物理因子在基因转染中的作用

基因转染是实现基因治疗的前提条件,也是分子生物学领域中一项重要技术。目前常用病毒转染方法缺乏靶向性,体内导入效率低,且存在安全隐患。非病毒载体中常用的脂质体体内导入除局部注入外,并无靶向性,即使直接注入,其效率仍然不高。寻找高效、安全的基因转染方法一直是基因治疗的热点问题。近年来,物理因子介导基因转移日益引起广大分子生物学家们的广泛关注。综述和探讨物理因子在基因转染中的作用必将为寻找新型基因转染方法,提高目前基因转染的效率和拓展物理因子的应用领域提供新的思路。     

物理因子在基因转染中的作用

基因转染是实现基因治疗的前提条件，也是分子生物学领域中一项重要技术。目前常用病毒转染方法缺乏靶向性，体内导入效率低，且存在安全隐患。非病毒栽体中常用的脂质体体内导入除局部注入外，并无靶向性，即使直接注入，其效率仍然不高。寻找高效、安全的基因转染方法一直是基因治疗的热点问题。近年来，物理因子介导基因转移日益引起广大分子生物学家们的广泛关注。综述和探讨物理因子在基因转染中的作用必将为寻找新型基因转染方法，提高目前基因转染的效率和拓展物理因子的应用领域提供新的思路。     

超声靶向破坏阳离子微泡介导SDF-1α基因转染提高外源性血管新生效应的研究

目的通过证实超声介导阳离子微泡能显著提高微泡的携基因量及体内转染后的血管新生效应来探讨阳离子微泡作为基因载体的价值。方法紫外分光光度法检测阳离子微泡与声诺维微泡在体外的基因携带效率。构建兔子急性心肌梗死模型并分组:阳离子微泡组(超声靶向破坏携SDF-1α基因的阳离子微泡并进行转染)、声诺维微泡组(超声靶向破坏携SDF-1α基因的声诺维微泡并进行转染)和对照组(不进行转染)。转染后3 d后3组各取3只兔子处死,ELISA检测目的基因蛋白表达情况;转染后4周行超声心动图和心肌超声造影,检测兔子心功能及心肌灌注情况,之后全部处死,免疫组化检测心肌血管新生效应。结果阳离子微泡组基因携带率是声诺维微泡组的11倍。阳离子微泡组SDF-1α蛋白表达是声诺维微泡组的4倍,对照组的9倍。阳离子微泡组在心肌毛细血管新生效应、心肌血流灌注和心功能方面均优于声诺维微泡组和对照组(P<0.05)。结论阳离子微泡能明显提高在体外的基因携带效率,从而使体内转染效率增强,更好地促进心肌血管新生,是一种高效的基因载体。     

超声介导声诺维提高人血管内皮细胞基因转染效率的体外实验研究

目的探讨使用超声破坏微泡造影剂-声诺维的方法对脂质体介导的增强型绿色荧光蛋白(EPGFP)基因转染人脐静脉血管内皮细胞(HUVEC)的作用。方法在单孔培养皿中加入HUVEC细胞悬液,每孔加入脂质体介导EPGFP4μg,加或不加微泡造影剂或不同条件的超声辐照。24h后,用荧光显微镜及流式细胞仪测量EPGFP在细胞内的表达及基因的转染效率。结果超声组转染率提高,超声 微泡组转染率明显提高。超声 微泡组,超声辐照时间60s机械指数(MI)1.4组转染效率最高;超声机械指数1.0辐照时间90s组转染率最高。结论声诺维在超声作用下能明显提高脂质体介导的基因对细胞转染的效率,可能作为基因治疗的载体。     

超声靶向破坏微泡介导腺相关病毒为载体的基因转染

腺相关病毒(AAV)是相对理想的基因治疗载体,但其转染效率低。超声靶向破坏微泡(UTMD)通过声孔效应、内吞作用和化学作用促进细胞摄取,是简单、易于操作、安全的提高基因转染率的方法。目前UTMD介导AAV为载体的基因转染已用于研究心脏、眼睛和肝脏的基因治疗。本文对AAV载体的转染特点、UTMD介导AAV为载体的基因转染机制及其应用进行综述。     

载基因微泡介导β-galactosidase质粒转染心肌细胞的对照研究

目的　观察不同的载基因微泡介导体外培养心肌细胞报告基因转染与表达,探讨微泡性质 与浓度在超声破裂微泡介导基因转染过程中的效应。方法　以β galactosidase质粒为报告基因,制备两 种不同性质的载基因微泡,利用诊断性超声破裂微泡进行体外心肌细胞基因转染,测定β galactosidase表 达水平并进行细胞活性检测。结果　超声载基因破裂氟烷气体微泡(PESDA)转染组心肌细胞β galactosidase表达约为单纯质粒转染组80倍(P<0.01),并在一定范围内随微泡浓度增加而增高。超声 破裂载基因PESDA转染组基因表达水平明显高于超声破裂载基因声振白蛋白微泡(ASDA)转染组(P< 0.05),而两组对细胞活性影响却无明显差异(P>0.05)。结论　超声微泡性质及浓度对超声破裂微泡介 导基因转染效率具有重要影响,低能量诊断性超声破裂载基因PESDA较超声破裂载基因ASDA能更为 有效地介导基因转染,是一种安全高效的基因传输方法。     

