EB病毒膜抗原BLLF1基因在转基因小鼠中的表达

目的:分析EB病毒膜抗原(membrane antigen, MA)BLLF1基因在转基因小鼠外周血淋巴细胞中的表达.方法:EB病毒膜抗原BLLF1基因转基因首建鼠4只,尾组织经PCR检测携带有BLLF1基因,用流式细胞仪分析MA在外周血淋巴细胞中的表达并用激光共焦显微镜确定MA在细胞中的表达部位.结果:4只首建鼠中有2只KM-Tg-EBV1和KM-Tg-EBV5外周血淋巴细胞中有MA的表达,表达部位在细胞浆中和细胞膜上.结论:表达EB病毒膜抗原的转基因小鼠可作为研究EB病毒致病机制的一个新的动物模型.     

TNF-SP-EGFP融合蛋白的构建表达及亚细胞定位

目的探讨肿瘤坏死因子信号肽（TNF-SP）在哺乳动物细胞的表达和亚细胞定位。方法采用聚合酶链反应（PCR）技术，以TNF-SP-pcDNA3.0质粒为模板扩增TNF-SP基因，采用PCR产物的粘端克隆法，将TNF-SP定向克隆入pEGFP-N1的多克隆位点，构建TNF-SP-EGFP重组质粒，酶切，PCR检测，序列分析鉴定，采用脂质体转染法，将TNF-SP-EGFP融合基因转染COS-7细胞进行表达，经碘化丙啶（PI）染色后以激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果PCR检测，酶切鉴定及测序证实目的基因TNF-SP正确连接到pEGFP-N1的多克隆位点。TNF-SP-EGFP重组体转染COS-7后，激光共聚焦显微镜显示TNF-SP-EGFP在细胞质表达。结论成功地构建了TNF-SP-EGFP融合蛋白真核表达质粒，在COS-7细胞中获得表达，并证实其定位于细胞质。     

转EB病毒BLLF1基因小鼠的SPF级净化

EB病毒是人群中普遍感染的病毒,初次感染后大多数人可转为终生潜伏感染。EB病毒在感染宿主细胞时,首先是病毒外膜上的膜抗原（membrane antigen,MA）与宿主细胞膜上的受体CR2（CD21）结合,然后经胞饮进入宿主细胞。EB病毒MA是相对分子质量约为22000～34000的糖蛋白（gp350）。CR2是主要表达于B细胞、T细胞和某些上皮细胞膜上的EB病毒受体和补体C3d受体,因此EB病毒主要感染B细胞、T细胞以及部分上皮细胞。大量研究表明,EB病毒感染与自身免疫病、淋巴瘤以及某些黏膜恶性变有关。EB病毒MA在复制型感染时表达,潜伏感染的EB病毒在体内外因子的刺激下可多次活化从而表达MA。另有研究表明灭活的EB病毒在体外亦可以活化淋巴细胞,显然MA与宿主细胞受体的结合在淋巴细胞的活化中具有重要意义。为了探讨EB病毒感染和人类免疫系统疾病的关系,特别是EB病毒膜抗原在这一过程中的作用,郭长占等用EB病毒膜抗原BLL F1基因制备了转基因小鼠。限于转基因小鼠制备时的条件,该转基因小鼠模型是用普通级昆明（KM）小鼠制备的。     

二甲基亚砜诱导HL-60细胞分化的评判

目的探讨一种可靠的肿瘤细胞分化模型，确立一套判定肿瘤细胞是否分化的简便、准确的方法。方法以分化诱导剂二甲基亚砜（dimethylsulphoxide，DMSO）影响下不同时间点的HL_60细胞为检测时象，应流式细胞术来分析细胞大小及细胞表面的分化标志物；经碘化丙啶（propidium iodide，PI）染色后，用激光共聚焦显微镜观察时已分化细胞进行形态学确认。结果随着药物诱导时间的延长，被诱导的HL-60细胞体积逐渐增大；在48h后，被DMSO诱导细胞开始表达分化标志物CD11b并出现细胞核型的变化。结论DMSO能诱导HL-60细胞分化；应流式细胞术来分析细胞大小及细胞表面的分化标志物，再用激光共聚焦显微镜观察已分化细胞核的形态变化是判定肿瘤细胞分化的简便、准确的方法。     

