电穿孔法基因转染K562细胞条件的优化

目的以绿色荧光蛋白（GFV）为观察指标优化K562细胞的电穿孔基因转染条件。方法通过改变悬液体积、温度及缓冲液、质粒浓度等转染条件，采用不同条件组合后用电穿孔法将质粒DNA转入K562细胞，通过流式细胞仪和荧光显微镜观察转染效率。结果①在电压250V、电容950μF的条件下，K562细胞得到58．6％的最大转染率。②应用0．4mL细胞悬液体积，当质粒浓度≥20μg／mL时可得到较高的转染效率。③温度和缓冲液中的血清成分对电穿孔效率影响不大。结论电穿孔是一种高效的基因转染法，通过优化条件，可提高转染率。     

绿色荧光蛋白标记的报告系统优化K562细胞转染条件的研究

本研究旨在提高基因转染人红白血病细胞株K562的转染效率，同时观察绿色荧光蛋白（GFP）作为报告系统用于转染条件优化的可行性。目的基因以GFP为报告系统，通过脂质体转染及电穿孔两种方式转染K562细胞，荧光显微镜下观察并计算转染效率。结果表明：电转染较脂质体转染K562细胞的效率高，电穿孔转染效率在10％左右，而脂质体转染效率小于1％。结论：电转染较脂质体转染K562细胞的效率高，GFP可作为报告系统优化K562细胞转染条件。     

稳定表达hermap及hermap-siRNA的K562细胞系的建立

本研究分别建立可稳定表达hermap和hermap-siRNA的K562细胞系。双酶切hermap的PCR扩增产物并与pEGFP-c1载体连接形成重组质粒;双酶切hermap-siRNA序列并与pRNAT载体连接形成重组质粒;使用电穿孔转染法将上述两质粒分别转染K562细胞,通过G418筛选建立可分别稳定表达hermap和hermap-siRNA的K562细胞系。结果显示,hermap-pEGFP-c1质粒及hermap-siRNA-pRNAT质粒转染K562细胞的效率分别为10.0%和9.3%;两种K562细胞系通过G418筛选均能稳定生长并表达目的基因。结论:成功构建了含hermap和hermap-siRNA的真核表达质粒,建立了稳定表达hermap和hermap-siRNA的K562细胞系,这为进一步研究HERMAP在红细胞分化发育过程中的作用奠定了基础。     

KLF4过表达对白血病K562细胞增殖、凋亡的影响

目的:建立稳定过表达人锌指转录因子Krüppel样因子4(Krüppe-like factor 4,KLF4)基因的K562细胞株,研究KLF4过表达对白血病K562细胞增殖、凋亡的影响。方法:将过表达KLF4的重组质粒p EGFP-KLF4电穿孔转染白血病K562细胞,G418压力筛选出稳定转染细胞克隆并扩大培养(K562/p EGFP-KLF4组),设p EGFP-C1空质粒转染K562细胞(K562/p EGFP-C1组)及空白K562细胞对照组。应用荧光显微镜观察EGFP-KLF4融合蛋白的表达,Western blot法检测各组细胞中KLF4蛋白表达水平,MTT比色法检测各组细胞的增殖能力,流式细胞术检测各组细胞的凋亡情况。结果:p EGFP-KLF4重组质粒转染K562细胞后,经G418压力筛选,获得了表达绿色荧光蛋白的稳定细胞株;与对照组相比,K562/p EGFP-KLF4组细胞KLF4的蛋白表达水平明显升高,其过表达率为74.07%(P0.05);过表达KLF4的K562细胞增殖活力被显著抑制(P0.05);过表达KLF4的K562细胞凋亡显著增加(P0.05)。结论:KLF4过表达可抑制K562细胞的增殖,促进其凋亡。     

一种新的离体培养大鼠海马神经元基因转染技术

目的:探讨一种新的离体培养大鼠海马神经元基因转染方法。方法:选用17d的胚胎SD大鼠,取海马组织分离成细胞悬液,离心去上清并用混有2μg CAG-RE质粒的电转液重悬,置Neucleofector电转染仪中,选用O-03程序进行电转染,后用基础神经元培养液稀释并接种于培养皿中,4h后,待细胞贴壁时换新的神经元维持培养液,继续培养,选择不同时间点在荧光显微镜下观察,并计算在转染后4d时神经元的转染效率。结果:电转染24h后可以观察到有绿色荧光蛋白表达的转染神经元,荧光可以持续表达到2周,转染效率可达60%～80%,神经元生长良好,神经突起显示清楚。结论:电穿孔基因转染技术可以作为离体培养海马神经元很好的基因导入方法。     