不同声学微泡对超声介导体外基因转染的作用

目的探讨超声介导基因转染时,不同声学微泡[脂质体微泡(LM)和SonoVue微泡]对体外基因转染的作用及其安全性。方法将红色荧光蛋白基因(DsRed)和LM加入培养的Hela细胞,后行超声辐照(US),对其他4种不同类型的细胞系(HepG_2、Ishikawa、MCF-7和B16-F10)行超声处理,并与PEI介导的基因转染进行比较分析,运用荧光显微镜、流式细胞术评估基因转染率和细胞损伤。结果培养的细胞经LM和超声辐照联合处理后(P+UTMI)),HepG_2、MCF-7的基因转染率略优于Sono Vue微泡转染时(P0.01),且未发现显著的细胞损伤;不同细胞类型对超声的反应性不同。与非辐照组相比,转染率和死亡率均有不同程度的增加。结论声学微泡对超声介导的体外基月转染有显著的增效作用,为基因治疗提供一种新颖、高效、安全的非病毒基因转染方法。     

超声微泡造影剂声诺维与基因转染的研究

超声微泡是一种新型基因转染的载体,超声辐照微泡（SonoVue）可增加基因在体内及体外的转染效率。若在不损伤基因和组织的前提下促进基因的转染,合适的辐照条件是非常重要的。     

实时PCR定量分析体内基因转染效率的可行性

为了探索实时PCR技术评价基因转染效率的可行性，利用克隆PCR检测逆转录病毒载体介导的neo基因导入原代成肌细胞的转染效率，同时行实时PCR检测，对二结果进行对比分析；利用线性扩增介导的PCR(LAM—PCR)技术和逆转录病毒5’LTR插入细胞基因组位点的分析，确定单细胞内基因转染的拷贝数。结果显示：①在低转柒率的情况下(＜36％)二的结果相近，但是在高转染率情况下二的结果差别明显；②逆转录病毒载体介导的转染，在原代成肌细胞中为单拷贝基因导入基因组内。结论：实时PCR可以比较精确地估计体内病毒载体介导的基因转染效率；而在体外由于多为高转染率，不适合用此方法进行检测。     

超声微泡破碎技术与基因转染研究进展

基因治疗是21世纪最具革命性的治疗手段，其用于人类各种疾病有许多潜在的优势，但基因治疗用于临床进展缓慢。问题在于缺乏安全有效的靶向性基因输送系统，且不能保证基因表达的长效性和稳定性。目前的载体中病毒载体转染效率高，但具有致突变性和免疫原性等潜在威胁，质粒和脂质体毒性低但转染效率低。近几年有报道证实超声微泡破碎技术（ultrasound microbubble destruction，UTMD）能有效增强基因转染效率。2000年有学者首次报道用超声微泡进行基因转染。     

超声声学造影剂介导肝细胞生长因子基因转染的体外实验

目的 研究超声声学造影剂促进肝细胞生长因子(HGF)基因在大鼠肝星状细胞株(HSC-T6)中的转染效果.方法 将HSC-T6接种在24孔板中,随机分为5组:①空白对照组(C);②单纯质粒组(P);③载基因超声声学造影剂组(M);④质粒+超声组(P+U);⑤载基因超声声学造影剂+超声组(M+U).荧光显微镜及流式细胞仪检测pIRES2-EGFP/HGF在细胞内的表达及转染效率;MTT法检测该方法 对HSC-T6生长活性的影响;Western Blotting检测HGF蛋白的表达.结果 M+U组的绿色荧光强度及转染率均高于其他各组,并对细胞活性无明显影响,且HGF蛋白的表达均高于其他各组(P＜0.05).结论 超声声学造影剂可提高基因在体外培养HSC-T6的转染效率,并对细胞活性无明显影响,为提高非病毒载体的转染效率提供一个新策略.     

Photochemical transfection: a new technology for light-induced, site-directed gene delivery

The development of methods for specific delivery of therapeutic genes into target tissues is an important issue for the further progress of in vivo gene therapy. In this article we report on a novel technology, named photochemical transfection, to use light to direct a precise delivery of therapeutic genes to a desired location. The technology makes use of photosensitizing compounds that localize mainly in the membranes of endosomes and lysosomes. On illumination these membrane structures will be destroyed, releasing endocytosed DNA into the cell cytosol. Using a green fluorescent protein gene as a model we show that illumination of photosensitizer-treated cells induces a substantial increase in the efficiency of transfection by DNA-poly-L-lysine complexes. Thus, in a human melanoma cell line the light treatment can increase the transfection efficiency more than 20-fold, reaching transfection levels of about 50% of the surviving cells. In this article various parameters of importance for the use of this technology are examined, and the potential use of the technology in gene therapy is discussed.     

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.     

