抗肿瘤的PF4及相关肽基因重组腺病毒的构建

利用细菌内同源重组法构建含PF4(血小板第四因子)全长1-70及PF4的小肽17-70基因的重组腺病毒。方法:将连有MCP-1信号肽的PF4全长及其C末端的改构体PF4-17-70两个基因片段克隆至腺病毒穿梭质粒pAdshuttle-CMV中,形成转移质粒pAdshuttle/PF4(1-70)和pAdshuttle/PF4(17-70),将之酶切线性化后与腺病毒骨架质粒pAdEasy-1共转化大肠杆菌BJ5183,抽提并鉴定含目的基因的重组体质粒,而后用磷酸钙共沉淀方法转染HEK293细胞,包装成重组腺病毒AdPF4(1-70)和AdPF4(17-70)。采用PCR方法对重组体腺病毒进行鉴定,扩增并纯化病毒,用TCID50方法测病毒滴度,利用带有CMV-LacZ的对照重组腺病毒(Ad-LacZ)对感染效率进行监测。结果:利用电穿孔法分别将质粒pAdshuttle/PF4(1-70),pAdshuttle/PF4(17-70)与质粒pAdEasy-1共转化大肠杆菌BJ5183获得70%以上的阳性重组体细菌克隆。PCR检测结果表明重组腺病毒已含有目的基因,滴度为1×1010pfu/ml。结论:利用细菌内同源重组法可以简便、快捷地构建含PF4(1-70)、PF4(17-70)cDNA的腺病毒载体,为两者在肿瘤基因治疗中的应用奠定了基础。     

The therapeutic effect of intratumoral injection of GM-CSF gene-modified allogenic macrophages on tumor-bearing mice

Both the antigen presenting ability and the cytotoxicity of macrophages can be enhanced by GM-CSF gene transfer. In the present study, the therapeutic effect of intratumoral injection with GM-CSF gene-modified allogenic macrophages on tumor-bearing mice observed. The peritoneal macrophages of C57BL/6 mice were transfected with GM-CSF gene mediated by recombinant adenovirus and the subcutaneous CT26 colon adeno-carcinoma-bearing BALB/c mice were treated by intratumoral injection of the above macrophages. The survival time of the tumor-bearing mice were prolonged significantly and some tumor mass disappeared completely. The necroses of the tumor cells and massive infiltration of inflammatory cells were observed 6 days after treatment 30 days after treatment, only the leftover of tumor cells and the inflammatory cells remained. The data indicated that introtumoral injection of GM-CSF gene-modified allogenic macrophages displayed more potent therapeutic effect on the preestablished tumor-bearing mice. Supported by National High Biotechnology Foundation (Z20-01-03). This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997).     

An adenoviral vector expressing the glucose transporter protects cultured striatal neurons from 3-nitropropionic acid

Considerable interest has focused on the possibility of using gene transfer techniques to introduce protective genes into neurons around the time of necrotic insults. We have previously used herpes simplex virus amplicon vectors to overexpress the rat brain glucose transporter, Glut-1 (GT), and have shown it to protect against a variety of necrotic insults both in vitro and in vivo, as well as to buffer neurons from the steps thought to mediate necrotic injury. It is critical to show the specificity of the effects of any such transgene overexpression, in order to show that protection arises from the transgene delivered, rather than from the vector delivery system itself. As such, we tested the protective potential of GT overexpression driven, in this case, by an adenoviral vector, against a novel insult, namely exposure of primary striatal cultures to the metabolic poison, 3-nitropropionic acid (3NP). We observed that GT overexpression buffered neurons from neurotoxicity induced by 3NP.     

Expression and Significance of Coxsackie and Adenovirus Receptor in Renal-Cell Carcinoma

OBJECTIVE To investigate the expression of Coxsackie and Adenovirus receptor (CAR) in renal-cell carcinoma and the relationship of the CAR to the biological behavior of the carcinomas.METHODS The immunohistochemical SP method was used to detect the expression of Coxsaekie and Adenovirus receptor in 48 cases of renalcell carcinoma and in 12 cases of normal renal tissue 2 cm away from the tumor tissue.RESULTS The positive rates of CAR were 100% in 12 cases of para-tumcr normal renal tissue and 35.4% in 48 cases of renal-cell carcinoma respectively. The difference of CAR expression between them was significant (P＜0.05). The grades of the tumor were as follows: 22 in Grade Ⅰ, 17in Grade Ⅱ and 9 in Grade Ⅲ with the CAR positive rate being 54.5%,23.5% and 11.1%, respectively. There was a negative correlation between CAR expression and tumor grading (P＜0.05). In addition, the number of the cases in stages Ⅰ to ⅣV were 19, 13, 11 and 5 respectively, with the respective positive rates being 57.9%, 30.8%, 18.2% and 0.0%, i.e. there also was a negative relationship between CAR expression and the stage (P＜0.05).CONCLUSION CAR expression is down-regulated in renal-cell carcinoma compared with normal tissue. The level of CAR may be a sensitive predictor of differentiation, invasion and metastasis. Loss of CAR expression correlates with the invasive phenotype in our analysis of renal-cell carcinoma.     

