Infection and growth of herpes simplex virus (HSV) in rabbit's corneal culture cells

With the view of making quantitative examination of the susceptibility of corneal epithelial cell and stromal cell to herpes simplex virus (HSV) infection, 6 HSV strains (4 of HSV type I, 2 of HSV type II) were inoculated in cultured rabbit's corneal cells (epithelial and stromal cells), and the following results were obtained: 1. When stock HSV was titrated with monolayer culture of each epithelial or stromal cells, strong CPE appeared within 24 hr in epithelial cell and the TCID50 titer of HSV was read as 10(7.5)-10(8.7)/ml at 120 hr. On the other hand, in stromal cell the TCID50 titer showed 10(6.4)-10(6.8)/ml at 72 hr and did not increase thereafter. 2. In terms of the growth curve for HSV type I, the eclipse period was about 4 hr for epithelial cell compared with about 4 hr for stromal cell. At 24 hr, postinfection of standard strain, about 10(7) TCID50/ml of virus was obtained from both epithelial and stromal cell culture. In the case of clinical isolate infection, however, about 10(7) TCID50 was obtained from epithelial cells and 1 in 10th lower titer of virus was obtained from stromal cells. The above results seemed to be of importance in the consideration of different onset mechanisms of epithelial type corneal herpes and stromal ones.     

以AdMax载体系统构建和制备肾癌相关抗原G250基因重组腺病毒表达载体

目的 构建肾癌相关抗原G250重组腺病毒载体,以期用于基因转染树突状细胞(DC)治疗肾癌的实验研究.方法 利用AdMax包装系统,首先构建穿梭质粒pDC316-G250,然后将所得的穿梭质粒和辅助质粒pBHGlox(delta)E1,3Cre以CaCL2法质粒共转染293细胞得到重组腺病毒Ad/G250,CsCl梯度离心纯化病毒,TCID50法测定病毒滴度.Ad/G250转染DC细胞,RT-PCR及流式细胞仪检测G250的表达.结果 采用双质粒共转染293细胞,Cre/loxP位点同源重组方法构建了E1和E3缺失的含外源基因的Ad/G250载体.经过PCR、RT-PCR和流式细胞仪证实腺病毒载体构建成功.病毒经过CsCl梯度离心纯化后,Ad/G250滴度达到5.6×109 U/ml.RT-PCR及流式细胞仪显示Ad/G250在DC中特异性表达.结论 成功构建了G250重组腺病毒载体.     

经典方法与LaSRT法测定绿色荧光蛋白标记重组病毒滴度的研究

OBJECTIVE: To compare large-scale real-time titration method (LaSRT) with standard titration method by flow cytometry (FACS) for determining the titers of green-fluorescence-protein (GFP)-marked recombinant retrovirus. METHODS: (1) Standard titration method: NIH3T3 cells were inoculated at 2x10(5) /well in 6-well plate, and after cell culture for 12 h, 0.5 ml, 50 microl and 5 microl GFP-marked recombinant retrovirus (n=3) were respectively used to infect the cells, with the final concentration of polybrene being 8 microg/ml. Forty-eight hours later, the cells were treated with trypsin and assayed for the positive rate of GFP by means of FACS. When the positive rate was lower than 10%, the titer was calculated according to the equation: virus titer (TU/ml) =2x10(5)xGFP positive rate/volume of virus stock solution used. (2) LaSRT method: The cells were inoculated at 5,000 cells/well in a 96-well plate, and after cell culture for 12 h, 90 microl/well complete culture medium was used with 8 microg/ml polybrene and 10% newborn bovine serum, 10 microl virus was added into the first well, and ten-fold dilution of the previous virus-containing solution was performed before the virus was added into the next well (8 wells in each group, altogether 3 groups). Forty-eight hours later, inverted fluorescence microscope was used to observe the fluorescence-positive cells in each well. The virus titer was calculated according to the equation: virus titer (TU/ml) =mx10(n+1), where n is the serial number of the reference well, and m the number of positive cells. (3) LaSRT was used to study the influence of freezing/thawing on the titers of recombinant retroviruses. RESULTS: The virus titer obtained with standard method by FACS was (1.54+/-0.38)x10(6) TU/ml, and that of LaSRT was (1.33+/-0.57)x10(6) TU/ml (P>0.05). After one cycle of freezing/thawing, the virus titer dropped to (18.1+/-9.9)% (n=7). CONCLUSION: LaSRT is more rapid and convenient as well as easier to determine the virus titer compared with standard method, and no significant difference is found between the two titration methods.     

