Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.     

Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation简

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.     

Asxl1在炎性微环境中对成骨细胞增殖分化的作用

背景:研究表明Asxl1的缺失可导致骨质发育不全、骨质缺损类疾病的发生,但目前在根尖周炎环境下该因子与骨破坏之间的关系暂无相关报道。目的:探讨炎性微环境下Asxl1对成骨细胞增殖分化的影响。方法:实验选用脂多糖刺激MC3T3-E1细胞建立体外炎性微环境,通过CCK-8实验筛取脂多糖最佳质量浓度和最佳作用时间,然后用20 mg/L脂多糖刺激MC3T3-E1细胞24 h,免疫荧光检测Asxl1的蛋白表达水平,Real Time-PCR检测Asxl1 mRNA的表达水平。为进一步验证Asxl1基因在炎性微环境中影响成骨细胞的增殖与分化,脂多糖刺激形成炎性微环境后转染Asxl1-SiRNA 24 h,采用CCK-8检测细胞增殖活性,RealTime-PCR检测Asxl1及成骨相关基因ALP和RUNX2 mRNA的表达水平。结果与结论:①脂多糖刺激MC3T3-E1细胞后,Asxl1蛋白和mRNA表达水平呈降低趋势;②脂多糖刺激MC3T3-E1细胞后,转染Asxl1-SiRNA 24 h,细胞增殖活性下降趋势明显,Asxl1基因及成骨相关基因ALP和RUNX2 mRNA的表达水平明显降低;③结果提示,Asxl1可能通过参与炎性反应过程,影响成骨细胞的增殖与分化,进而参与骨破坏进程。     

Fluorinated poly(propylenimine) dendrimers as gene vectors

The use of poly(propylenimine) (PPI) dendrimers as gene delivery vectors is limited by their low transfection efficacy and serious cytotoxicity. In this study, we prepare a series of efficient and low cytotoxic gene vectors based on PPI dendrimers using a facile fluorination strategy. Fluorinated G3, G4, and G5 PPI dendrimers show comparable or superior transfection efficacies to six representative transfection reagents such as Lipofectamine 2000, jetPEI, branched poly(ethyleneimine), SuperFect, PolyFect, as well as arginine-modified dendrimer on both HEK293 and HeLa cells. These fluorinated PPI dendrimers exhibit low cytotoxicity on the transfected cells because they achieve optimal transfection efficacy at extremely low nitrogen to phosphorus (N/P) ratios. In addition, the synthesized materials show high transfection efficacy on 3D multicellular spheroids. These results demonstrate that fluorination is a promising strategy to improve the transfection efficacy of PPI dendrimers while reducing the cytotoxicity of these cationic polymers.     

表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫的构建

H9N2亚型禽流感病毒已经被证实可以传播到人类,对它存在的潜在危害已受到越来越多的关注。因此,研究安全而有效的H9N2禽流感病毒疫苗已迫在眉睫。为研究和缓艾美耳球虫作为活载体表达及运输禽流感病毒抗原的潜能,本研究构建了可稳定表达H9N2禽流感病毒HA1蛋白的转基因和缓艾美耳球虫。利用黄色荧光蛋白（YFP）及融合的乙胺嘧啶抗性基因（DHFR-TSm2m3）作为报告基因及筛选基因,我们将表达H9N2禽流感病毒HA1抗原的质粒载体核转球虫子孢子,接种鸡后在乙胺嘧啶药物的选择压力下进行筛选并连续传代获得转基因和缓艾美耳球虫系。利用染色体步移和免疫印迹证实了HA1基因的成功插入和表达;利用间接免疫荧光证实HA1蛋白定位于球虫子孢子细胞膜表面和头部。研究进一步发现,转基因球虫的繁殖力与野生球虫相当,感染后亦于第6d达到排卵囊高峰。该转基因和缓艾美耳球虫株具有作为疫苗活载体的潜能。     

重组腺病毒Ad-APN-EGFP的构建与促进种植体周围成骨

目的 构建重组腺病毒Ad-APN-EGFP,研究其转染效率和对种植体周围成骨的影响。方法 构建含脂联素（APN）基因的的重组腺病毒Ad-APN-EGFP,并测序鉴定和酶切鉴定。体外转染大鼠骨髓间充质干细胞（BMSCs）,检测转染效率和APN基因mRNA表达;建立大鼠股骨干骺端羟磷灰石涂层种植体植入动物模型。植入种植体前,实验组于植入体窝注入10 μL的Ad-APN-EGFP悬浮液;对照组注入10 μL的PBS。4周后取材,光镜下观察种植体周围成骨。结果 测序鉴定和酶切鉴定表明,成功构建重组腺病毒Ad-APN-EGFP;转染大鼠BMSCs效率可达90%以上,并能高表达APN基因mRNA;体内实验显示实验组种植体周围成骨明显高于对照组。结论 重组腺病毒Ad-APN-EGFP可以提高大鼠BMSCs的APN基因表达,具有促进种植体周围成骨的作用。     

