N-[4-(N,N,N-Trimethylammonium)Benzyl]Chitosan Chloride as a Gene Carrier: The Influence of Polyplex Composition and Cell Type

Polyplex-based gene delivery systems are promising substitutes for viral vectors because of their high versatility and lack of disadvantages commonly encountered with viruses. In this work, we studied the DNA polyplexes with N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS) of various compositions in different cell types. Investigations of the interaction of TMAB-CS with DNA by different physical methods revealed that the molecular weight and the degree of substitution do not dramatically influence the hydrodynamic properties of polyplexes. Highly substituted TMAB-CS samples had a high affinity for DNA. The transfection protocol was optimized in HEK293T cells and achieved the highest efficiency of 30–35%. TMAB-CS was dramatically less effective in nonadherent K562 cells (around 1% transfected cells), but it was more effective and less toxic than polyarginine.     

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.     

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。     

Smad2特异性siRNA转染肝星状细胞的效率检测

[目的]提高siRNA表达载体在肝星状细胞(hepatic stellate cells,HSCs)中的转染效率。[方法]pEGFP-C1-CR4转染原代HSCs、HSC-T6细胞株,实验根据质粒DNA和阳离子脂质体的不同比例分为4组:1μg/0μL、1μg/1μL、1μg/2μL、1μg/4μL,1μg/0μL为对照组,其余3组为实验组。转染48 h后,使用荧光显微镜观察转染情况,同时流式细胞仪计算转染效率。[结果]质粒DNA和阳离子脂质体的比例为1μg/2μL和1μg/4μL时,原代HSCs转染效率为(56.55±3.12)%和(58.62±2.03)%(P>0.05),HSC-T6细胞株转染效率为(24.52±3.55)%和(25.49±2.33)%(P>0.05)。同样比例条件下,原代HSCs的转染效率显著高于HSC-T6细胞株的转染效率(P<0.05)。在实验组质粒、脂质体比例为1μg/2μL和1μg/4μL时,同种细胞的转染效率均无显著差别(P>0.05)。[结论]采用质粒DNA和阳离子脂质体的比例为1μg/2μL、1μg/4μL的条件,均能够在HSCs和HSC-T6中获得最佳转染效率,但前者更为经济。     

HBV全基因组克隆转染细胞系的建立

目的:建立HBV体外细胞培养体系. 方法:构建HBV全基因克隆质粒pcDNA3-3HBV,稳定转染HepG2细胞,G418筛选. ELISA检测细胞上清液HBsAg,HBeAg的表达,免疫组化检测细胞内HBcAg的表达,RT-PCR检测细胞S基因mRNA的表达,PCR检测细胞上清液DNA. 结果:成功构建了HBV全基因克隆质粒pcDNA3-3HBV,稳定转染HepG2后,培养上清HBsAg,HBeAg阳性,细胞内HBcAg呈核浆型分布,且以浆型分布为主. RT-PCR证实有HBV S基因 mRNA 的表达,上清中HBV S及前S基因阳性,荧光定量PCR检测显示培养上清中HBV 滴度达1×108拷贝/L. 结论:重组质粒pcDNA3-3HBV能在HepG2细胞中表达、转录、复制,该细胞培养体系能产生较高水平的HBV.     

节律基因mPeriod2过表达对体外Lewis肺癌细胞γ射线敏感性的影响

目的评价mPeriod2(mPer2)基因转染后过表达对小鼠Lew is肺癌细胞(LLCs)60Coγ-射线放射敏感性的影响。方法采用脂质体介导方法将mPer2基因转染至LLC细胞,以空白组及空载体质粒pcD-NA3.1为对照,采用RT-PCR及流式细胞术检测mPer2基因在LLC细胞中的表达,给予60Coγ-射线4Gy单次照射后,流式细胞仪检测细胞周期分布及凋亡,采用增殖细胞核抗原(PCNA)免疫组化、细胞集落形成实验检测细胞增殖及存活情况。结果60Coγ-射线照射后,与转染空载体质粒组及空白组比较,转染mPer2基因细胞表现为G2/M期阻滞,凋亡率下降(contro l:8.10±0.03;pcDNA3.1-vector:11.20±0.07;pcDNA3.1-mPer2:4.40±0.02;P<0.05),细胞克隆形成增加(contro l:14%;pcDNA3.1-vec-tor:11%;pcDNA3.1-mPer2:32%;P<0.01)及PCNA阳性表达增加(contro l: ,pcDNA3.1-vector: ,pcDNA3.1-mPer2: ,P<0.05)。结论节律基因mPer2过表达使60Coγ-射线照射的小鼠Lew is肺癌细胞凋亡下降,细胞增殖明显,降低了该细胞对放射损伤的敏感性。     

多聚胺阳离子脂质体转染效率及细胞毒性的评价

目的评估四种多聚胺阳离子脂质体的转染效率及细胞毒性,筛选高效低毒的阳离子脂质体。方法多聚胺阳离子脂质TC-Chol、DC-Chol与中性磷脂DOPE分别以1：1、3：1摩尔比制备多聚胺阳离子脂质体,转染以增强型绿色荧光蛋白为报告基因的质粒PIRES2-EGFP入Hela细胞、Hep2细胞,倒置荧光显微镜下检测转染细胞的报告基因表达,筛选出高效的阳离子脂质体,通过MTT法检测转染细胞毒性,从而筛选相对高效低毒的阳离子脂质体。结果所制备的多聚胺阳离子脂质体在透射电镜下呈圆形、椭圆形囊泡样结构,少数呈管状、不规则状,粒径50-200nm,可将质粒PIRES2-EGFP有效转染入Hela细胞、Hep2细胞,其中多聚胺阳离子脂质TC-Chol与DOPE以3：1摩尔比制备的多聚胺阳离子脂质体是一种相对高效低毒的阳离子脂质体,转染效率39．5％～42．1％。结论多聚胺阳离子脂质TC-Chol、DC-Chol与中性磷脂DOPE以一定的摩尔比可制备高效低毒的多聚胺阳离子脂质体,其转染效率和细胞毒性与阳离子脂质的结构和中性磷脂DOPE的比例有关,在基因转染和基因治疗方面具有较广阔的应用前景。     

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。     

脑源性神经营养因子基因转染可促进弥漫性轴突损伤神经元修复和轴突再生

应用脂质体将外源脑源性神经营养因子基因导入弥漫性轴突损伤模型大鼠脑内,力图通过脑源性神经营养因子促进神经元再生及修复的作用,促进损伤大鼠的形态功能恢复。结果显示基因转染后弥漫性轴突损伤额叶皮质神经元的形态得到改善,额叶皮质组织神经丝蛋白表达增加,证实脑源性神经营养因子可促进弥漫性轴突损伤后神经元的修复及轴突的再生。