顺铂联合重组人5型腺病毒腹腔灌注治疗胃癌合并恶性腹腔积液的临床疗效

目的探讨顺铂联合重组人5型腺病毒(H101)腹腔灌注治疗胃癌合并恶性腹腔积液的临床疗效。方法采用随机数字表法将68例胃癌合并恶性腹腔积液患者分为顺铂组(n=34,顺铂腹腔灌注)和顺铂联合H101组(n=34,顺铂联合H101腹腔灌注)。比较两组患者的腹腔积液缓解情况、腹腔积液间隔时间、生存情况、生活质量及不良反应发生情况。结果顺铂联合H101组患者腹腔积液总缓解率为73.5%,高于顺铂组患者的50.0%,差异有统计学意义(P﹤0.05)。顺铂联合H101组患者腹腔积液间隔时间为(34.02±9.79)天,明显长于顺铂组患者的(27.56±9.36)天,差异有统计学意义(P﹤0.01)。顺铂联合H101组患者的中位总生存期长于顺铂组(P﹤0.05)。两组患者的不良反应总发生率比较,差异无统计学意义(P﹥0.05)。结论顺铂联合H101腹腔灌注治疗胃癌合并恶性腹腔积液患者,能够安全有效地控制腹腔积液生成,改善患者的生活质量,延长患者的生存期。

表达SFTSV Gn基因的重组5型腺病毒的构建与鉴定

为了构建出表达新布尼亚病毒(SFTSV)Gn基因的重组5型腺病毒,试验根据GenBank上发表的SFTSV Gn基因序列(登录号为ADZ04482.1)设计合成引物,采用特异性PCR方法在Gn基因前添加了KOZAK序列和tPA信号肽序列;然后通过酶切、连接等方法构建重组穿梭质粒PGA-KOZAK-tPA-Gn,与腺病毒骨架质粒pAd5-ΔE1ΔE3-5E4在BJ5183感受态细胞中进行同源重组,得到重组腺病毒质粒rAd5-KOZAK-tPA-Gn,用PacⅠ酶酶切重组腺病毒质粒,并将线性化的质粒转染至HEK 293细胞中进行病毒包装、扩繁和纯化;采用PCR扩增、Western-blot等技术检测病毒基因的表达情况,并测定重组腺病毒效价。结果表明:通过PCR扩增获得了1 546 bp的KOZAK-tPA-Gn基因,并成功构建了重组穿梭质粒;SFTSV Gn基因在重组腺病毒传代过程中稳定存在;重组腺病毒在HEK 293细胞中表达出分子量约为61 ku的SFTSV Gn蛋白;测得重组腺病毒效价为1×10^(-7.63)/0.1 mL TCID_(50)。说明试验成功构建出表达SFTSV Gn基因的重组5型腺病毒。

Transarterial chemoembolization combined with recombinant human adenovirus type 5 H101 prolongs overall survival of patients with intermediate to advanced hepatocellular carcinoma: a prognostic nomogram study

Background: Patients with intermediate to advanced hepatocellular carcinoma(HCC) are most commonly treated with transarterial chemoembolization(TACE). Previous studies showed that TACE combined with recombinant human adenovirus type 5(H101) may provide a clinical survival benefit. In the present study, we aimed to determine the survival benefit of TACE with or without H101 for patients with intermediate to advanced HCC and to develop an e ective nomogram for predicting individual survival outcomes of these patients.Methods: We retrospectively collected data from 590 patients with intermediate to advanced HCC who were treated at Sun Yat?sen University Cancer Center between January 2007 and July 2015. After propensity score matching, 238 patients who received TACE with H101(TACE with H101 group) and 238 patients who received TACE without H101(TACE group) were analyzed. Overall survival(OS) was evaluated using the Kaplan–Meier method; the nomogram was developed based on Cox regression analysis. Discrimination and calibration were measured using the concordance index(c?index) and calibration plots.Results: Clinical and radiologic features were similar between the two groups. OS rates were significantly lower in the TACE group than in the TACE with H101 group(1?year OS rate, 53.8% vs. 61.3%; 2?year OS rate, 33.4% vs. 44.2%; 3?year OS rate, 22.4% vs. 40.5%; all P < 0.05). Multivariate Cox regression analysis for the entire cohort showed that alpha?fetoprotein level, alkaline phosphatase level, tumor size, metastasis, vascular invasion, and TACE with or without H101 were independent factors for OS, all of which were included in the nomogram. Calibration curves showed good agreement between nomogram?predicted survival and observed survival. The c?index of the nomogram for predict?ing OS was 0.716(95% confidence interval 0.686–0.746).Conclusions: TACE plus H101 extends the survival of patients with intermediate to advanced HCC. Our proposed nomogram provides individual survival prediction and stratification for patients with intermediate to advanced HCC who receive TACE with or without H101.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