肝癌细胞纤维肌动蛋白的共聚焦激光扫描显微镜光学切片观察

目的：建立培养细胞共聚焦激光扫描显微镜光学切片的方法。探讨肝癌细胞纤维肌动蛋白（F-actin)的空间结构。方法：采用共聚焦激光扫描显微镜光学切片技术结合异硫酸氢荧光素-鬼笔环肽（FTTC-phalloidin)标记纤维肌动蛋白和碘化丙啶（PI）标记细胞核的荧光探针双重标记技术对肝癌细胞纤维肌动蛋白进行形态学观察和图像分析。结果：肝癌细胞内纤维肌动蛋白微丝形成束状纤维。粗壮而密集,平行排列或纵横交错成网状贯穿整个细胞和细胞突起。结论：（1）共聚焦激光扫描显微镜的光学切片技术结合FTTC-phalloidin和PI荧光探针双重标记技术是观察和分析细胞骨架系统三维立体结构的良好方法;（2）肝癌细胞内纤维肌动蛋白微丝粗壮、形成束状或网状结构。     

转染MIP-1α和B7-1基因增强小鼠抗淋巴瘤效应的初步研究

【摘要】 目的 观察转染趋化因子MIP-1α和共刺激分子B7-1基因增强小鼠抗淋巴瘤的效应。方法 将MIP-1α和B7-1基因慢病毒重组载体转染小鼠EL-4淋巴瘤细胞,应用RT-PCR检测MIP-1α和B7-1基因mRNA表达,Western blot法检测MIP-1α和B7-1蛋白表达;转基因EL-4细胞注入小鼠右腋皮下,观察成瘤情况;灭活的转基因EL-4细胞注入成瘤小鼠体内,观察其增强小鼠抗淋巴瘤效应。结果RT-PCR检测发现EL-4/MIP-1α+B7-1细胞内有MIP-1α及B7-1mRNA表达,Western blot显示MIP-1α及B7-1蛋白表达;MIP-1α组、B7-1组较对照组成瘤时间延长,成瘤率降低,肿瘤平均体积较小,而联合组成瘤性消失;MIP-1α和B7-1治疗组的肿瘤平均体积、重量及肿瘤器官转移率明显小于对照组（P＜0.05）,而联合组的肿瘤平均体积及重量明显小于MIP-1α和B7-1组（P＜0.05）,而且联合组小鼠平均生存期,均较单基因组、对照组明显延长（P＜0.05）。结论 转染MIP-1α和B7-1基因能够明显增强小鼠机体抗淋巴瘤效应,明显延长荷瘤小鼠的平均生存期,为淋巴瘤的基因治疗提供了新的思路。     

扶正抑瘤颗粒对小鼠肝癌H22细胞凋亡率及线粒体膜电位的影响

目的:观察扶正抑瘤颗粒对小鼠肝癌H22细胞凋亡率及线粒体膜电位的影响。方法:48只荷瘤小鼠随机分为模型组、环磷酰胺治疗组、扶正抑瘤颗粒大剂量治疗组和扶正抑瘤颗粒小剂量治疗组,分别予以相应药物治疗14 d。碘化丙啶染色,流式细胞术检测各组小鼠肝癌H22细胞的凋亡率。采用荧光探针罗丹明123负载,激光扫描共聚焦显微镜下观察H22细胞的荧光强度,分析H22细胞线粒体膜电位的大小。结果:扶正抑瘤颗粒可以提高小鼠肝癌H22细胞的凋亡率,降低H22细胞的线粒体膜电位,与模型组比较,差异有统计学意义。结论:扶正抑瘤颗粒的抗肿瘤机制可能与降低肿瘤细胞线粒体膜电位,诱导肿瘤细胞凋亡有关。     