遗传性铁粒幼细胞贫血致病的ALAS2基因表达载体的构建

X连锁遗传性铁粒幼细胞贫血(XLSA)是红系特异性δ-氨基-γ酮戊酸合酶(δ-amionlevulinic acid syntase 2，ALAS2)基因突变造成的，本研究构建ALAS2基因的真核表达载体，观察表达栽体转染真核细胞后蛋白表达的情况。将获自人胎肝的ALAS2基因插入到红色荧光蛋白质粒pDs—red2-N1中，命名为pDs—red2-N1／ALAS2；采用电穿孔方法将质粒转染K562细胞；转染后48小时提取细胞总RNA，进行RT—PCR检测ALAS2基因表达；流式细胞术检测红色荧光蛋白表达。再采用脂质体方法将质粒转染COS7细胞，转染后72小时提取细胞总RNA，RT—PCR检测ALAS2基因表达，荧光显微镜观察COS7细胞红色荧光蛋白表达情况。结果表明：构建了一种pDs—red2-N1／ALAS2真核表达载体，提取质粒DNA经酶切、电泳可以看到在4700bp和1764bp处存在两条电泳条带；全长测序证实构建的栽体序列正确；质粒转染K562和COS7细胞后均出现阳性ALAS2基因转录产物：流式细胞术检测转染后K562细胞红色荧光蛋白表达的阳性细胞百分率为19．2％；荧光显微镜显示转染后COS7细胞红色荧光蛋白，表达高峰出现在转染后第3天，阳性细胞百分率为10．7％，并可持续表达10天左右。红色荧光蛋白的表达可以间接说明ALAS2基因的表达。结论：ALAS2基因的真核表达载体构建成功，可以通过电穿孔和脂质体的方法转染真核细胞中并在其胞浆中表达，有可能用于XLSA患者基因替代治疗。     

siRNA特异性抑制K562细胞Livin基因表达及其诱导凋亡作用

本研究探讨小分子RNA干扰技术抑制livin基因表达对白血病细胞系K562细胞凋亡的影响。设计合成livin特异性小干扰RNA(siRNA),核转染K562细胞,培养转染后的K562细胞,用RT-PCR检测livin mRNA的表达,Western blot检测Livin蛋白的表达。以未转染细胞作对照,同时转染带有增强型绿色荧光蛋白的载体作为阳性对照,用流式细胞术检测其细胞绿色荧光以确定转染效率。用膜联蛋白Ⅴ及碘化丙锭双染法检测细胞凋亡率。结果表明,电穿孔的转染效率可达50%。siRNA既可以抑制livin mRNA表达,也可以抑制livin蛋白表达。特异性siRNA转染细胞后48 h细胞凋亡率为(27.41±2.30)%,与对照组(9.63±0.89%)比较明显提高(P<0.05)。结论:SiRNA可以抑制livin基因的表达,并能抑制livin基因的抗凋亡作用。     

CD44基因沉默对K562/A02细胞逆转耐药及生物学活性的研究

本研究旨在探讨CD44基因表达抑制对白血病多药耐药细胞株K562/A02耐药性及生物学活性的影响。根据CD44基因表达序列设计有效的RNA干扰片段,将其插入质粒表达载体中,获得针对CD44mRNA的特异性siRNA真核质粒(pGCsiRNA-CD44),转染K562/A02细胞。用实时荧光定量RT-PCR检测K562/A02细胞CD44、mdr-1和bcl-2mRNA的表达;用MTT法检测阿霉素对K562/A02细胞的半数抑制浓度(IC50);流式细胞术(FACS)检测细胞周期;Hochest33258染色荧光显微镜观察细胞凋亡的形态学变化。结果表明:针对CD44基因的siRNA真核质粒可有效沉默K562/A02细胞的CD44基因:与对照组相比,转染了4条pGCsiRNA-CD44质粒的K562/A02细胞CD44表达均明显受抑,以转染了siCD44-1的细胞中抑制最强。转染pGCsiRNA-CD44质粒48小时后,K562/A02细胞CD44mRNA表达水平下降64.1%(p0.05),同时mdr-1与bcl-2mRNA的表达量较对照组分别下降了25.6%与50.8%;K562/A02细胞IC50为(17.97±1.61)μg/ml,无义序列质粒转染后阿霉素对K562/A02细胞的IC50为(16.95±1.72)μg/ml,pGCsiRNA-CD44转染后IC50明显降低为(8.77±1.63)μg/ml(p0.01)。转染48小时后,G0/G1期细胞比例提高了10.7%;细胞出现核固缩、核边集、凋亡小体等改变。结论:特异性CD44siRNA可显著抑制K562/A02细胞CD44基因的表达,抑制细胞增殖,诱导其凋亡,并有效逆转K562/A02细胞的多药耐药性。     