Recombinant adenovirus-p53(Gendicine) sensitizes a pancreatic carcinoma cell line to radiation

Objective： In this study, we examine the effects of recombinant adenovirus-p53 （rAd-p53） on the pancreatic carcinoma cell line SW1990. Specifically, we determine if expression of rAd-p53 sensitizes these cells to radiation. Methods： Following transfection of SW1990 cells with rAd-p53, we measured expression of P53, P21 and Bax by immunocytochemistry. Both transfected and control cell lines were irradiated with a range of doses, and the survival fractions （SF） were calculated. Dose survival cttrves were constructed and modeled for comparison. Results： Transfection of SW1990 cells with rAd-p53 resulted in increased expression of P53, P21 and Bax in a time-dependent manner. At 96 h after transfection, 89.92% of cells expressed P53, 56.8% expressed P21, and 76.50% expressed Bax. The SF following radiation was lower in the rAd-p53 transfected cells compared to the control cells, suggesting that rAd-p53 sensitizes SW1990 cells to radiation （Do for the experimental and control groups was 2.199 and 2.462, respectively）. Conclusions： Use of the adenoviral vector is an effective means of transfecting SW1990 cells with wild-type P53, and this sensitizes the cell line to irradiation. This work suggests that combining rAd-p53 with radiation therapy in pancreatic cancer may be therapeutically beneficial.

Adenovirus-mediated Transfer of p53 and p16Inhibiting Proliferating Activity of Human Bladder Cancer Cell EJin vitro and in vivo

To evaluate the effects of adenovirus (Ad) - mediated transfer of p5 3and p16 on hum an bladder cancer cells EJ,EJwere transfected with Ad- p5 3and Ad- p16 . Cell growth,m orphologi- cal change,cell cycle,apoptosis were measured using MTT assay,flow cytom etry,cloning form a- tion,im munocytochemical assays.Ad- p16 or Ad- p5 3alone could inhibit the proliferating activity of EJcells in vitro.Ad- p5 3could induce apoptosis of partial EJcells.G1arrest was observed72 h after infection with Ad- p16 ,but apoptosis was not obvious.The transfer of Ad- p16 and Ad- p5 3 could significantly inhibit the growth of EJcells,decrease the cloning formation rate and induce apoptosis of large num ber of EJcells. The occurrence time of subcutaneous tumor was delayed and the tum or volume in 4 weeks was dim inished by using Ad- p5 3com bined with Ad- p16 and the dif- ference was significant com pared with using Ad- p5 3or Ad- p16 alone.It was suggested that the transfer of wild- type p5 3and p16 could significantly inhibit the growth of human bladder cancer in vitro and in vivo.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