用显微注射法制备携带Epstein-Barr病毒膜抗原基因--BLLF1的转基因小鼠

目的 :用显微注射法制备携带EB病毒 (Epstein Barrvirus,EBV)膜抗原 (membraneantigen ,MA)基因 BLLF1的转基因小鼠。方法 :对 38只昆明种小鼠进行超排卵 ,收集受精卵 ,将EBVMA基因 BLLF1显微注射到受精卵的原核内。将注射后成活且健康的受精卵移植到假孕母鼠的输卵管内使其发育直至分娩。PCR分析检测仔鼠基因组中转基因的整合。结果 :显微注射 6 77个受精卵 ,其中成活且健康的 32 0个 ,卵的成活率为 47.2 %。将其移植到 12只假孕母鼠的输卵管内 ,其中 2只鼠怀孕并产仔 12只 ,出生率为 3 .75 %。PCR分析 5只仔鼠基因组整合有BLLF1基因 ,整合率为 41.7%。结论 :携带EBVMA基因 BLLF1的转基因小鼠已制备成功     

检测Herceptin诱导乳腺癌细胞凋亡的多种显微技术比较

目的:比较多种显微技术在检测细胞凋亡中的应用.方法:以Herceptin诱导乳腺癌SKBR-3细胞凋亡,应用Gieamsa染色光镜,EB和Hoechst染色荧光显微镜,Annexin V/PI染色激光共聚焦显微镜以及扫描和透射电镜分别检测细胞凋亡的发生.结果:在Herceptin作用下,Hoechst染色荧光显微镜、激光共聚焦显微镜、扫描和透射电镜分别观察到乳腺癌SKBR-3细胞出现不同方面的明显凋亡特征.结论:不同显微技术各有优缺点,要客观地证实凋亡的发生,应联合运用多种检测方法.     

新藤黄酸对黑色素瘤B16细胞凋亡的作用

目的探讨新藤黄酸(gambogenic acid,GNA)对小鼠黑色素瘤B16细胞凋亡的影响。方法用不同浓度的GNA培养B16细胞24h,倒置显微镜下观察细胞形态;采用四甲基偶氮唑蓝(methyl thiazolyl tet-razolium,MTT)法测定GNA对B16细胞增殖的抑制作用;荧光显微镜观察吖啶橙/溴化乙锭(acridine or-ange/ethidium bromide,AO/EB)染色后细胞凋亡;采用膜联蛋白Ⅴ-异硫氰酸荧光素/碘化丙啶双染,流式细胞仪检测B16细胞凋亡。结果 GNA作用B16细胞后,细胞的抑制率随着药物浓度的增加而升高;AO/EB染色荧光显微镜观察显示GNA处理的B16细胞出现了典型的凋亡特征;流式细胞仪检测显示GNA处理的B16细胞随着药物浓度的增大凋亡率随之上升。结论 GNA在一定的范围内,能够浓度和时间依赖性地通过诱导细胞凋亡抑制小鼠黑色素瘤B16细胞的增殖。     

腮腺淋巴上皮瘤样癌中EB病毒基因的表达

采用原位杂交和免疫组化技术，检测２０例腮腺淋巴上皮瘤样癌（ＬＥＬＣ）组织中ＥＢ病毒编码的小ＲＮＡ（ＥＢＥＲｓ）、潜伏感染膜蛋白（ＬＭＰｌ）、溶解感染期立即早期基因编码蛋白ＺＥＢＲＡ、早期基因编码蛋白ＥＡ－Ｄ、晚期基因编码蛋白ＶＣＡ和ＭＡ。结果ＥＢＥＲｓ阳性率为１００％（２０／２０），ＬＭＰｌ阳性率为８５％（１７／２０），ＺＥ－ＢＲＡ均为阴性，ＥＡ－Ｄ阳性率８５％（１７／２０），ＶＣＡ阳性率６０％（１２／２０），ＭＡ阳性率为０．５％（１／２０）。阳性信号限于癌细胞。癌周正常组织和间质细胞均阴性。说明在我国南方鼻咽癌高发区，腮腺ＬＥＬＣ的发生发展与ＥＢ病毒感染密切相关，本文还对ＥＢ病毒基因的腮腺ＬＥＬＣ中表达的生物学意义作了初步探讨。     

