胰腺癌细胞 BXPC-3转染条件的优化

目的：探讨实验室常用的人胰腺癌细胞株BXPC-3的有效转染方法及最佳转染参数。方法通过脂质体法和电穿孔法将表达绿色荧光蛋白的质粒（ pGFP-N3）导入人胰腺癌细胞株BXPC-3中,24 h后观察并比较细胞的存活率及瞬转效率,确定最佳转染参数。结果常规脂质体法BXPC-3细胞瞬转阳性率为1．36％,细胞存活率为87．45％;而电穿孔法在电压120 V电击时程70μs条件下,BXPC-3细胞瞬转阳性率为54．78％,细胞存活率为64．01％,2种转染方法的瞬转阳性率差异有统计学意义（P＜0．05）。结论转染BXPC-3细胞,电穿孔法可取得比常规脂质体法更高的转染效率。通过条件的优化,电穿孔法可以用于某些脂质体法难以有效转染的细胞株。     

Electroporation and Shock-Induced Transmembrane Potential in a Cardiac Fiber During Defibrillation Strength Shocks

Experimental studies have shown that the magnitude of the shock-induced transmembrane potential (V_m) saturates with increasing electric field strength. This study uses a mathematical model to investigate the effects of electroporation and membrane kinetics on V_m in a cardiac fiber. The model consists of the core conductor equation for a one-dimensional fiber, where excitability is represented by the Luo–Rudy dynamic model (1994–1995) and electroporation is described by a membrane conductance that increases exponentially with V_m squared. For shocks delivered during the plateau of an action potential, the model reproduces the experimentally observed saturation of V_m with a root mean square error of 4.27% and a correlation coefficient of 0.9992. For shocks delivered during diastole, the saturation of V_m is qualitatively reproduced even when the sodium and calcium channels are inactivated. Quantitative replication of the response to diastolic shocks is hindered by the choice of electroporation parameters (optimized for shocks delivered during the plateau) and differences in the membrane kinetics between model and experiment. The complex behavior of V_m during large shocks is due to a combination of electroporation, electrotonus, propagation, and active membrane kinetics. The modeling results imply that the experimentally observed saturation of V_m is due to electroporation of the lipid bilayer. © 1998 Biomedical Engineering Society. PAC98: 8722Fy, 8710+e, 8722Jb     

在家蝇中表达人胰岛素基因的研究

目的 研究人胰岛素基因在家蝇中的表达，建立家蝇生物反应器。方法 以家蝇作为实验动物，利用BAEKON6000型基因转移仪将胰岛素基因组基因导入家蝇进行实验研究。收集家蝇新产卵(1—1．5h内)，将环状质粒pCMV—mINS、pCMV-ImINS、pEF1α—mINS、pEF1α—ImINS以10μg/ml浓度分别与新采集的家蝇卵混合后进行电转移。经人工孵化发育为成虫(R)，G418(3．0mg／ml)筛选Fl代，待F1代发育至成虫并产卵后，采用PCR、Tricine-SDS-PAGE和Western-blot技术对F2代蝇蛆进行整合与表达水平检测。结果 四种质粒均能在家蝇中表达人胰岛素，表达量各占蝇蛆总蛋白0．207％、0．357％、0．473％和0．831％。结论 人胰岛素基因能够在蝇蛆中表达，即蝇蛆机体可对人胰岛素原进行翻译后加工。     

The use of an electroporation apparatus for the production of murine hybridomas

Antigen-directed electrofusion was carried out using biotin-streptavidin to bridge antigen-specific splenocytes to myeloma cells. Electrofusion was performed using a commercial electroporation apparatus. Electrofusion conditions were optimized by measuring the survival of myeloma cells after a range of electrical pulse conditions. The procedure was tested with antigens of high and low immunogenicity. The yields of hybridomas secreting antibodies specific for both antigens were considerably increased by the use of the electrofusion procedure.     

Use of electroporation in genetic analysis of enterococcal virulence

We present two methods for electroporation for the gram positive bacterium Enterococcus faecalis that can also be used as guidelines for work with other gram positive species. We demonstrate the use and the advantages of this technique for investigating genes, both chromosomal and plasmid-linked, encoding surface structures. Electroporation was used to deliver constructs created on shuttle vectors for insertional inactivation of a chromosomal gene involved in binding substance formation as well as for the expression of Aggregation Substance in strains with different chromosomal backgrounds. The influence of defects in lipoteichoic acid synthesis and the expression of Aggregation Substance on virulence was shown in a rabbit endocarditis model.     

Successful application of targeted electrochemotherapy using novel flexible electrodes and low dose bleomycin to solid tumours

Electroporation is the application of very brief electric pulses to cells or tissues to render the cell membranes transiently and reversibly permeable, facilitating cellular uptake of otherwise impermeant molecules. Flexible electrode arrays were developed which may be used with endoscopic and laparoscopic devices for delivery of therapeutic electroporation. Their efficacy in enhancing the delivery of bleomycin, an impermeant drug, was assessed in vitro and in vivo in both human and murine cancer cell lines, and growing tumours (xenografts). These flexible electrodes consistently and predictably deliver the permeabilising electric pulses requisite for in vivo electroporation, and would be suitable for electrochemotherapy of endoluminal tumours when incorporated into an endoscopic delivery system.     

Supramembrane potential-induced electroconformational changes in sodium channel proteins: a potential mechanism involved in electric injury

Effects of imposed large supraphysiological transmembrane potential (TP) pulses on channel proteins, particularly on the voltage-gated Na channels, were investigated. Voltage clamp techniques were used to deliver both shock and stimulation pulses, and to monitor changes in the channel functions. Our experimental results indicated that more than one 4 ms duration TP shock of -450 mV resulted in electroconformational denature of voltage-gated Na channels. This resulted in functional reductions in muscle cells' excitability. We quantified the TP shock-induced decrease in the Na channel currents, compared the pre- and post-shocked Na channel currents' voltage dependency, and studied the reversibility of the electroconformationally denatured ion channel proteins. These observations are particularly relevant to the problem of explaining the neuromuscular damage following high voltage electrical shock injuries despite no evidence of a thermal injury component.     

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 μg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.     

The use of an in vitro 3D melanoma model to predict in vivo plasmid transfection using electroporation

A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a “tissue-like” spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids delivered using a 6-plate electrode applying 6 pulses with nominal electric field strength of 500 V/cm and pulse-length of 20 ms produced significant increase of GFP (7.3-fold) and IL-15 (3.0-fold) expression compared to controls. This in vitro 3D model demonstrates the predictability of cellular response toward delivery techniques, limits the numbers of animals employed for transfection studies, and may facilitate future developments of clinical trials for cancer therapies in vivo.     

Membrane electroporation theories: a review

Electroporation, the transient increase in the permeability of cell membranes when exposed to a high electric field, is an established in vitro technique and is used to introduce DNA or other molecules into cells. When the trans-membrane voltage induced by an external electric field exceeds a certain threshold (normally 0.2–1 V), a rearrangement of the molecular structure of the membrane occurs, leading to pore formation in the membrane and a considerable increase in the cell membrane permeability to ions, molecules and even macromolecules. This phenomenon is, potentially, the basis for many in vivo applications such as electrochemotherapy and gene therapy, but still lacks a comprehensive theoretical basis. This article reviews the state of current electroporation theories and briefly considers current and potential applications in biology and medicine.