血清TGR5 mRNA和BNIP3 mRNA在急性心肌梗死患者中的表达水平及临床意义

目的 探究血清G蛋白偶联胆汁酸受体5(TGR5)mRNA与Bcl-2/腺病毒QE1B-19kDa相互作用蛋白3(BNIP3)mRNA在急性心肌梗死(AMI)患者中的表达水平情况,以及二者对于术后心脏不良事件(MACE)发生的预测价值。方法 选取首都医科大学门头沟教学医院2018年1月至2020年1月收治的98例进行经皮冠状动脉介入术(PCI)的AMI患者[AMI患者包括急性非ST段抬高型心肌梗死(NSTEMI)46例与急性ST段抬高型心肌梗死(STEMI)52例]为研究组,另选取同期90例体检健康者作为对照组。采用实时荧光定量PCR(qRT-PCR)检测血清TGR5 mRNA和BNIP3 mRNA表达水平,根据PCI术后随访结果将研究组分为MACE组(46例)和非MACE组(52例)。收集两组患者临床资料,采用Pearson法分析术后MACE组血清TGR5 mRNA与BNIP3 mRNA表达水平的相关性,采用受试者工作特征(ROC)曲线分析血清TGR5 mRNA和BNIP3 mRNA对于术后AMI患者MACE发生的预测价值,采用Logistic回归分析AMI患者术后MACE发生的影响因素。结果 研究组血清TGR5 mRNA表达水平显著低于对照组,血清BNIP3 mRNA表达水平显著高于对照组,差异有统计学意义(P<0.05);术后MACE组左心室射血分数(LVEF)、TGR5 mRNA表达水平显著低于非MACE组,血清肌酐(SCr)、红细胞分布宽度(RDW)、Killip分级Ⅲ+Ⅳ级比例、BNIP3 mRNA表达水平显著高于非MACE组,差异有统计学意(P<0.05);经Pearson相关性分析,血清TGR5 mRNA与BNIP3 mRNA表达水平呈负相关(r=-0.543,P<0.05);ROC曲线分析显示,血清TGR5 mRNA预测AMI患者MACE发生的曲线下面积(AUC)为0.704(95%CI:0.601~0.808),血清BNIP3 mRNA预测AMI患者MACE发生的AUC为0.762(95%CI:0.696~0.883),血清TGR5 mRNA与BNIP3 mRNA联合预测AMI患者术后MACE发生的AUC为0.867(95%CI:0.783~0.932),优于二者单独预测(Z_(二者联合-TGR5)=2.346,Z二者联合-BNIP3=1.715,P=0.019、0.043);Logistic回归分析结果显示,TGR5 mRNA、BNIP3 mRNA、RDW是AMI患者术后MACE发生的影响因素(P<0.05)。结论 TGR5 mRNA在AMI患者血清中呈低表达水平,BNIP3 mRNA在AMI患者血清中呈高表达表达,二者对于预测术后MACE发生具有重要意义。

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo

AIM To investigate the anticancer effect of a recombinant adenovirus-mediated p53(r Ad-p53) combined with 5-fluorouracil(5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of r Ad-p53 in reversal of 5-FU resistance.METHODS nude mice bearing human colon cancer SW480/5-FU(5-FU resistant) were randomly assigned to four groups(n = 25 each): control group, 5-FU group, r Ad-p53 group, and r Ad-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C(PKC), permeability-glycoprotein(P-gp) and multidrug resistance-associated protein 1(MRP1)(Western blot) and apoptosis(TUNEL) were determined.RESULTS The area ratios of tumor cell apoptosis were larger in the r Ad/p53 + 5-FU group than that in the control, 5-FU and r Ad/p53 groups(P < 0.05), and were larger in the r Ad/p53 group than that of the control group(P < 0.05) and the 5-FU group at more than 48 h(P < 0.05). The p53 expression was higher in the r Ad/p53 and the r Ad/p53 + 5-FU groups than that of the control and 5-FU groups(P < 0.05), and were higher in the r Ad/p53 + 5-FU group than that of the r Ad/p53 group(P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the r Ad/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups(P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and r Ad/p53 groups at more than 48 h(P < 0.05). In the r Ad/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h(P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h(P < 0.05).CONCLUSION5-FU combined with r Ad-p53 has a synergistic anticancer effect in SW480/5-FU(5-FU resistance), which contributes to reversal of 5-FU resistance.

AB_5-type Hydrogen Storage Alloy Modified with Ti/Zr Used as Anodic Materials in Borohydride Fuel Cell

Fuel cell using borohydride as the fuel has received much attention. AB5-type hydrogen storage alloy used as the anodic material instead of noble metals has been investigated. In order to restrain the generation of hydrogen and enhance the utilization of borohydride, Ti/Zr metal powders has been added into the parent LmNi4.78Mn0.22 （where Lm is La-richened mischmetal） alloy （LNM） by ball milling and heat treatment methods. It is found that the addition of Ti/Zr metal powders lowers the electrochemical catalytic activity of the electrodes, at the same time, restrains the generation of hydrogen and enhances the utilization of the fuel. All the results show that the hydrogen generation rate or the utilization of the fuel is directly relative to the electrochemical catalytic activity or the discharge capability of the electrodes. The utilization of the fuel increases with discharge current density. It is very important to find a balance between the discharge capability and the utilization of the fuel.