Immunogenicity and Reactogenicity of Ad26.COV2.S in Korean Adults: A Prospective Cohort Study

BackgroundAs the coronavirus disease 2019 (COVID-19) pandemic continues, there are concerns regarding waning immunity and the emergence of viral variants. The immunogenicity of Ad26.COV2.S against wild-type (WT) and variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) needs to be evaluated.MethodThis prospective cohort study was conducted between June 2021 and January 2022 at two university hospitals in South Korea. Healthy adults who were scheduled to be vaccinated with Ad26.COV2.S were enrolled in this study. The main outcomes included anti-spike (S) IgG antibody and neutralizing antibody responses, S-specific T-cell responses (interferon-γ enzyme-linked immunospot assay), solicited adverse events (AEs), and serious AEs.ResultsFifty participants aged ≥ 19 years were included in the study. Geometric mean titers (GMTs) of anti-S IgG were 0.4 U/mL at baseline, 5.2 ± 3.0 U/mL at 3–4 weeks, 55.7 ± 2.4 U/mL at 5–8 weeks, and 81.3 ± 2.5 U/mL at 10–12 weeks after vaccination. GMTs of 50% neutralizing dilution (ND50) against WT SARS-CoV-2 were 164.6 ± 4.6 at 3-4 weeks, 313.9 ± 3.6 at 5–8 weeks, and 124.4 ± 2.6 at 10–12 weeks after vaccination. As for the S-specific T-cell responses, the median number of spot-forming units/10⁶ peripheral blood mononuclear cell was 25.0 (5.0–29.2) at baseline, 60.0 (23.3–178.3) at 5-8 weeks, and 35.0 (13.3–71.7) at 10–12 weeks after vaccination. Compared to WT SARS-CoV-2, ND50 against Delta and Omicron variants was attenuated by 3.6-fold and 8.2-fold, respectively. The most frequent AE was injection site pain (82%), followed by myalgia (80%), fatigue (70%), and fever (50%). Most AEs were grade 1–2, and resolved within two days.ConclusionSingle-dose Ad26.COV2.S was safe and immunogenic. NAb titer and S-specific T-cell immunity peak at 5–8 weeks and rather decrease at 10–12 weeks after vaccination. Cross-reactive neutralizing activity against the Omicron variant was negligible.     

组织型纤溶酶原激活物重组腺病毒载体的构建及其转染ECV304细胞后对血管内皮细胞生长因子表达的影响

目的 评估外源性组织型纤溶酶原激活物(t-PA)对ECV304细胞中血管内皮细胞生长因子(VEGF)表达的影响.方法 应用细菌内同源重组技术快速构建Adt-PA腺病毒重组质粒,转染不同病毒滴度Adt-PA至ECV304细胞,转染比率(MOI)分别为1∶10、1∶50、1∶100,选取合适MOI值;病毒转染后检测ECV304细胞内t-PA蛋白的表达.然后将培养的ECV304细胞分为二组,在培养液中加入Adt-PA混合培养24 h和48 h分别作为实验组1和实验组2,加入空病毒Ad混合培养48 h作为对照组,比较各组细胞中VEGF mRNA的转录和蛋白表达水平.结果 成功构建了重组腺病毒Adt-PA.当MOI为1∶50时,细胞转染效率为(69.6±21.2)%,且对ECV304细胞增殖具有一定的促进作用;Adt-PA转染ECV304细胞后在72 h内随着时间的延长其蛋白表达量逐渐升高(P<0.01);细胞内VEGFmRNA的转录水平和蛋白表达量在实验组1和2中较对照组均有明显升高(P<0.01).结论 构建的t-PA腺病毒表达载体可有效感染ECV304细胞并可显著增加其VEGF的表达水平.     

以AdMax载体系统构建携带HCV NS5B基因的重组腺病毒表达载体

目的构建表达丙型肝炎病毒(HCV)非结构基因NS5B的重组腺病毒表达载体,并对其相关指标进行鉴定,为进一步研究防止丙型肝炎感染的基因免疫和基因治疗奠定实验基础。方法应用基因工程技术将HCVNS5B基因定向克隆至穿梭质粒pDC316上,利用脂质体介导的方法将AdMax腺病毒包装系统的骨架质粒pBHGloxΔE1、3Cre和穿梭质粒pDC316-NS5B共转染293细胞,进行同源重组,产生重组腺病毒pAd-NS5B并进行鉴定、反复感染293细胞进行扩增,扩增后测定重组病毒滴度。结果重组病毒颗粒pAd-NS5B经双引物PCR、凝胶电泳证明插入片段与HCV非结构基因NS5B片段大小相符;经测序证明其插入序列与设计HCV NS5B基因序列完全一致,扩增后重组病毒滴度达到2.3×1013IU/L。结论成功构建了表达HCV NS5B基因的重组腺病毒载体。     

昆明市幼儿病毒性腹泻病原学监测分析

目的 了解云南省昆明市病毒性腹泻的病原谱特征,探索其流行规律,以期为病毒性腹泻患儿临床治疗及制定有针对性的防控措施提供参考.方法 采集2018年1月至2019年12月因腹泻就诊的昆明市5岁以下患儿粪便标本,用实时荧光定量PCR方法进行核酸检测,检测轮状病毒、诺如病毒、腺病毒、星状病毒及札如病毒,并分析各种病毒的流行病学...