非病毒载体聚乙烯亚胺介导DNA转染的研究

目的研究新型非病毒类载体聚乙烯亚胺(PEI)的最适转染条件。方法分析各种因素对25 ku线性PEI转染效率的影响,优化实验条件。结果PEI与DNA的质量比≥2能保证DNA与PEI完全结合,最佳混合时间为10 min,但血清的存在将降低其结合效率。在一定范围内提高PEI与DNA的质量比有利于提高转染效率。293T细胞最适转染密度为培养面积的80%。结论确定了PEI转染细胞的最优条件。     

细小病毒非结构蛋白基因转染及表达对胃癌细胞的作用

细小病毒非结构蛋白基因转染人人低分化胃癌细胞株MKN.45后诱导其表达。研究发现该基因的表达能明显影响MKN-45细胞的生物特性。表现为部分细胞脱落死亡;存活细胞间粘附力增强;细胞倍增时间延长;细胞形态变圆、胞浆丰富,核/浆比例缩小;体外克隆形成率及裸小鼠体内成瘤能力下降。提示:1.转染的细小病毒非结构蛋白基因能在胃癌细胞内诱导表达。2.非结构蛋白基因表达对部分胃癌细胞具有直接细胞毒作用。3.该基因表达能干扰胃癌细胞生长,可能影响细胞增殖和分化过程而改变胃癌细胞的一些生长特征。     

非诺贝特与过氧化体增殖物激活型受体α对人肝瘤细胞1型纤溶酶原激活物抑制剂表达的影响

目的探讨非诺贝特对人肝瘤细胞株(HepG2)细胞1型纤维酶原激活物抑制剂(PAI-1)表达的影响及机制。方法用不同浓度非诺贝特刺激HepG2细胞,采用半定量逆转录聚合酶链反应(RT-PCR)法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化。构建4个荧光素酶报告基因质粒,分别由PAI-1启动子序列从-804至+17间不同长度片段驱动,体外转染HepG2细胞,检测荧光素酶的活性。结果非诺贝特能使HepG2细胞PAI-1mRNA表达及蛋白活性显著降低,且呈一定剂量依赖性;还可使PAI-1转录活性显著降低;当转染质粒含有PAI-1启动子序列-636~+17、-449~+17-、276~+17 bp 3个片段时,荧光素酶活性显著增高;共转染过氧化体增殖物激活型受体α(PPARα)表达质粒(PPAR-αpSG5)的细胞在非诺贝特诱导下PAI-1转录活性显著降低。结论非诺贝特可以抑制HepG2细胞PAI-1mRNA表达及其活性,调节PAI-1的基因转录,PPARα参与非诺贝特对PAI-1基因的表达调控。     

p15INK4B基因转染联合亚砷酸对人食管鳞癌的协同抑制作用

目的探讨p15^INK4B（p15）基因转染与亚砷酸（As2O3）联合应用对人食管鳞癌细胞系EC109细胞增殖和凋亡的影响。方法脂质体介导将pcDNA3．1（＋）-p15转染EC109细胞，稳定筛选后加入2μmol／LAs2O3。PCR检测外源p15基因cDNA，Western印迹法检测转染细胞P15蛋白的表达。用MTT、集落形成实验和透射电镜检测p15基因转染联合As2O3对EC109细胞增殖和凋亡的影响，流式细胞仪检测EC109细胞周期分布和凋亡率。结果与二者单独应用相比，联合应用可明显抑制EC109细胞体外生长速度，集落形成率明显降低（P〈0．01），联合作用3d后发生更为明显的G1／S阻滞，G1期比例显著升高（P〈0．05），S期比例明显降低（P〈0．05），凋亡率明显升高（P〈0．01）。透射电镜发现二者联合应用诱导EC109发生更明显的细胞凋亡。结论p15基因转染与As2O3联合应用可以进一步增强对人食管鳞癌EC109细胞的抑制作用和诱导凋亡作用。