表达呼吸道合胞病毒融合前F蛋白的重组人5型腺病毒的构建

目的筛选并构建成功表达A亚型人呼吸道合胞病毒(Human Respiratory Syncytial Virus,HRSV)融合前构象F蛋白的重组人5型腺病毒(Recombinant Human type5 adenovirus,rHADV-5),为制备和研发HRSV重组腺病毒载体疫苗奠定基础。方法在真核表达载体pcDNA3.1(+)上插入具有不同突变位点的HRSV F蛋白编码序列,利用融合前F蛋白单克隆抗体AM14和D25筛选HRSV F蛋白突变体;通过同源重组将筛选出的前构象F蛋白突变体编码序列插入非复制型人5型腺病毒载体,在HEK293细胞中进行重组病毒的包装和扩增,经氯化铯密度梯度法获得纯化的重组病毒;应用夹心ELISA法和Western blot鉴定F蛋白的表达及构象。结果通过AM14和D25单克隆抗体筛选出了2个稳定维持在融和前构象的HRSV F蛋白序列(SC-3M和SC-5M)。将SC-3M和SC-5M编码序列插入非复制型人5型腺病毒载体,对两种重组人5型腺病毒(pAd5-SC-3M和pAd5-SC-5M)和空载体(pAd5-vector)纯化,病毒滴度分别达到2.016×10^(10),2.52×10^(10)和2.948×10^(11)IU/mL。pAd5-SC-3M和pAd5-SC-5M感染HEK293后均能高效表达HRSV融合前构象的F蛋白并维持三聚体结构,且pAd5-SC-5M感染细胞中F蛋白表达量高于pAd5-SC-3M。结论成功在体外传代细胞中获得可高效表达A型HRSV融合前F蛋白的重组人5型腺病毒,为进一步通过动物试验评价该HRSV重组腺病毒载体疫苗奠定了基础。

The Role of NK Cell in T Cell Recruitment in Murine Liver Infected with Adenovirus

To study the role of natural killer (NK) cells in T cell recruitment in murine liver infected with virus, mice were intravenously injected daily with anti-NK1.1 + antibody to deplete NK cells. Lymphocytes in the liver tissue of mice infected with type 5 adenovirus depleted in the E1 and E3 regions were assessed by fluorometric activated cell sorting (FACS). Expression of chemokine IP-10 and its receptor CXCR3 mRNA in the liver, hepatic lymphocytes and spleen tissue were examined by reverse transcription polymerase chain reaction (RT-PCR). Serum alanine aminotransferase (ALT) was measured as an indicator of liver injury. It was found that infection of adenovirus and anti-Fas monoclonal antibody (mAb) into mice caused liver injury and high expression of interferon-γ inducible protein-10 (IP-10) mRNA in the liver. Anti-NK1.1 + mAb, which was intraperitoneally injected into the mice infected with adenovirus, suppresses T cell recruitment and expression of IP-10 mRNA in the liver. Slighter liver injury was also observed. After virus infection, expression of CXCR3 mRNA in spleen and liver tissue was observed at different time. The results suggested that T cell recruitment was initiated by NK cell dependent chemokine IP-10, which induced activated T cells priming in the spleen to the liver of the mouse. NK cells played a key role in T cell recruitment in the liver of mouse infected with adenovirus.

Microstructure and electrochemical characteristics of nonstoichiometric La-rich AB_5-type alloy for Ni/MH battery electrode

The nonstoichiometric La-rich mischmetal (designated by MI)-based hydrogen storage alloy with a composition of MI(Ni0.64Co0.2Mn0.12Al0.04)(4.76) was prepared by arc melting and annealed at 1173 K for 10 h to investigate the effect of annealing treatment on the microstructure and electrochemical characteristics of the alloy. X-ray diffraction analysis showed that annealing can cause a release of the crystal lattice strain and an increase in amounts of the La2Ni7-type second phase in MI(Ni0.64Co0.20Mn0.12Al0.04)(4.76) alloy. Scanning electron microscopy and electron probe microanalysis examinations indicated that annealing leads to disappearance of the dendrite structure in the as-cast alloy, growth of crystal grain, and decrease of composition segregation. The annealing at 1173 K for 10 h flattened and extended the potential plateau and increased the maximum discharge capacity to 328 mA center dot h/g from 310 mA center dot h/g and the cycling life. The mechanism of the improvement in electrochemical characteristics was discussed based on the alloy microstructure change induced by annealing.

