电脉冲介导基因转移法在真核细胞表达CD4和CD19抗原

应用电脉冲介导基因转移法,将人白细胞分化抗原CD_4、CD_(19)。基因转入Cos细胞,间接免疫荧光及APAA(?)。联酶标染色表明CD_4和CD_(19)。基因获得高效转移和表达。这为进一步研究其分子结构,功能及筛选、鉴定相应的单克隆抗体提供了新的途径。     

电化学治疗肿瘤研究进展

利用细胞电穿孔结合抗肿瘤药物治疗肿瘤 ,称为肿瘤的电化疗 (Electrochemotherapy ,ECT)。离体肿瘤细胞和活体肿瘤组织电穿孔的最低电场强度分别为 4 5 0～ 6 5 0V/cm和 4 0 0～ 6 0 0V/cm。治疗肿瘤时常用脉冲个数 8个 ,脉冲宽度 10 0 μs,场强 6 0 0～130 0V/cm .,频率 1Hz的电场。已经有 10余种抗肿瘤药物用于电化学治疗肿瘤的研究 ,其中博莱霉素的效果最好 ,其次为顺式铂氨等。电化学治疗肿瘤 ,从体外培养细胞株到体内实体瘤 ,从实验室到临床 ,都已经取得了一些成果。电场参数、电极针的直径、电场磁力线的几何分布、肿瘤的大小、给药方式和药物种类等影响电化学治疗的效果。电化学治疗肿瘤运用于临床治疗还面临着一些问题 ,比如临床药物的选择和剂量、给药方式、对正常组织的影响、治疗机理等。     

EB病毒潜在膜蛋白对鼻咽癌细胞系CNE1生长及HLA表达的影响

目的研究ＥＢＶ－ＬＭＰ对高分化鼻咽癌细胞株ＣＮＥ１生长及ＨＬＡ表达的影响。方法以人高分化鼻咽癌细胞株（ＣＮＥ１）为对象，采用电穿孔基因转染技术，将重组ＥＢＶ－ＬＭＰ表达质粒ｐＣＭＶａ－ＬＭＰ转染ＣＮＥ１细胞。用细胞体外增殖试验、免疫组化和流式细胞术等方法，观察细胞生长及ＨＬＡ表达的变化。结果ＥＢＶ－ＬＭＰ在体外可明显促进ＣＮＥ１细胞的增殖，增殖吸光度（Ａ）比值试验组与空白组及阴性对照组比较有显著性差异（Ｐ＜０．０１）；实验组细胞软琼脂克隆形成率显著高于空白及阴性对照组（Ｐ＜０．０１）；细胞ＤＮＡ含量明显增高（Ｐ＜０．０１／０．０５）；ＦＣＭ法测定细胞角蛋白表达，实验组比空白及阴性对照组阳性率显著降低（Ｐ＜０．０５）；ＨＬＡ免疫组化检测结果表明，ＬＭＰ表达细胞系ＨＬＡⅠ类及Ⅱ类抗原的表达都明显下降（Ｐ＜０．０１）。结论ＥＢＶ－ＬＭＰ对ＣＮＥ１细胞生长有明显的促进作用且可明显抑制细胞分化，ＬＭＰ可致鼻咽癌细胞ＨＬＡ抗原的表达改变。     

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation

Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes.     

The Combined Effect of Electroporation and Borocaptate in Boron Neutron Capture Therapy for Murine Solid Tumors

¹⁰B-Enriched borocaptate (BSH) was administered intraperitoneally to SCCVII tumor-bearing C3H/He mice. Electroporation (EP) was conducted by using a tweezers-type electrode. The ¹⁰B contents in tumors were measured by prompt γ-ray spectrometry. The colony formation assay was applied to investigate the antitumor effects of boron neutron capture therapy (BNCT) and thereby to estimate the intratumor localization of BSH. The ¹⁰B concentrations in tumors decreased with time following BSH administration, falling to 5.4(±0.1) ppm at 3 h, whereas EP treatment (3 repetitions) 15 min after BSH injection delayed the clearance of BSH from tumors, and the ¹⁰B level remained at 19.4(±0.9) ppm at 3 h. The effect of BNCT increased with the ¹⁰B concentration in tumors, and the combination with EP showed a remarkably large cell killing effect even at 3 h after BSH injection. The effect of BNCT, i.e., slope coefficient of the cell survival curve of tumors, without EP was proportional to tumor ¹⁰B level ( r =0.982), and that of BSH-BNCT combined with EP lay close to the same correlation line. However, tumors subjected to EP after BSH injection did not show high radiosensitivity when irradiated after conversion to a single cell suspension by enzymatic digestion. This indicates that the increase of the BNCT effect by EP was a consequence of enclosure of BSH in the interstitial space of tumor tissue and not within tumor cells. This is different from a previous in vitro study. The combination of EP and BNCT may be clinically useful, if a procedure to limit EP to the tumor region becomes available or if an alternative similar method is employed.     

