TALEN质粒转染HEK-293T细胞的条件优化

本研究探讨了TALEN质粒转染HEK-293T细胞的最佳转染条件。以TALEN质粒右臂中的绿色荧光蛋白为报告基因,分别研究转染试剂、质粒DNA的量、转染试剂与质粒DNA的比例对转染效果的影响,转染24 h后荧光显微镜下统计具有代表性视野的绿色荧光细胞,计算绿色荧光细胞/总细胞数的比例得出转染率,并利用CCK-8试剂盒检测细胞存活率。实验结果表明,TALEN质粒转染293T细胞,采用Fugene HD转染试剂比Lipofectamine 2000转染试剂细胞存活率高;以6孔板为例,转染质粒DNA量为4μg时转染效率最高(49.0±8.0)%;Fugene HD与质粒DNA比例(μg/μL)为3:1时转染效率最高。本研究建立TALEN表达质粒转染HEK-293T细胞的最佳转染条件,可作为相关研究或应用提供参考。     

鸡原始生殖细胞转染条件优化

为获得鸡原始生殖细胞(primordial germ cells,PGCs)的最佳转染效率,本研究比较不同质粒用量和不同细胞数在3种转染试剂(Lipofectamine 2000、3000和LTX&Plus Reagent)中PGCs的转染效率,利用荧光激活细胞分选技术(fluorescence activated cell sorting technology,FACS)辅助优化Lipofectamine 3000转染试剂,经FACS进一步分选获得带绿色荧光蛋白(GFP)的PGCs,继续培养3周后,移植回注到受体鸡胚中,移植3.5 d后分离性腺拍照观察。结果显示,转染试剂Lipofectamine 3000的转染效率最高,质粒、Lipofectamine 3000转染试剂和PGCs细胞数的配比为3μg:4μL:0.5×10⁴个,转染5 h转染效率最高,达到23.4%,与现有的研究结果相比提高了2倍以上。移植回注PGCs到受体鸡胚中,荧光显微镜观察到鸡胚性腺中有GFP阳性细胞。本研究综合考虑转染试剂、质粒用量和细胞数量的影响因素以优化PGCs的转染条件,为高效制备转基因鸡及基因编辑鸡的研究奠定基础。     

NK细胞中过表达Livin的实验研究

目的:将携带Livin的质粒pIRES2-EGFP-Livin进行扩增,转染自然杀伤细胞(NK)及胃癌细胞株SGC-7901,并检测其在NK及胃癌细胞株SGC-7901中的表达。方法:将携带Livin基因的质粒p IRES2-EGFP-Livin进行扩增,鉴定质粒纯度与浓度;从健康人外周血中获得NK细胞,应用HP转染试剂将质粒pIRES2-EGFP-Livin转染体外培养的NK及胃癌细胞,对比分析NK及胃癌细胞株SGC-7901中基因转染效率及目的基因的表达情况。结果:用无血清培养基在体外成功的扩增大量的NK细胞;质粒提取试剂盒抽提得到大量无内毒素的质粒,质粒DNA基因序列并未发生突变,浓度和纯度较高。胃癌细胞株SGC-7901中观察到明显的质粒pIRES2-EGFP-Livin绿色荧光表达;而NK中未观察到绿色荧光表达。结论:质粒pIRES2-EGFP-Livin能使Lvin蛋白表达于胃癌细胞株SGC-7901中,而在NK中未表达。     

慢病毒转染对鼻咽癌细胞5-8F增殖迁移的影响

目的:探讨慢病毒转染对鼻咽癌细胞株5-8F增殖、迁移的影响,以验证慢病毒转染是否能有效的应用于鼻咽癌细胞的增值及迁移相关研究。方法:以红色荧光标记的慢病毒为转染载体,选定不同的MOI值转染鼻咽癌5-8F细胞株,扩大培养后筛选纯化,流式细胞仪检测转染效率。以最佳MOI值转染后的5-8F(RFP-5-8F)细胞为实验组,未转染的亲代5-8F为空白对照组,取对数生长期未转染的亲代5-8F和红色荧光标记的慢病毒转染的5-8F(RFP-5-8F)细胞进行MTT、划痕实验,观察细胞镜下形态,了解细胞转染前后生长曲线,细胞迁移能力的变化。结果:流式细胞仪检测5-8F细胞慢病毒转染效率大于95%,转染最佳MOI值为30,镜下荧光强度适中。实验组与对照组比较,转染前后5-8F细胞光镜形态相似,生长曲线一致,差异无统计学意义(P=0.997),划痕实验显示5-8F与RFP-5-8F细胞迁移能力一致,差异无统计学意义(P0.05)。结论:慢病毒转染后鼻咽癌细胞能真实有效的的反应原细胞的增值及迁移能力,可以很好的应用于鼻咽癌增殖及其转移机制的相关研究。     