Lung-Targeted Transgene Expression of Nanocomplexed Ad5 Enhances Immune Response in the Presence of Preexisting Immunity

Recombinant adenovirus serotype 5(Ad5)vector has been widely applied in vaccine development targeting infectious diseases,such as Ebola virus disease and coronavirus disease 2019(COVID-19).However,the high prevalence of preexisting anti-vector immunity compromises the immunogenicity of Ad5-based vaccines.Thus,there is a substantial unmet need to minimize preexisting immunity while improving the insert-induced immunity of Ad5 vectors.Herein,we address this need by utilizing biocompatible nanoparticles to modulate Ad5–host interactions.We show that positively charged human serum albumin nanoparticles((+)HSAnp),which are capable of forming a complex with Ad5,significantly increase the transgene expression of Ad5 in both coxsackievirus–adenovirus receptor-positive and-negative cells.Furthermore,in charge-and dose-dependent manners,Ad5/(+)HSAnp complexes achieve robust(up to227-fold higher)and long-term(up to 60 days)transgene expression in the lungs of mice following intranasal instillation.Importantly,in the presence of preexisting anti-Ad5 immunity,complexed Ad5-based Ebola and COVID-19 vaccines significantly enhance antigen-specific humoral response and mucosal immunity.These findings suggest that viral aggregation and charge modification could be leveraged to engineer enhanced viral vectors for vaccines and gene therapies.

Phase structure and electrochemical properties of La_(0.7)Ce_(0.3)Ni_(3.75)Mn_(0.35)Al_(0.15)Cu_(0.75-x)Fe_x hydrogen storage alloys

La0.7Ce0.3Ni3.75Mn0.35Al0.15Cu0.75-xFex （x=0-0.20） hydrogen storage alloys were synthesized by induction melting and subsequent annealing treatment, and phase structure and electrochemical characteristics were investigated. All alloys consist of a single LaNi5 phase with CaCu5 structure, and the lattice constant a and the cell volume （V） of the LaNi5 phase increase with increasing x value. The maximum discharge capacity gradually decreases from 319.0 mA？h/g （x=0） to 291.9 mA？h/g （x=0.20） with the increase in x value. The high-rate dischargeability at the discharge current density of 1200 mA/g decreases monotonically from 53.1% （x=0） to 44.2% （x=0.20）. The cycling stability increases with increasing x from 0 to 0.20, which is mainly ascribed to the improvement of the pulverization resistance.

腺病毒介导GRIM19对恶性胶质瘤CHG-5细胞增殖及分化的影响

目的观察增强外源性GRIM19(genes associated with retinoid-IFN-induced mortality 19)基因表达对恶性胶质瘤CHG-5细胞生物学特性的影响,初步探讨其机制。方法利用已成功构建的Ad-GRIM19腺病毒感染CHG-5细胞,通过形态学、细胞生长曲线、软琼脂克隆形成等观察增强GRIM19基因表达后CHG-5细胞形态、增殖和体外成瘤能力的变化,台盼蓝染色法检测细胞活力,Real-timePCR及Western blot检测STAT3与PCNA基因表达。结果Ad-GRIM19在体外感染CHG-5细胞后,明显改变CHG-5细胞生长形态,细胞增殖及体外成瘤减低,STAT3、PCNA基因表达下调。结论GRIM19过表达可显著抑制CHG-5细胞恶性表型,提示GRIM19基因在调控胶质瘤细胞分化程度及恶性生物学行为方面具有重要作用。

化学发光法中和抗体检测技术对广州地区人5型腺病毒的流行病学研究

目的建立高通量、可定量中和抗体检测方法,并且调查广州健康成人及儿童人群中人5型腺病毒中和抗体存在情况。方法采用以带有分泌型碱性磷酸酶报告基因的5型重组腺病毒使用化学发光法,调查116份成人血清和94份儿童血清中5型腺病毒中和抗体情况。结果该方法是一种适合高通量调查的血清学调查方法,广州地区成人中人5型腺病毒中和抗体阳性比例占26.72%,儿童中阳性比例为17.02%。结论5型腺病毒在广州人群中具有一定的免疫程度,结合中和实验结果来应用该种病毒载体并同时开发其他血清型的腺病毒载体很有必要。