High Ca(2+)-phosphate transfection efficiency enables single neuron gene analysis

Introducing exogenous genes into cells is one of the most important molecular techniques to study gene functions. Comparing to other type of cells, neurons are more difficult to transfect with cDNAs because they are very sensitive to microenvironmental changes. Among various gene transfer techniques, the Ca(2+)-phosphate transfection method is one of the most popular tools in neuroscience research because of its low cell toxicity and easiness to use. However, it is well known that the Ca(2+)-phosphate transfection efficiency in neurons is very low, typically in the range of 1-5%, which has limited its applications in gene functional analyses. Here we report a novel Ca(2+)-phosphate transfection protocol that dramatically increased the transfection efficiency by 10-fold, up to 60%, while maintaining low cell toxicity. The critical factors are the formation of homogenous snow-like precipitate with particle size about 1-3 microm and the subsequent removal of the precipitate. Using this new transfection protocol, we were able to routinely transfect single autaptic neurons in hippocampal microisland cultures and combine it with electrophysiology and fluorescent imaging methods to study gene functions. This high efficiency, low toxicity, and simple to use gene transfer method will have a broad application in gene research at the single cell level.     

Intracytoplasmic gene delivery for in vitro transfection with cytoskeleton-specific immunoliposomes.

A novel and highly efficient method of in vitro gene transfection has been developed. This method employs direct intracytoplasmic gene delivery into embryonic cardiocytes using neutral cytoskeletal-antigen specific immunoliposomes (CSIL). These immunoliposomes target cardiocytes specifically under reversible hypoxic conditions. Two independent reporter genes, pGL2 and pSV-beta-galactosidase, were used to verify CSIL-transfection (CSIL-fection). The efficiency of CSIL-fection with firefly luciferase pGL2 vector was 30+ times greater than controls consisting of hypoxic cardiocytes treated with plain liposomes (PL) or normoxic cardiocytes treated with CSIL, PL or naked DNA. CSIL-fection was also compared to cationic liposome transfection. Net cationic liposome transfection appeared to be more efficient than CSIL-fection for pGL2 vectors. However, a smaller number of viable cells was observed in the cationic liposome treated cultures than in the CSIL treated cultures. Therefore, to determine whether more cells were transfected with cationic liposomes or CSIL, pSV-beta-galactosidase vector was used. CSIL-fection with pSV-beta-galactosidase vector produced at least 40 times more transfected cells than those transfected with cationic liposomes. No transfection with pSV-beta-galactosidase vectors was obtained with IgG-liposome, PL or naked DNA treatments. Targeted enhanced efficiency of transfection by this novel method could have practical therapeutic applications in the genetic modification of cells ex vivo that could then be reimplanted into patients for gene therapy.     

Influence of needle gauge on in vivo ultrasound and microbubble-mediated gene transfection

Ultrasound and microbubble-mediated gene transfection are potential tools for safe, site-selective gene therapy. However, preclinical trials have demonstrated a low transfection efficiency that has hindered the progression of the technique to clinical application. In this paper it is shown that simple changes to the method of intravenous injection can lead to an increase in transfection efficiency when using 6-MHz diagnostic ultrasound and the ultrasound contrast agent, SonoVue. By using needles of progressively smaller gauge, i.e., larger internal diameter (ID), from 29 G (ID 0.184 mm) to 25 G (ID 0.31 mm), the transfection of a luciferase plasmid (pGL4.13) was significantly increased threefold in heart-targeted female CD1 mice. In vitro work indicated that the concentration and size distribution of SonoVue were affected by increasing needle gauge. These results suggest that the process of systemic delivery alters the bubble population and adversely affects transfection. This is exacerbated by using high-gauge needles. These findings demonstrate that the needle with the largest possible ID should be used for systemic delivery of microbubbles and genetic material.     

Biosurfactant MEL-A enhances cellular association and gene transfection by cationic liposome.

Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy.     

治疗性超声介导体外基因转染的参数优化

目的 探讨不同治疗性超声(TUS)参数对基因转染的作用,优化TUS参数以实现高效率的转染,减少对细胞活力和质粒完整性的影响.方法 将质粒和SonoVue微泡加入培养的Hela细胞后行超声辐照,改变超声强度、占空比以及辐照时间等参数,比较不同的TUS辐照策略对细胞活力及红色荧光蛋白(DsRed)表达效率的影响,以确定最佳辐照参数,并对质粒完整性进行分析.结果 低超声强度(0.4 W/cm2、1.0 W/cm2)、低占空比(10%和20%)时,细胞存活率较高(>80%),辐照1 min和3min之间的细胞活力差异不明显(P>0.05).当增加超声强度(1.6 W/cm2、2.2 W/cm2)和占空比(50%)时,细胞活力显著下降(P<0.05).当20%占空比,1.0 W/cm2的TUS辐照3min时,可实现最高的转染率.质粒DNA结构的完整性不受优化的TUS参数影响.结论 TUS是一种有效的基因输送方法 ,优化的参数在无显著细胞死亡和DNA损伤的前提下增加转染,这种非侵袭性的基因转染方法 可能是临床基因疗法的一种有用工具.