CONSTRUCTION OF HU-PBL／SCID CHIMERAS AND DEVELOPMENT OF EBV-RELATED LYMPHOMAS

Objective To construct hu-PBL/SCID chimeras and to investigate the development of lymphoma and oncogenicity of the Epstein-Barr virus (EBV). Methods Human peripheral blood lymphocytes (PBLs) were isolated from healthy adult donors and transplanted intraperitoneally into severe combined immunodeficient (SCID) mice. Mice with hu-PBL engraftment from healthy EBV seronegative donors were injected intraperitoneally with EBV-containing supematant from suspension culture of B95-8 cell line (active infection), whereas mice receiving lymphocytes from healthy EBV seropositive donors were not re-infected with B95-8 derived EBV (latent infection). Pathological examination and molecular analysis were performed on experimental animals and induced neoplasms. Results In the early stage of this experiment, 12 mice died of acute graft-versus-host disease, mortality was 34.3% (12/35 mice) with an average life span of 17.5 days. In 19 survival hu-PBL/SCID chimeric recipients from 12 healthy donors,tumor incidence was 84.2% (16/19 mice). The average survival time of tumor-bearing mice was 65.5 days. EBV-related neoplasms in SCID mice were nodular tumors with aggressive and fatal features. Histological morphology of tumors exhibited diffuse large cell lymphomas. Immunohistochemistry revealed that LCA (CD45) and L26 (CD20) were positive, but both PS1 (CD3) and UCHL-1 (CD45RO) were negative, and EBV products ZEBRA, LMP1, and EBNA2 were expressed in a small number of tumor cells. EB virus particles were seen in the nuclei of some tumor cells by electron microscopy, and EBV DNA could be amplified in the tumor tissues by PCR. In situ hybridization indicated that the nuclei of tumor cells contained human-specific Alu sequence. Conclusions EBV-induced tumors were human B-cell malignant lymphomas. We obtained direct causative evidence dealing with EBV-associated tumor deriving from normal human cells.     

肠道B细胞淋巴瘤Epstein-Barr病毒感染与癌基因

目的:研究肠道非霍奇金淋巴瘤B细胞型的Epstein-Barr(EB)病毒感染、p53、p21ras基因表达及其相关性.方法 :应用单克隆抗体、免疫组化方法对瘤细胞进行免疫表型研究,CD20、CD45RA、CD79a 鉴定瘤细胞的B细胞分化,CD45RO、CD3除外瘤细胞T细胞分化.同时用免疫组化方法,检测瘤细胞p53基因表达、p21ras基因表达.EB病毒寡核苷酸探针(EBER)原位杂交,观察瘤细胞EB病毒感染情况.结果:34例肠道B细胞淋巴瘤好发部位为回肠和结肠,以单发瘤结节多见,常伴有表面溃疡形成.34例中,弥漫性大B细胞淋巴瘤9例(26.5%),19例为惰性的边缘区B细胞淋巴瘤黏膜相关淋巴组织型(MZL-MALT型)(55.9%),6例为MZL-MALT型伴高度恶性转化(17.6%).EBV- EBER 原位杂交检测,全部病例为阴性.p53蛋白表达共有23例,占全部病例的67.6%(其中高度恶性占47.8%);16例有p21ras的表达,为47.1%(其中高度恶性占43.8%).有15例同时检出p53蛋白和p21ras的表达,占病例数的44.1%.结论 :34例肠道B细胞淋巴瘤以惰性为多见,但恶性转化明显.EBV的研究提示肠道B细胞淋巴瘤有别于T细胞淋巴瘤,与EBV感染无关.p53蛋白的表达高于p21ras,p53和p21ras的共同表达检出率较高,提示癌基因p53和p21ras在肠道B细胞淋巴瘤的发生中可能起一定作用.     