古尼拟青霉诱生及促诱生干扰素的实验研究

本文报导古尼拟青霉诱生及促诱生干扰素的实验研究。以VSV为攻击病毒,人羊膜细胞FL株为测定细胞,结果显示古尼拟青霉提取液可诱导产生干扰素。一定浓度范围内,诱生能力随浓度增加而增强。诱生动态试验表明,2h即可出现较低效价干扰素,6h达最大值并持续到12h。如先用NDV-F处理FL细胞1h再加提取液,可将其诱生产量由4000IU/ml提高到8000IU/ml。     

重组腺病毒Ad-sTNFRI-IgGFc转染人气道上皮细胞及其表达的实验研究

目的 构建携带人可溶性肿瘤坏死因子受体I胞外区和IgGFc融合基因的重组腺病毒载体(Ad-sTNFRI-IgGFc),体外转染正常人气道上皮细胞株(HBE135-E6E7),探讨重组腺病毒Ad-sTNFRI-IgGFc转染气道上皮治疗支气管哮喘的可行性.方法 将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316,与辅助质粒pBHGlox△E1,3Crc共转染HEK293细胞,重组产生Ad-sTNFRI-IgGFc,PCR鉴定毒种正确后,进行扩增、纯化和滴度测定,转染培养HBE135-E6E7,流式细胞术检测转染效率,RT-PCR、免疫组化检测转染后细胞中sTNFRI-IgGFc的表达和转录.结果 成功构建了Ad-sTNFRI-IgGFc,感染性滴度达3×1010TCID50/ml,转染后HBE135-E6E7在mRNA和蛋白水平均有sTNFRI-IgGFc表达.结论 构建的Ad-sTNFRI-IgGFc可有效转染HBE135-E6E7,并在其中高效表达,并能拈抗TNF活性,为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础.     

鸡胚成纤维细胞培养抗肿瘤NDV疫苗的实验研究

目的用细胞培养法研制高效价人用抗肿瘤NDV。方法采用鸡胚成纤维细胞低血清培养法,在接种NDV之前的细胞培养中,少量使用优质小牛血清（2%）,让单层细胞基本铺满培养瓶底时,用无血清培养液多次洗涤单层细胞,以尽量降低残余牛血清含量。接种NDV之后,以人白蛋白代替小牛血清继续滋养细胞,培养病毒。结果NDV收获液LogPFU≥5.5,血凝效价1：640,经反向间接血凝试验检测,残余牛血清蛋白含量≤50ng/ml。结论NDV效价和活性符合人用疫苗要求。     

Tween 80: a marker for differentiation of hog cholera and bovine viral diarrhea viruses.

Markers for differentiating hog cholera and bovine viral diarrhea viruses were studied using Tween 80, chloroform, trichlorotrifluoroethane and tri (n-butyl) phosphate. Attenuated A and virulent Ames strains of hog cholera virus were employed. Moreover, the NADL PK-15 cell culture adopted strain and low cell culture passaged Purdue strain of bovine viral diarrhea virus were used. These viruses were reacted with 2,500 micrograms/ml of Tween 80 for one hour at 37 degrees C. When attenuated A and virulent Ames strains of hog cholera virus with titers greater than 10(6) and 10(5) plaque forming units respectively, were reacted with Tween 80 the titer of each strains was reduced by approximately 10(4) plaque forming units of virus. When either strain of bovine viral diarrhea virus was reacted with Tween 80, virus was not detected.     