脂质体转染反义脱氧寡核苷酸对K562细胞α珠蛋白基因表达及细胞增殖的影响

本研究查明脂质体转染反义脱氧寡核苷酸(ASON)对K562细胞α珠蛋白基因表达的抑制作用及对细胞增殖的影响,探讨基因调控治疗β地中海贫血的新思路。设计合成靶向α珠蛋白基因的ASON,并与正义寡核苷酸(SON)和空白对照进行比较。采用脂质体转染技术将ASON、SON与K562细胞共同培养,用荧光显微术、流式细胞术检测脂质体转染效率,用实时荧光定量RT-PCR法检测K562细胞中α珠蛋白基因表达情况,并通过Cell Counting Kit-8(CCK-8)法观察脂质体转染ASON技术对细胞增殖的影响。结果表明:脂质体转染效率在ASON作用24h达到最高,脂质体转染ASON组的α珠蛋白基因表达水平显著低于SON和空白对照组(p<0.01),并对细胞的增殖有明显的抑制作用,上述效应呈浓度依赖性。结论:脂质体转染ASON能特异性的抑制K562细胞中α珠蛋白基因表达,可能会成为β地中海贫血基因治疗的一个新靶点。     

EGFP作为报道基因在研究WT1基因调控元件中的应用

本研究调查EGFP基因作为报道基因在研究WT1基因调控功能中应用的可行性。利用基因重组技术分别将WT1基因启动子和增强子的功能片段构建到质粒pEGFP-1载体中,转化感受态E.coli菌细胞,筛选了阳性转化菌落,通过限制性酶切鉴定重组质粒插入片段正确的菌落,抽提重组质粒DNA;同时将重组质粒转染K562细胞,通过荧光显微镜检测重组质粒的功能。结果表明：通过基因重组技术分别构建了含有WT1基因启动子的载体（pEWP）和含有WT1基因启动子和增强子的载体（pEWPE、pEWPA和pEWPD）。转染48小时后,pEWP、pEWPE、pEWPA和pEWPD的K562细胞在荧光显微镜下能够显示荧光,而转染了空载体pEGFP-1的K562细胞未出现荧光。结论：EGFP基因作为报道基因可以用于WT1基因调控功能的研究,为进一步开展基因治疗创造了条件。     

RNA干扰对慢性粒细胞白血病bcr/abl融合基因表达的抑制作用

目的研究RNA干扰(RNAi)效应对慢性粒细胞白血病(CML)bcr／abl融合基因表达的抑制作用。方法体外化学合成针对bcr／abl融合基因的小干扰RNA(siRNA)，并用电穿孔方法将siRNA转染K562细胞株，以未转染的细胞及非特异性的siRNA转染细胞作对照；同时转染带有增强型绿色荧光蛋白(EGFP)的载体作为阳性对照，流式细胞术检测其绿色荧光以确定转染效率；实时定量RT—PCR及Western blot法检测siRNA的抑制效应。MTT法检测细胞增殖抑制率。膜联蛋白V(Annexin V)及碘化丙锭双染法测细胞凋亡率。结果电穿孔的转染效率可达70％；化学合成的siRNA可以抑制bcr／abl融合基因在mRNA和蛋白水平的表达；特异性siRNA可以抑制细胞增殖，转染24h细胞增殖抑制率达47％，48h达56％；特异性siRNA转染细胞24h组凋亡率为15．05％．48h组为19．47％，与对照组(1．00％)比较明显提高。结论在细胞水平上，化学合成的siRNA可以抑制bcr／abl融合基因的表达，为利用RNA干扰作为基因治疗的研究奠定基础。     