新型纳米转染试剂转染PNP自杀基因体外杀伤实验

将壳聚糖纳米粒包裹的报告基因pEGFP-N1质粒转染至HEK293细胞,并在HEK293细胞中成功表达荧光蛋白的基础上,进一步将本室自行构建的PNP基因的真核高效表达载体质粒pcDNA3-PNP转染至HEK293细胞。转染72h后,对转染的HEK293细胞给予前体药6-MPDR至终浓度40μg/ml,一天后,采用MTT比色法测定药物对细胞增值的影响,并进行统计学处理。实验结果表明采用壳聚糖纳米粒转染试剂转染并给予前体药6-MPDR的实验组活细胞数,与用壳聚糖转染但不给前体药6-MPDR的对照组活细胞数相比,有显著差异(P<0.05),说明新筛选出的壳聚糖纳米粒转染试剂可以将PNP自杀基因递送至靶细胞中,并在细胞中进行表达,从而使PNP/6-MPDR自杀基因系统发挥杀伤细胞的作用。分别采用相同工作浓度的脂质体与壳聚糖纳米粒转染试剂转染相同浓度的基因质粒,壳聚糖纳米粒对靶细胞生长数量影响很小,说明的壳聚糖纳米粒细胞毒性大大低于阳离子脂质体的细胞毒性。     

尘螨变应原Der f1 真核表达载体的构建及转染CHO 细胞

目的:构建尘螨变应原Der f1真核表达载体,转染真核细胞并进行蛋白表达.方法:根据Genebank中Der f1基因的核酸序列(AB034946),设计引物,采用PCR法,从保存的JM109工程菌中扩增Der f1编码基因,克隆到真核表达质粒pcDNA3.1/myc-hisA上,以脂质体法转染CHO细胞,经G418筛选,进行稳定表达细胞株的筛选和鉴定.结果:将目的基因Der f1成功连接到pcDNA3.1/myc-hisA-Derf1并转染CHO细胞,获得稳定表达的CHO细胞株.结论:成功构建了尘螨变应原Der f1真核表达载体,并转染CHO细胞表达蛋白质.     

小鼠骨样细胞MLO-Y4转染方法的研究

为了建立质粒转染小鼠骨样细胞MLO-Y4的方法,分别采用阳离子脂质体法和电转染法将增强型绿色荧光蛋白（EGFP）质粒pEGFP-C1转染小鼠骨样细胞MLO-Y4,正常培养48h后检测并统计转染率和死亡率。结果显示,脂质体法转染,当质粒与脂质体比例为1∶4时,转染效率可达到（36.8±3.7）%,细胞死亡率为（18.4±1.9）%;电转染法转染,脉冲电压240 V,脉冲时间300μs,脉冲次数3次时,转染率最高,可达到（23.8±2.3）%,细胞死亡率为（14.1±1.1）%。而后MTT实验显示脂质体转染法相对于电转染法对MLO-Y4细胞的增殖有一定的抑制作用,但对后续实验研究影响不大。脂质体转染法转染小鼠骨样细胞MLO-Y4优于电转染法。     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

多聚赖氨酸在悬浮细胞转染中的应用

目的:悬浮细胞的转染较贴壁细胞存在一定难度,用多聚赖氨酸包被的细胞培养板培养悬浮细胞使其贴壁,用脂质体2000按照贴壁细胞转染的方法转染悬浮细胞,提供一种高效的转染悬浮细胞的方法。方法:悬浮细胞Jurkat或CCRF-CEM培养于包被了0.1 mg/mL多聚赖氨酸的细胞培养板,16 h后洗掉未贴壁的细胞,用脂质体2000分别将pWPXLd质粒或靶向人ABL1基因的小干扰RNA(siRNA)转染细胞,24 h后于荧光显微镜下观察绿色荧光蛋白印迹鉴定siRNA的干扰效率。结果:pWPXLd成功转染2种细胞,siRNA成功抑制了ABL1的表达。结论:质粒和siRNA均能成功转染,提供了一种高效可行的转染悬浮细胞的方法。     

Xfect介导重组质粒pEGFP-C2/LAT-ORF3高效转染Vero细胞的方法及其条件的优化

旨在用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,以期获得转染效率较高的方法,并检测目标片段的表达,从而为进一步研究HSV-2 LAT基因及其ORF3片段的生物学功能奠定基础。用Xfect试剂介导重组质粒pEGFP-C2/LAT-ORF3转染Vero细胞,48 h后观察荧光表达情况,并计算不同转染方法分别在有无血清及质粒与Xfect Polymer比例不同时的转染效率。用RT-PCR检测ORF3片段在Vero细胞中的表达。结果显示,采用贴壁转染法,在有血清及质粒与Xfect Polymer比例为5μg/2μL时转染效率较高;RT-PCR可以获得目的条带,证明ORF3片段在Vero细胞中得到表达。本试验获得的优化条件可以显著提高Xfect Polymer对Vero细胞的转染效率;以绿色荧光蛋白作为标签鉴定目的基因在细胞中表达的方法切实可行。     

Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments

pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays.     

LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

High cell density transient transfection of CHO cells for TGF‐β1 expression

High cell densities for transient transfection with polyethyleneimine (PEI) can be used for rapid and maximal production of recombinant proteins. High cell densities can be obtained by different cultivation systems, such as batch or perfusion systems. Herein, densities up to 18 million cells/mL were obtained by centrifugation for transfection evaluation. PEI transfection efficiency was easily determined by transfected enhanced green fluorescence protein (EGFP) reporter plasmid DNA (pDNA). A linear correlation between fluorescence intensity and transfection efficiency was improved. The transfection efficiency of PEI was highly dependent on the transfection conditions and directly related to the level of recombinant protein. Several factors were required to optimize the transient transfection process; these factors included the media type (which is compatible with low or high cell density transfection), the preculture CHO‐K1 suspension cell density, and the pDNA to PEI level. Based on design of experiment (DoE) analyses, the optimal transfection conditions for 10 × 10⁶ cells/mL in the CHOMACS CD medium achieved 73% transfection efficiency and a cell viability of over 80%. These results were confirmed for the production of transforming growth factor‐beta 1 (TGF‐β1) in a shake flask. The purified TGF‐β1 protein concentration from 60 mL supernatant was 27 µg/mL, and the protein was biologically active.     

小鼠精原干细胞不同转染方法效率的比较

本研究采用腺病毒感染、慢病毒感染、脂质体转染和电穿孔转化方法将含有绿色荧光蛋白（GFP）的质粒转入经过差异贴壁法初步分离纯化的小鼠精原干细胞(SSCs)中,转染48 h后通过流式细胞仪检测GFP阳性细胞比例比较4种方法在体外转染精原干细胞的效率.结果显示,脂质体转染效率最高仅为8.64%,不能满足对精原干细胞进一步实验的要求;电穿孔法效率最高达到25.27%,但转化后细胞大量死亡;腺病毒转染细胞的效率达到了32.4%;慢病毒转染效率最高,达到74.25%. 因此,慢病毒转染法是体外转染小鼠精原干细胞的有效方法.     

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single‐cell bioluminescence imaging

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single‐cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single‐cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay. Copyright © 2013 John Wiley & Sons, Ltd.     

食管鳞癌血管生成拟态的组织学和细胞学研究

目的:探讨血管生成拟态(vasculogenic mimicry,VM)与食管鳞癌临床病理特征的关系及其对患者预后的影响,并分析食管癌血管生成拟态的形成机制。方法:收集57例食管鳞癌石蜡包埋样本,进行过碘酸雪夫氏(PAS)及CD34免疫组织化学双重染色,结合HE染色,观察食管鳞癌血管生成拟态的发生情况。对患者临床病理和预后信息进行单因素分析,Kaplan-Meier生存比较和Cox风险模型分析。通过食管鳞癌细胞株Eca-109三维培养建立,观察RNAi沉默VE-cadherin对食管鳞癌Eca109血管生成拟态形成的影响。结果:食管鳞癌中VM表达的阳性率为54.3%,显著高于正常食管黏膜组织;VM在病理分型为低分化食管鳞癌的阳性表达率为78.9%,显著高于中高分化组(P0.05);III-Ⅳ期食管鳞癌患者VM阳性率显著高于Ⅰ-Ⅱ期食管鳞癌患者(P0.05);有淋巴结转移的食管鳞癌者VM阳性率明显高于无淋巴结转移者(P0.05)。单因素分析结果显示食管鳞癌VM的发生率与肿瘤的分化程度、TNM分期和淋巴转移显著相关。Kaplan-Meier生存分析显示有VM组食管鳞癌患者的生存期明显短于无VM组(P0.05);Cox分析显示VM是影响食管鳞癌患者预后的独立危险因素(RF=0.67)。三维培养结果显示Eca-109细胞在基质胶上形成典型的血管网状样结构,VE-cadherin-siRNA可有效抑制VE-cadherin在Eca109的表达,抑制体外培养的Eca109细胞VM的形成。结论:血管生成拟态是食管鳞癌一种独特的血液供应模式,与食管鳞癌的分化程度、TNM分期、淋巴转移密切相关,是食管鳞癌患者术后生存期的独立危险因素。