下培养6h的细胞环氧合酶2(COX-2)和血栓素合酶(TXS)的mRNA。结果:HIF-1诱导剂CoCl2可放COX-2和TXS基因转明显增强2,向细胞导入野生EPO3'增强子片断可阻断CoCl2诱  相似文献   

9.
内皮素对培养心肌细胞内游离钙浓度的作用   总被引:5,自引:0,他引:5  
Wang TH  Wu B  Zhu XN  Pan JY 《生理学报》1999,51(4):391-396
实验用培养新生SD大鼠心室肌细胞,以Fura-2/AM荧光指示剂负载检测收肌细胞内游离钙浓度(「Ca^2+」)的变化,探讨内皮素-1(ET-1)对「Ca^2+」i的作用及其机制。结果显示:ET-1引起心肌细胞「Ca^2+」i升高有两个时相,瞬时相持续相。ET-1诱导的瞬时相「Ca^2+」i升高呈浓度依赖性,预先用ETA特异性受阻断剂BQ123处理,可阻断ET-1引起的「Ca^2+」i升高,揭示上述  相似文献   

10.
一种新的恒河猴T淋巴细胞活化抗原PTA1   总被引:1,自引:0,他引:1  
本文应用抗人T细胞活化抗原CD25和PTA1McAb对恒河猴PBMC进行间接免疫荧光染色和流式细胞仪分析,结果发现,恒河猴静止PBMCCD25和PTA1均为阴性,而PHA活化后或经连续2个月注射rHuTNF-α恒河猴PBMC均表达高比率的CD25和PTA1阳性细胞。此结果表明,PTA1抗原可作为恒河猴活化T淋巴细胞的一种有用标志,为PTA1分子结构和功能研究以及采用恒河猴作为灵长类动物模型,观察细胞免疫功能状态提供了有用的资料。  相似文献   

11.
一株释放逆转录病毒样颗粒的人恶性T淋巴细胞株的建立   总被引:1,自引:0,他引:1  
蓝祥英  王得新 《病毒学报》1992,8(2):187-190
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12.
13.
Cell lines which are infected with retrovirus are resistant to superinfection by a related retrovirus. Packaging of whole virions within synthetic lipid vesicles allows efficient infection of such resistant cell lines. This system is more efficient in introducing encapsulated virus into infected cells than into uninfected cells.  相似文献   

14.
Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2‐like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L ‐domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.  相似文献   

15.
In the mid-1960s the #620 cell passage line of a murine lymphoma-leukemia was developed at the Section of Clinical Tumor Virology and Immunology, Department of Medicine, The University of Texas M.D. Anderson Hospital in Houston, TX. The diploid lymphoma cells released a retrovirus and were antigenic in young adult Swiss (YAS) mice. Small lymphoma cell inocula were rejected with immunity acquired against large inocula of lymphoma cells. Tissue sections revealed the "starry sky" configurations. In one of the tissue cultures set up from lymphoma #620, a cell line consisting of large round poly- or tetraploid cells arose and was referred to as lymphoma cell line #818. The #818 cells grew in suspension cultures and in the form of large, lethal ascitic tumors in YAS mice. Diploid #620 lymphoma cells stained for retroviral antigens; #818 cells stained both for retroviral antigens and immunoglobulins. Fluids withdrawn from #818 cultures neutralized the leukemia virus in spleen focus assays. Immunoglobulin precipitated from #818 suspension culture fluids strongly and specifically neutralized the leukemia virus. The growth of #620 or #818 cells in YAS mice treated with rabbit anti-lymphoma cell immune sera was strongly inhibited but culture fluids of #818 cells showed weak and insignificant inhibition against leukemia-lymphoma #620 (in one experiment, unpublished). In two experiments #620 lymphoma cells were co-inoculated with immune spleen cells into the peritoneal cavities of YAS mice. The immune spleen cells derived from mice that rejected #620 cell inocula or were actively immunized with a photodynamically inactivated mouse leukemia virus vaccine. In the peritoneal cavities of mice co-inoculated with #620 cells and immune spleen cells, clones of large round cells emerged with tetra- or polyploid chromosomal modes. These cells stained for leukemia viral antigens and immunoglobulins. When passaged in YAS mice these cells induced lethal ascites tumors. It was concluded as early as in 1968-69 that an immune plasma cell can fuse with a lymphoma cell, if the lymphoma cell expresses retroviral antigens against which the plasma cell is producing a specific antibody. Some human lymphoma-leukemia cells express retroviral antigens and/or budding retroviral particles, whether due to the acquisition of new env sequences by incomplete resident endogenous retroviral genomes or due to the entry of exogenous retroviruses into lymphopoietic stem cells. In the Discussion illustrations are provided for the human cell line #778 established from a patient with "lymphosarcoma cell leukemia" in 1966. The malignant cells released unidentified retrovirus-like particles and fused with one another and with reactive lymphoid cells of the host. It should be investigated further if human lymphoma-leukemia cells could fuse with an immune plasma cell of the host and thus alter the clinical course of the disease.  相似文献   