WT1基因表达下调对K562/A02细胞阿霉素敏感性的影响

目的 探讨WT1基因表达下调对人红白血病耐药细胞系K562/A02阿霉素敏感性的影响.方法 将WT1mRNA的短发夹RNA(short hair RNA,shRNA)构建至真核表达载体后转染K562/A02细胞,流式细胞术检测转染效率;荧光定量RT-PCR和Western blot法分析WT1基因在转染前后表达的差异;pWT1shRNA转染K562/A02细胞48 h并经阿霉素作用后,MTT法检测各组细胞阿霉素IC50值;流式细胞术检测细胞阿霉素累积量及细胞凋亡率.结果 与未转染对照组及空载体组相比,转染WT1shRNA质粒的K562/A02细胞WT1mRNA和蛋白水平均明显降低;经阿霉素诱导24 h后,其对阿霉素的敏感性明显增高,相对逆转率为71.5%,细胞内阿霉素的累积量较各对照组明显增高(P值均<0.05),细胞凋亡率明显升高(P<0.05).结论 靶向WT1shRNA干扰质粒可有效抑制K562/A02细胞WT1基因表达,增强K562/A02细胞对阿霉素的敏感性.     

New technique for gene transfection using laser irradiation.

BACKGROUND: We have developed a gene transfection system using laser beams. The principle of this procedure is that a small hole is made in a cell membrane by pulse laser irradiation, and a gene contained in a medium is transferred into the cytoplasm through the hole. This hole disappears immediately with the application of laser irradiation of the appropriate power. METHODS: A pulse-wave Nd:YAG laser with a wavelength of 355 nm was used to make a hole in a cell membrane. To trap a cell, a continuous-wave Nd:YAG laser with a wavelength of 1015 nm was used. Plasmids that encode the enhanced green fluorescent protein (EGFP) gene were contained in a medium and transferred to HuH-7 and NIH/3T3 cells with pulse laser irradiation. We evaluated transfection efficiency on the basis of the number of cells that expressed EGFP. Stimulatory protein 2 cells in suspension were fixed using a trapping laser and the neomycin-resistance gene was transfected by pulse laser irradiation. We examined cell proliferation in the selection medium. RESULTS: Cells that expressed EGFP were recognized in the group that was irradiated by pulse laser. No cells expressed EGFP without irradiation. Transfection efficiency was approximately 10% at a plasmid concentration of 10.0 microg/mL. At concentrations greater than 20 microg/mL, the transfection rate reached a plateau. We also successfully transfected neomycin-resistance genes to cells floating in suspension after fixation that was achieved with trapping laser irradiation. CONCLUSIONS: This method enables us to transfect targeted cells, ie, cells in suspension as well as attached cells, with a simple technique that does not involve harmful vectors. The present method is very useful for gene transfection in cellular biotechnology.     

低频超声联合微泡造影剂增强脂质体介导pEGFP-N1基因转染体外前列腺癌细胞

目的探讨低频超声联合微泡造影剂增强脂质体介导增强型绿色荧光蛋白质粒(pEGFP-N1)转染前列腺癌细胞的可行性,并对超声微泡浓度参数进行优化。方法将前列腺癌PC-3细胞悬液分为空白对照组、超声组、微泡组、微泡+超声组、脂质体组、脂质体+微泡组、脂质体+超声组、脂质体+微泡+超声组,其中脂质体+微泡+超声组根据微泡体积浓度不同分为(0、10%、20%、30%、40%和50%)6个亚组。经超声辐照,24h后用荧光显微镜观察细胞中基因表达情况,并用流式细胞仪检测转染率。结果脂质体+微泡+超声组基因转染效率最高,与其他组比较差异均有统计学意义(P均<0.05);在脂质体+微泡+超声亚组中,微泡浓度为20%亚组基因转染率最高。结论低频超声联合微泡能有效增强脂质体介导pEGFP-N1基因在体外前列腺癌细胞中的转染率,20%是体外基因转染前列腺癌细胞的最佳微泡浓度。     