Primary nasopharyngeal non-Hodgkin lymphoma and its relationship with Epstein-Barr virus infection

Objectives To investigate the immunophenotypes of primary nasopharyngeal non-Hodgkin lymphoma(NPL) and their relationship to Epstein-Barr virus (EBV) infection.Methods The clinical data and biopsies of 73 patients with NPL were collected in Guangzhou. In situ hybridization was performed to detect the EBV-encoded small non-polyadenylated nuclear RNAs(EBERs) on biopsy slides. Immunohistochemistry was used to classify the immunophenotypes of NPL and detect EBV antigen expression.Results Forty-four (60. 27%) of the 73 NPLs were of B cell lineage (CD79α^ /CD3^-/CD56^-)while the 29 others (39.73%) were of non-B cell lineage. Seventy-three NPLs could be classified into 3 major immunophenotypes: B cell (CD79α^ /CD3^-/CD56 ^-, 44 cases), peripheral T cell (CD79α^-/CD3^ /CD56^-, 22) and NK/T cell (CD79α^-/CD3^ /CD56^ , 7). The percentages of EBV infection differed among the 3 major immunophenotypes ( B cell : 11.36%, 5/44 ; peripheral T cell: 81.82%,18/22; NK/T cell: 100%, 7/7). Both CD56-positive and CD56-negative immunophenotypes could further be divided into 4 subtypes: CD8^-/CD4^-, CD8^ /CD4^- , CD8^-/CD4^ and CD8^ /CD4^ All the CD8^-/CD4^- NPLs with CD56-positivity (7) or CD56-negativity (2) were infected with EBV. The neoplastic cells of a nasopharyngeal Burkitt‘ s lymphoma expressed EBV nuclear antigen 1 (EBNA1)and EBV RNA (EBERs) only. In the other 29 EBV-infected NPLs, most of the lymphoma cells harboring EBV also expressed EBNA1 and EBERs; 21 of the 29 NPLs had a considerable number of neoplastic cells expressing latent membrane protein 1 ( LMP1 ) (21/29, 72.41% ) and 23 of 29 NPLs expressed latent membrane protein 2A (LMP2A) (23/29, 79. 31%). A few lymphoma cells in 17( 17/29, 58. 62% ), 23 (23/29, 79.31% ) and 22 NPLs (22/29, 75.86% ) expressed Zta ( Barn HI Z transactivator), viral capsid antigen (VCA) and membrane antigen (MA), respectively.Conclusions The prevalence ratio of the 3 immunophenotypes, namely, B cell, peripheral T cell and NK/T cell lymphoma, is about 6:3: 1. However, the EBV infection ratio is reversed, 1:8: 10. All the NK/T cell (CD56^ ) and peripheral immature T cell (CD3^ /CD8^-/CD4^-) NPLs were EBV-infected.Except for one Burkitt‘ s lymphoma, the EBV harbored in both B cell and non-B cell NPLs was mainly latent infection, type It, expressing EBNA1, LMP1 and LMP2A. However, the EBV found in a few lymphoma cells could become replicative, expressing lytic proteins.     