细胞死亡调控基因GRIM19重组腺病毒的构建及在恶性胶质瘤CHG-5细胞中的表达

目的构建细胞死亡调控基因GRIM19(genes associated with retinoid-IFN-induced mortality 19)重组腺病毒载体,观察GRIM19对病毒包装细胞是否具有细胞毒作用,并验证其对脑胶质瘤CHG-5细胞的感染效率。方法采用PCR方法分别将GRIM19以及EGFP基因亚克隆至穿梭质粒pAdTrack-CMV,在BJ5183(pAdeasy)菌内同源重组,筛选阳性克隆,酶切测序鉴定,线性化后转染293T细胞进行包装、扩增、纯化,PCR检测病毒上清,TCID50法测定病毒滴度并感染CHG-5细胞后Western免疫印迹法验证GRIM19蛋白表达。结果连接重组后经酶切和测序法筛选出pAd-GRIM19;转染293T包装细胞,观察到绿色荧光蛋白(GFP)明显表达,细胞生长状态良好,未见明显细胞毒作用。Ad-GRIM19及Ad-EGFP初纯病毒经氯化铯梯度离心纯化最终获得约5×1010U/ml与8×1010U/ml滴度的重组病毒;将其体外感染胶质瘤CHG-5细胞,均能达到90%左右的感染率,Western免疫印迹法表明Ad-GRIM19感染组GRIM19蛋白表达明显升高。结论成功构建了携带GRIM19基因的重组腺病毒,为体内外进一步研究GRIM19对脑胶质瘤的作用奠定了基础。

重组腺病毒Ad5/F11p-GFP对不同肿瘤细胞系感染效率的比较研究

目的以Ad5-GFP及Ad5/F35-GFP为对照,评价Ad5/F11p-GFP对不同肿瘤细胞系的感染效率。方法制备并纯化Ad5-GFP、Ad5/F11p-GFP及Ad5/F35-GFP三种重组腺病毒,以不同的感染复数(MOI)感染乳腺癌细胞系SKBR-3、MCF-7及肾癌细胞系786O。48 h后,荧光显微镜观察GFP的表达,流式细胞仪检测感染效率及不同细胞表面柯萨奇腺病毒受体(CAR)、CD46和桥粒芯糖蛋白质2的表达。结果 Ad5/F35-GFP对SKBR-3、MCF-7及786O均具有很高的感染效率,且其在较低的MOI时就具有较高的感染效率;Ad5/F11p-GFP对CD46高表达的MCF-7及786O具有较高的感染效率,5 MOI感染即可达到60%以上的感染效率;而Ad5-GFP仅对CAR高表达的细胞系786O具有较高的感染效率,且感染效率随着MOI的增加而升高。Ad5/F11p-GFP和Ad5/F35-GFP对三种肿瘤细胞系的感染效率明显高于Ad5-GFP。结论对5型腺病毒进行纤维顶球的改造,可增强对肿瘤细胞的感染效率。

人5型腺病毒六邻体蛋白单克隆抗体的制备及鉴定

目的制备抗人5型腺病毒(HAdV5)六邻体(Hexon)蛋白的单克隆抗体并对其进行鉴定。方法用纯化的Hexon蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞融合,经ELISA间接法筛选和克隆化培养,获得4株分泌抗HAdV5的Hexon蛋白单克隆抗体的杂交瘤细胞。纯化获得单克隆抗体,使用ELISA和Western blot鉴定单克隆抗体的敏感性和特异性。结果 ELISA和Western blot结果表明这4株单克隆抗体与HAdV5的Hexon蛋白可以特异性结合。单克隆抗体株4A9-HRP标记和8D6建立的ELISA夹心法具备较高灵敏度和特异性。结论获得4株抗HAdV5的Hexon蛋白的单克隆抗体,为HAdV5相关的疫苗研制和HAdV5感染性疾病的早期快速诊断奠定了基础。