重组腺病毒临床级基因治疗制品的质量控制

目的　建立并完善我国重组腺病毒临床级基因治疗制品重组人 p5 3腺病毒注射液的质量控制标准和方法。方法　采用组织培养半数感染剂量 (TCID50 )方法测定病毒的感染滴度 ;采用HPLC进行病毒的纯度分析 ;采用A5 4 9细胞进行样品的复制型病毒检测 ,其他质量控制方法按照中国生物制品规程进行。结果　重组人p5 3腺病毒注射液 (Lot 2 0 0 10 70 1)成品的病毒颗粒数为 1 0 3×10 12 VP/ml,病毒滴度为 5 0 1× 10 10 IU/ml;比活性为 4 86 % ,纯度达到 98 6 2 % ,复制型腺病毒(RCA)水平小于 1RCA/ 3× 10 10 VP ,符合中国食品药品监督管理局 (SFDA)的标准。结论　建立了重组腺病毒临床级基因治疗制品的全套质量标准 ,达到SFDA的质量要求 ,可有效地控制制品的质量 ,保证重组腺病毒制品在临床应用中安全有效。     

用浓缩地鼠肾细胞狂犬病毒抗原免疫马匹获得高效价血浆

目的制备高效价马抗狂犬病免疫血浆。方法通过细胞制备、病毒的接种、培养、收获、浓缩等步骤，研制了浓缩地鼠。肾细胞狂犬病毒抗原，并与羊脑狂犬病毒抗原作马匹免疫对比试验。结果浓缩地鼠。肾细胞抗狂犬病免疫血浆的效价明显高于羊脑抗狂犬病免疫血浆。试验组血浆平均效价为157IU／ml，对照组为113IU／ml，两组差异有统计学意义。结论已成功制备高效价抗狂犬病免疫血浆。     

基因工程IFNα_(2a)对肿瘤细胞生长的抑制作用

采用α－干扰素抑制各种肺癌细胞生长的MTT法。结果：不同品牌的10000,1000,100IU／ml的IFNα2a对人脑胶质瘤细胞（SHG—44）、人骨肉瘤细胞（HOS－8603）、人白血病细胞（HL－60）、红白血病细胞（K562）和淋巴瘤细胞（Raji）的生长抑制率分别为20％,10％,5％左右。结论：进口和国产的不同品牌IFNα2a。对上述5种肿瘤细胞株均有较明显的生长抑制效应,且抑制率相近。     

RSV感染对人支气管上皮细胞分泌IL-8和Eotaxin-1的影响

目的探讨呼吸道合胞病毒（RSV）感染对人支气管上皮细胞（NHBE）分泌白介素（IL）-8、嗜酸细胞趋化因子Eotaxin-1的影响。方法用Hep-2细胞扩增病毒,并进行毒力鉴定。建立并鉴定RSV感染NHBE的体外细胞模型。实验分RSV感染组和正常细胞对照组,感染组以不同滴度分为1000、500、100、50 TCID504个组,酶联免疫吸附（ELISA）法检测12、24、48和72h各组IL-8、Eotaxin-1水平。结果实时荧光定量RT-PCR法鉴定细胞模型建立成功。相同感染时间内（12、24、48和72h）,随着感染滴度增加（50、100、500、1000 TCID50）,各感染组IL-8分泌水平均呈上升趋势,与正常细胞对照组比较差异均有统计学意义（P均〈0.05）。相同感染滴度内（50、100、500、1000 TCID50）,随着感染时间增加（24、48和72h）,各感染组IL-8分泌水平均呈上升趋势,与12h组比较均有统计学差异（P均〈0.05）;相同感染时间内（12、24、48和72h）,随着感染滴度增加（50、100、500、1000 TCID50）,各感染组Eotaxin-1分泌水平较正常细胞对照组差异无统计学意义。50、100、500 TCID50相同感染滴度内,随着感染时间增加（24、48和72h）,各感染组Eotaxin-1分泌水平较12h组差异无统计学意义。结论 RSV感染人支气管上皮细胞可显著上调IL-8分泌水平,进一步引起以中性粒细胞浸润为主的气道炎症,而早期的RSV感染对Eotaxin-1影响不大。     