电脉冲化疗治疗人前列腺癌裸鼠移植瘤

目的　探讨平阳霉素（ＰＹＭ） 与电脉冲（ＥＰ） 相结合的电脉冲化疗对人前列腺癌细胞系ＰＣ３ｍ 裸鼠皮下移植瘤的作用。方法　将左右侧颈背部荷有ＰＣ３ｍ 肿瘤的裸鼠随机分为５ 组：按左右不同分别给予如下治疗：（１）Ｐ－ Ｅ－ ｜Ｐ＋ Ｅ－ ,（２）Ｐ－ Ｅ－ ｜Ｐ－ Ｅ＋ ,（３）Ｐ－ Ｅ－ ,｜Ｐ＋ Ｅ＋ ,（４）Ｐ＋ Ｅ－｜Ｐ＋ Ｅ＋ ,（５）Ｐ－ Ｅ＋ ｜Ｐ＋ Ｅ＋ 。Ｐ－ Ｅ－ 代表未治疗,Ｐ＋ Ｅ－ 代表肿瘤内注射平阳霉素,Ｐ－ Ｅ＋ 代表单给电脉冲,Ｐ＋ Ｅ＋ 代表电脉冲化疗,即肿瘤内注射平阳霉素后给予电脉冲。结果　亚治疗量的平阳霉素肿瘤内注射后给予电脉冲,１００％ 有效,其中６２．５ ％ 部分缓解、３７．５％ 完全缓解,即Ｐ＋ Ｅ＋ 有效,而其他组合无效。结论　平阳霉素介导的电脉冲为前列腺肿瘤及其他肿瘤的局部治疗提供了新思路。     

Electro-gene transfer to skin using a noninvasive multielectrode array

Because of its large surface area and easy access for both delivery and monitoring, the skin is an attractive target for gene therapy for cutaneous diseases, vaccinations and several metabolic disorders. The critical factors for DNA delivery to the skin by electroporation (EP) are effective expression levels and minimal or no tissue damage. Here, we evaluated the non-invasive multielectrode array (MEA) for gene electrotransfer. For these studies we utilized a guinea pig model, which has been shown to have a similar thickness and structure to human skin. Our results demonstrate significantly increased gene expression 2 to 3 logs above injection of plasmid DNA alone over 15 days. Furthermore, gene expression could be enhanced by increasing the size of the treatment area. Transgene-expressing cells were observed exclusively in the epidermal layer of the skin. In contrast to caliper or plate electrodes, skin EP with the MEA greatly reduced muscle twitching and resulted in minimal and completely recoverable skin damage. These results suggest that EP with MEA can be an efficient and non-invasive skin delivery method with less adverse side effects than other EP delivery systems and promising clinical applications.     

A novel plasmid DNA electroporation method allows transfection of murine DC

Under steady state conditions dendritic cells (DC) exert tolerogenic function, but acquire potent immunogenic function due to strong upregulation of costimulatory molecules and proinflammatory cytokines. In numerous studies the potential of modified DC to induce tolerance or immune reactions towards a distinct antigen has been demonstrated. However, DC are refractory to transfection with plasmid DNA by non-viral methods. In this study we have tested the suitability of a newly developed electroporation device to transfect immature murine bone-marrow derived DC (BM-DC). Transfected BM-DC expressed reporter molecules at considerable extent which renders this method suitable to perform all kinds of promoter studies. While electroporation did not alter the low allostimulatory capacity of immature BM-DC, it impaired the stimulation-associated increase in allostimulatory potency of transfectants. However, stimulated transfected BM-DC pulsed with myelin oligodendrocyte protein (MOG)-derived peptide induced proliferation of MOG-reactive CD4⁺ T cells as potently as did non-transfected MOG peptide-pulsed BM-DC. BM-DC transfected with an expression construct encoding MOG efficiently stimulated MOG peptide-specific T cell proliferation. Transfection of BM-DC with an IL-10 encoding expression construct resulted in high IL-10 expression and strongly diminished allogeneic T cell proliferation. Therefore, this method also allows to study functional properties of genetically altered DC.     

Comparison of red blood cell membrane microstructure after different physicochemical influences: Atomic force microscope research

Purpose

After the influence of different actions on the blood, the erythrocytes may change their macrostructure. At the same time, the microstructure of cell membrane will be changed as well. This study provides the results of comparison of red blood cell membrane microstructure after they have been affected by different factors.

Materials and Methods

Images and spatial profiles of the cell surface were obtained by atomic force microscope. It was proposed to use spatial Fourier transform to decompose the initial complex profile into series of simple ones. This made it possible to compare surface parameters after exposure of red blood cells to different external actions.

Results

Quantitative differences between membrane profile harmonic composition parameters (amplitude and spatial period) after physical impact (impulse electrical field, osmotic swelling) and after chemical impact (the fixing fluid glutaraldehyde and the drug Esmeron) were experimentally confirmed.

Conclusions

Such experimental and theoretical approach may lay down the foundations of mechanisms of different factors' effect on red blood cells both in research and in clinics.     

电穿孔对原代大鼠肝细胞生物学性状影响

目的 探讨不同电穿孔条件对原代大鼠肝细胞生物学性状的影响,优化外源基因转染原代肝细胞的条件。方法 以低电压（220－400V）、高电容（500－950μF）单脉冲电击原代大鼠肝细胞后接种培养,分别于36h-9d不同时点采用台盼蓝拒染法与MTT法观测肝细胞活力,并检测肝细胞培养上清液中白蛋白（ALB）、ALT与乳酸脱氢酶（LDH）含量。以未电穿孔肝细胞为对照组。结果 培养36h-9d各组肝细胞存活率均高于90％,贴壁率高于95％。培养36h肝细胞上清液中ALB、ALT与LDH值,电穿孔B组（220V,950μF）,C组（400V,950μF）明显高于对照组;但MTT法检测各电穿孔组肝细胞活力与对照组比较差异无显著性。结论 该电穿孔条件不影响肝细胞的存活能力,还能促进肝细胞ALB等蛋白的合成与释放,可作为外源基因转染原代肝细胞的优选方法之一。