THP-1单核细胞不同转染方法的比较

背景：人单核细胞系THP-1细胞是典型的悬浮细胞系,其转染并瞬时表达外源蛋白比一般贴壁细胞相对困难。其中DEAE-葡聚糖介导的细胞转染方法虽然建立较早,但在国内应用很少。目的：采用不同质粒转染方法介导质粒绿色荧光蛋白真核表达质粒（pEGFP-N1）转染人THP-1单核细胞,以获得较高转染效率的方法。方法：分别采用DEAE-葡聚糖、Lipofectamine 2000、FuGENE 6、梭华-Sofast转染试剂不同方法介导质粒绿色荧光蛋白真核表达质粒进行转染,测定不同方法的转染效率、及其对细胞活力的影响。结果与结论：荧光显微镜下观察及流式细胞仪检测结果均显示DEAE-Dextran介导的转染效率最高,Lipfectamine 2000脂质体次之,FuGENE 6和梭华-Sofast则明显低于前二者。将质粒绿色荧光蛋白真核表达质粒体外成功转染人THP-1单核细胞中,通过优化转染方法提高转染效率、降低对细胞活力的影响。     

Nonviral transfection of suspension cells in ultrasound standing wave fields

Ultrasound-induced cavitation has been widely used for delivering DNA vectors into cells. However, this approach may seriously disrupt cell membranes and cause lethal damage when cells are exposed to the inertial cavitation field. In this study, instead of using sonoporation, ultrasound standing wave fields (USWF) were explored for nonviral transfection of suspension cells. Acoustic resonance in a tubular chamber was generated from the interference of waves emitted from a piezoelectric transducer and consequently reflected from a borosilicate glass coverslip. The suspended K562 erythroleukemia cells were transfected by polyethyleneimine (PEI)/DNA complexes with and without exposure to 1-MHz USWF for 5 min. During USWF exposure, K562 cells moved to the pressure nodal planes first and formed cell bands by the primary radiation force. Nanometer-sized PEI/DNA complexes, circulated between nodal planes by acoustic microstreaming, then used the cell agglomerates as the nucleating sites on which to attach. After incubation at 37 degrees C for 48 h, the efficiency of nonviral transfection based on EGFP transgene expression was determined by fluorescent microscopy and fluorometry. Both studies showed that USWF brought suspended K562 cells and PEI/DNA complexes into close contact at the pressure nodal planes, yielding an approximately 10-fold increment of EGFP transgene expression compared with the group without ultrasonic treatment.     

Gene packaging with lipids, peptides and viruses inhibits transfection by electroporation in vitro.

To develop improved methods of gene delivery, packaging DNA in chemical or viral vectors could increase electroporation-mediated transfection. To test this hypothesis, electroporation was applied to DU145 prostate cancer cells incubated with green fluorescent protein-encoded DNA plasmid either naked or packaged with cationic lipid (Lipofectin), polycationic peptide (salmon protamine) or retroviral vectors (Moloney murine leukemia viruses) and then assayed for gene expression and cell viability. Cationic lipid or electroporation alone each significantly increased transfection, but their combination was less effective. Addition of protamine peptide during electroporation was also less effective than electroporation alone. The combination of retroviral vectors and electroporation transfected fewer cells than retrovirus alone. We conclude that the combination of electroporation with chemical or viral vectors does not improve gene transfection in vitro.     

Improving electrotransfection efficiency by post-pulse centrifugation.

We have demonstrated that the viability of electrotransfected adherent CHO and suspended NK-L, K-562, L1210 and MC2 cells is improved if pelleting by centrifugation is performed immediately after pulsing. The protection effect on cell viability is cell line- and pellet thickness-dependent. For forming CHO cell pellets, centrifugation force (300-13,000 g) and duration are not crucial; about five to 10 cell layers in the pellet provide the optimal protection effect. NK-L, K-562, L1210 and MC2 cell pellets are optimally formed by centrifugation at 13,000 g in an Eppendorf desktop centrifuge. Pelleting improves the cell viability over the whole range of the NK-L, K-562, L1210 and MC2 cell concentrations studied. When this pelleting method is applied to load CHO cells with FITC-dextran (41,000 MW), not only is the success rate close to 100%, but the growth rate is similar to the control, which is far better than the conventional electroporation method. Furthermore, the transfection efficiency of the five cell lines in pellet is significantly higher than that in suspension.