高糖负荷下人蛋白激酶Cβ_2基因过表达内皮细胞模型的构建及鉴定

目的 应用AdMax系统构建含人蛋白激酶Cβ_2(protein kinase Cβ_2,PKCβ_2)基因的重组腺病毒载体,转染人脐静脉内皮细胞(HUVEC),高糖刺激下观察蛋白量的表达,构建进一步功能研究的细胞模型.方法 从pMD18-T-PRKCB1质粒中,扩增出人PRKCB1.所得片段定向克隆至穿梭质粒pDC315上,后与骨架质粒pBHGlox△E1,3Cre共转染293细胞,经同源重组构建腺病毒Ad-PRKCB1,TCID50测定重组病毒的滴度,免疫细胞化学和RT-PCR方法进行鉴定.构建成功后转染HUVEC,激光共聚焦显微镜(LSCM)下,观察高糖刺激下荧光蛋白转位及表达量,鉴定细胞模型构建.结果 通过酶切鉴定目的 片段正确插入穿梭质粒.倒置显微镜下观察到细胞的病变效应.测得病毒滴度为7.9×10~9IU/ml,免疫细胞化学和RT-PCR验证了目的 蛋白表达.LSCM下观察到高糖负荷下荧光蛋白转位及过表达.结论 成功构建了含PRKCB1基因的重组腺病毒载体并在高葡萄糖负荷下观察到其活化及蛋白过表达,为下一步信号蛋白组学研究提供分子工具.     

特异性溶瘤重组腺病毒KH901的体外特异性抗肿瘤作用研究

目的 研究特异性溶瘤重组腺病毒KH901在体外培养的人肿瘤细胞中的选择性复制和杀伤作用.方法 以感染复数(MOI)=2 PPC的KH901和野生型腺病毒(Ad5)感染人肿瘤和正常细胞,72 h后采用病毒滴定法测定病毒滴度;以MOI＝10 PPC和1 PPC的KH901感染人肿瘤和正常细胞,24 h后通过ELISA法和TF-1依赖细胞株/MTT比色法测定粒细胞-巨噬细胞集落刺激因子(GM-CSF)的免疫活性和生物活性;以MOI分别为0、0.1、1、10、100、1000 PPC的KH901和野生型Ad5感染人肿瘤和正常细胞,采用MTT法测定各细胞活力,计算半数药物有效浓度(EC50).结果 野生型Ad5在正常细胞和肿瘤细胞中均大量复制,均表现出明显的杀伤作用;KH901在肿瘤细胞中大量复制[(2526.4±136.8)～(2796.6±104.6) TCID50/cell],表达大量的人GM-CSF[(1177.793±6.62)～(3924.497±17.79) IU/(106细胞·24 h)],并显示出明显的杀伤作用[EC50为(0.31±0.06)～(0.19±0.01) pfu/cell],在正常细胞中KH901复制较差[(56.8±9.2)～(90.1±14.4) TCID50/cell],只有少量的GM-CSF产生[(13.397±0.82) IU/(106细胞·24 h)],杀伤作用轻微[EC50为(92.33±9.12)～(121.20±19.94) pfu/cell],两者间差异有统计学意义(P＜0.05).结论 KH901具有肿瘤选择性复制和杀伤作用,并能表达具有生物活性的GM-CSF,显示出治疗实质性肿瘤的潜力.     

Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus

目的：研究乙脑病毒变异的特性以及变异株性状与E蛋白表达的关系。方法：通过应用乙脑病毒的野生株以及人肝癌细胞KN73建立持续感染模型。采用胰蛋白酶消化技术每周进行细胞传代。经反复冻融收集持续感染细胞内病毒。采用BHK细胞空斑实验方法进行病理滴定。采用间接免疫荧光方法检测E和NS3蛋白抗原。采用Western印迹杂交检测E和NS3蛋白的表达。结果：感染早期（24小时-36小时）野生株感染的KN73细胞的培养液中病毒滴度为10^6PFU/ml。在感染后期（3年）滴度为10^3-4PFU/ml。病毒重叠感染实验发现在急性重叠感染野生株的持续感染KN73细胞中培养液的病毒滴度比在同一时相急性感染的正常KN73细胞培养液的滴度要低得多。间接免疫荧光检测表明,在持续感染的KN73细胞中病毒抗原的存在低于急性感染的KN73细胞。Western印迹杂交检测表明,E、NS3蛋白的分子量分别为53KD和73KD。在持续感染的KN73细胞中NS3蛋白的表达很稳定,但E蛋白的表达却明显受抑。结论：从持续感染的KN73细胞中获得的病毒具有缺陷干扰病毒的一些特性且参与持续感染,其毒力和增殖均低于亲本病毒。这些变异可能与E蛋白表达减少有关。     

慢性乙型肝炎患者病毒载量短期自发波动分析

目的通过监测未抗病毒治疗的处于免疫清除期的慢性乙型肝炎患者的HBV DNA载量,评价其HBV DNA短期自发波动情况及影响其波动的相关因素。方法入选123例ALT>2×ULN的未抗病毒治疗的HBeAg阳性慢性乙型肝炎患者,收集首次就诊的HBV DNA定量(罗氏COBAS)、HBsAg定量、ALT、AST结果,第二次就诊(间隔4周以内)的HBV DNA定量结果;对于首次就诊4周内行肝脏穿刺的受试者评价其肝组织学结果(Knodell坏死炎症评分和Ishak组织纤维化评分)。结果 93例(75.6%)受试者HBV DNA波动≤0.5 Log IU/ml,30例(24.4%)受试者HBV DNA波动>0.5 Log IU/ml。应用二元Logistic多因素回归分析提示,HBV DNA的波动程度与Knodell坏死炎症评分及HBV DNA载量相关。Knodell坏死炎症评分≥10的患者其发生HBV DNA显著波动的概率显著高于Knodell坏死炎症评分<10的患者(50.0%vs 18.3%,P=0.042);HBV DNA载量<7 LogIU/ml的患者,其发生HBV DNA显著波动的概率显著高于HBV DNA载量≥7 Log IU/ml的患者(42.9%vs 20.6%,P=0.030)。结论慢性乙型肝炎患者在免疫清除期,其HBV DNA在短期内存在波动,其中约25%的受试者的HBV DNA波动在0.5 LogIU/ml以上;HBV DNA波动的程度与肝脏炎症程度以及HBV DNA载量相关。     

不同滴度噬菌体D29对气道上皮细胞9HTE的生长及功能影响研究

目的 研究噬菌体D29不同滴度对气道上皮细胞株9HTE的生长及功能影响.方法 分别用高滴度(109 PFU/mL)和低滴度(107 PFU/mL)噬菌体D29及噬菌体缓冲液组作用于9HTE细胞株后,用四甲基偶氮唑蓝比色法(MTT)检测细胞生长情况;用酶联免疫吸附试验(ELISA)法测量细胞上清液白细胞介素(IL)-6、IL-8水平;用逆转录PCR(RT-PCR)检测细胞间黏附分子-1(ICAM-1)mRNA的表达,用流式细胞技术检测细胞凋亡率.结果 高、低滴度噬菌体D29作用气道上皮细胞一段时间后,对细胞生长活性、细胞因子IL-6、IL-8和ICAM-1 mRNA的表达,以及细胞凋亡率的影响与噬菌体缓冲液组比较差异无统计学意义(P>0.05).结论 高、低滴度噬菌体D29对体外培养的气道上皮细胞生长及功能无明显影响.     