人多聚免疫球蛋白受体转基因鼠的建立

目的 构建携带有多聚免疫球蛋白受体(pIgR)的转基因鼠，使其能以近乎自然的状态感染EBV，观察EBV在鼻咽部病变过程中的作用。方法 采用角质上皮特异性启动子ED—L2调控的pIgR基因，用显微注射方法将其导入受精卵中，使该基因能在F0代转基因鼠鼻咽部特异性表达。结果 PCR检测F0代pIgR基因阳性率达37．5％(6／16)，Southem杂交F0代pIgR基因阳性率为12．5％(2／16)。结论 表达多聚免疫球蛋白受体的转基因动物有望成为研究EBV病毒致病机制的一种新的动物模型。     

用单克隆抗体研究EB病毒的株间差异

为研究新分离的H_18EB病毒株的抗原性与其它已知EB病毒的差异,建立产生抗H_18病毒膜抗原的单克隆抗体杂交瘤。其中T_14-11杂交瘤株所产生的McAb只对H_18株呈阳性反应,能中和H_18EB病毒的感染性.Western Blot检测表明,T_14-11 McAb可识别H_18病毒蛋白的三个多肽P220、P175和P140.这些多肽分布于EB病毒感染的细胞膜和胞浆内,属于EB病毒基因的早期产物(早期膜抗原)。     

Transgenic mice with overexpression of human scavenger receptor A on endothelial cells

Objectives To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR- A) in atherosclerosis in vivo. Methods Human scavenger receptor minigene- driven mouse tie- 1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used t o screen the positive transgenic mice. RT- PCR and immunohistochemical analysis were used to detect the level and location of human SR- AⅠ expression in transgenic mice. The activity of human SR- AⅠ was determined by morphologi c observation of aortic endothelial cells of transgenic mice under transmission electron microscopy. Results The electrophoresis assay showed the expected 4 fragments of 0. 9 kb, 1. 1 kb, 1. 2 kb and 4. 2 kb in the SmaⅠ digest and 2 fragments of 0. 8 kb and 6 . 7 kb in BglⅡ digest of plasmids pTie- 1/hSR- A. The fragment sequence of tie - 1 p romoter and human SR- A cDNA in plasmids pTie- 1/hSR- A was correct and no A TG b efore the translation initiation sites of human SR- A was found by sequence ana lysis. 561 injected and surviving embryos with the purified human SR- A minigen e were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Sou thern blot analysis. The results of RT- PCR and immunohistochemical analysis sh owed human SR- A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in tran sgenic mice. Transmmion electron microscope (TEM) of aorta of transgenic mice s howed that a large number of vesicles, multivesicle bodies and swollen mitochond ria filled the plasma of endothelial cells. Conclusions A transgenic mouse model with overexpression of human SR- A in endothelial cells was successfully established. The transgene was integrated and transmitted int o the chromosome of transgenic mice. Tie- 1 promoter controlled the transgene t o express in endothelial cells in mice. Pinocytic activity of aortic endothelia l cells in transgenic mice was higher than that of C57BL/6J mice. Our studies w ill provide a new transgenic model for investigation of atherosclerosis and func tions of human SR- A.     

抗EB病毒膜抗原单克隆抗体的制备及鉴定

本文将sp_2/o细胞与经脾内注射用0.2mMH2O2激活的B95—8全细胞免疫的BALB/C小鼠脾细胞,用50％PEG4000进行融合,其平均融合率为51.79%,抗体分泌为43.48％。经亚克隆后获得一株能持续分泌抗EB病毒脱抗原的单克隆抗体(以下简称抗EBV—MA单抗)的杂交瘤细胞—B3B7,该细胞的染色体数为111.7±4.76。经ELISA法,间接免疫酶法和间接免疫荧光法证实该单抗不能与EBV—MA(一)的人Rajì细胞发生特异性免疫反应,只能与EBV—MA(+)的人P3HR—1和绒猴的B95—8细胞的反应,而且与H_2O_2激活的B95—8和P_3HX—1细胞的反应强度非常显著地高于与未经H_2O_2激活的B95—8和P_3HR—1细胞的反应强度(P<0.001),同时亦证实特异性荧光主要集中在EBV—MA(+)细胞的细胞膜上,从而表明B_3B_7确实是能分泌抗EBV—MA单抗的杂交瘤细胞。