16.
Human APOBEC3G (huAPOBEC3G), also known as CEM15, is a broad antiretroviral host factor that deaminates dC to dU in the minus strand DNA of human immunodeficiency virus type 1 (HIV-1), other lentiviruses, and murine leukemia virus (MLV), thereby creating G-to-A hypermutation in the plus strand DNA to inhibit the infectivity of these viruses. In this study, we examined the antiretroviral function of a murine homologue of APOBEC3G (muAPOBEC3G) on several retrovirus systems with different producer cells. MuAPOBEC3G did not suppress the infectivity of murine retroviral vectors produced from human or murine cells, whereas it showed antiviral activity on both wild-type and Deltavif virions of HIV-1 in human cells. In contrast, huAPOBEC3G showed broad antiviral activity on HIV-1 and murine retroviral vectors produced from human cells as well as murine cells. These data suggested that muAPOBEC3G does not possess antiretroviral activity on murine retroviruses and has a different target specificity from that of huAPOBEC3G and that huAPOBEC3G works as a broad antiviral factor not only in human cells but also in murine cells. A functional interaction study between human and murine APOBEC3G supported the former hypothesis. Furthermore, studies on the expression of APOBEC3G in producer cells and its incorporation into virions revealed that muAPOBEC3G is incorporated into HIV-1 virions but not into MLV virions. Thus, muAPOBEC3G cannot suppress the infectivity of murine retrovirus because it is not incorporated into virions. We suggest that murine retroviruses can replicate in murine target cells expressing muAPOBEC3G because they are not targets for this enzyme.  相似文献   

17.
The Fv-1b-mediated restriction of N-tropic retrovirus vector infection of BALB/3T3 cells was partially abrogated by prior infection with N-tropic murine leukemia virus. Likewise, abrogation of the Fv-1b restriction of N-tropic murine leukemia virus replication was accomplished by prior infection with genome-deficient virions produced by an N-tropic murine leukemia virus packaging cell line. The latter observation suggests that the Fv-1 target in genome-deficient virions abrogates Fv-1 restriction in the absence of any viral genome-directed processes.  相似文献   

18.
19.
We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.  相似文献   

20.
目的 构建表达重组人骨形成蛋白7 (bone morphogenic protein 7, BMP7)基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并探讨其作用机制。方法 克隆BMP7基因,以loxP同源重组法构成逆转录病毒载体pLP-LNCX-BMP7(pLLBMP7),转染包装细胞PT67进行病毒包装并测定病毒滴度;将逆转录病毒感染人成骨细胞,MTT法检测细胞生长变化,琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;Western blotting检测BMP7,caspase-3和bcl-2蛋白表达。结果 重组逆转录病毒载体pLLBMP7经鉴定连接正确,转染PT67细胞后上清液中可得到病毒,滴度达1×109pfu;MTT检测见pLLBMP7病毒组48和72h细胞抑制率高于对照组(35.1% vs. 5.3%,68.5% vs.18.3%,均p<0.05),48h可见BMP7蛋白高表达。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达14.42%;BMP7蛋白高表达时caspase-3蛋白的表达亦有显著升高,但bcl-2蛋白未见表达差异。结论 构建了BMP7逆转录病毒,在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能是通过激活caspase-3而发生作用。  相似文献   

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