纤维素与明胶微载体微重力培养肝细胞的比较

目的 探讨商品化的多孔微载体:cytopore 2和cultispher S何种更适合于微重力条件下培养CL-1细胞.方法 采用RCCS旋转培养系统.在50 ml水平微重力旋转培养CL-1细胞.分别对接种至cytopore 2和cultispher S的细胞进行动态形态学观察、细胞计数和功能学检测.结果 CL-1细胞在两种载体上均可粘附生长,并于第9天达到生长高峰.细胞计数结果提示cultispher S的细胞密度可达到4×10 6个/ml.在第10、11、12天,cytopore 2组的上清中自蛋白浓度明显高于cultispher S组.有显著差异(P<0.05),在第10、11天,cytopore 2组的上清中尿素浓度明显高于cultispher S组,有显著差异(P<0.05).结论 Cytopore 2培养的CL-1细胞计数低于Cultispher S组,但功能好于Cultispher S组.     

Experimental oral and nasal transmission of rabies virus in mice.

Weanling female white Swiss mice were exposed to challenge virus standard rabies virus and street virus isolates from various domestic and wild animals. Virus was given free choice as suspension or as infected mouse brain by stomach tube, by single injection of suspension into the oral cavity of unanesthetized mice, by repeated injection into the oral cavity of anesthetized mice and by single application to the external nares of anesthetized mice. Challenge virus standard virus in mouse brain suspension and a suspension of skunk salivary glands infected with street virus (titers greater than or equal to 10(6)MICLD50/0.03 ml) consistently produced high rates of infection in mice exposed intranasally, low to high rates of infection in mice exposed by forced feeding and other artificial methods of oral exposure and very low rates of infection when given free choice. Street virus isolates passaged intracerebrally in mice had titers less than or equal to 10(4.5) MICLD50/0.03 ml and rarely caused rabies in mice exposed orally or nasally by any method. The results indicate that with the isolates used, virus of high titer (greater than or equal to 10(6)MICLD50/0.03 ml) is required to consistently produce infection in mice by the nasal route and that the mucosa of the nasal cavity probably is the chief route of infection even after oral administration.     

Isolation of a strain of Hantaan virus from peritoneal exudate cells of a patient with hemorrhagic fever with renal syndrome.

A new strain of Hantaan virus (HTNV), GH716, was isolated from the peritoneal exudate cells (PEC) of a patient with severe hemorrhagic fever with renal syndrome (HFRS). The isolate was propagated in Vero E6 cells. At each passage the virus-infected cells were examined for HTNV with immunofluorescence technique using monoclonal antibodies (McAb) 25-1 and 84-1 against HTNV. From passage 5 on, fluorescent intensity tended to stabilize at (+++) and infectivity titer reached 10(5) TCID50/ml. Using 14 McAb to 76-118 and BI strains of HTNV, we found that strain GH716 was antigenically similar to strain 76-118 (Apodemus type) and different from strain R22 (Rattus type), suggesting that GH716 may fall into the Apodemus type of HTNV. This is the first isolation of an HTNV from human PEC collected on the tenth day of illness. The successful isolation of strain GH716 may provide an alternative source of obtaining HTNV during the later stages of HFRS.

     

霍乱毒素B亚单位基因的克隆和表达

用DNA重组技术构建CT-B表达性质粒pXB1及进一步修饰的pXB2,使CT-B基因在大肠杆菌中得到较高水平的表达(280～350ng/ml),且有71.5％分泌率。表达动力学研究阐明,克隆子培养24h产物收率较高。一定浓度的Mg~(2+)、L-组氨酸和天冬酰胺能刺激CT-B的表达。GM1-ELISA、CHO细胞测毒结合间接免疫荧光检测和家兔肠段结扎试验证实,表达的CT-B能与游离的或活细胞膜上的GM1受体结合,但无细胞和肠段毒性。这种细胞测毒结合免疫荧光序贯试验,为客观证实CT-B的生物学活性开拓了新途径。