The ACIST power injection system reduces the amount of contrast media delivered to the patient, as well as fluoroscopy time, during diagnostic and interventional cardiac procedures

The ACIST injection system is an automatic power injection device that allows for online control of injection rate and volume of contrast. Limited data is available whether this technology allows reducing use of contrast and fluoroscopy time. Accordingly, we compared the use of this system to manual injection among 450 consecutive patients who underwent diagnostic coronary angiography and/or angioplasty who were randomly assigned to either manual contrast injection (control; n=198) or to the ACIST system (study group; n=252). The amount of contrast, fluoroscopy and total procedural times were recorded for each patient. In the diagnostic group, the mean total amount of contrast (including wasted) was reduced by 63% when the ACIST was used compared to control (100+/-42 ml versus 163+/-56 ml; P<0.001, respectively). When only the net amount of contrast delivered to the patient was considered, the differences were smaller (20%, P=0.004). During angioplasty, the amount of contrast was also lower in the ACIST group (206+/-65 versus 230+/-69, P=0.008), whereas no difference were noted in net amount of contrast. Fluoroscopy time was significantly shorter in the ACIST group compared to control both during diagnostic catheterization (4.7+/-3.5 min versus 6.3+/-5.5 min, respectively; P=0.014), and angioplasty (16.7+/-9.1 min versus 19.6+/-12.4 min, respectively; P=0.05). Routine utilization of the ACIST system during diagnostic and interventional procedure significantly reduced the total amount of contrast media used and fluoroscopy time.     

Optimization of 25 kDa linear polyethylenimine for efficient gene delivery.

A 25-kDa linear polyethylenimine (25 kDa L-PEI) has proven to be efficient and versatile agent for gene delivery. Therefore, we determined the optimal transfection conditions of 25 kDa L-PEI and examined whether it has comparable transfection efficiency with other commercially available reagents, ExGen 500, LipofectAMINE 2000, and Effectene by using EGFP expression vector in different cell lines. Transfection efficiency and cytotoxicity were measured by flow cytometry. First of all, we determined the optimal ratio of nitrogen to phosphorous (N/P) and DNA concentration. With the increase of N/P ratio and DNA amounts, transfection efficiency increased with a slight variation in cell types. The optimal amounts of 25 kDa L-PEI were determined at N/P ratio 40 and DNA concentration varied among the cell types. In addition, 25 kDa L-PEI worked efficiently and was less toxic than other reagents. However, the efficiency and toxicity of all these reagents varied according to cell types as well as the ratio of DNA to reagents and the amounts of DNA. Our finding illustrates the importance of optimal transfection conditions of 25 kDa L-PEI to obtain maximal transgene expression with less cytotoxicity. Importantly, the optimization of those conditions may make possible to perform transfection cost-effectively and efficiently.     

Blocking the MyD88-dependent pathway protects the myocardium from ischemia/reperfusion injury in rat hearts

We examined whether blocking the MyD88 mediated pathway could protect myocardium from ischemia/reperfusion (I/R) injury by transfecting Ad5-dnMyD88 into the myocardium of rats (n=8) 3 days before the hearts were subjected to ischemia (45min) and reperfusion (4h). Ad5-GFP served as control (n=8). One group of rats was (n=8) subjected to I/R without transfection. Transfection of Ad5-dnMyD88 significantly reduced infarct size by 53.6% compared with the I/R group (15.1+/-3.02 vs 32.5+/-2.59) while transfection of Ad5-GFP did not affect I/R induced myocardial injury (35.4+/-2.59 vs 32.5+/-2.59). Transfection of Ad5-dnMyD88 significantly inhibited I/R-enhanced NFkappaB activity by 50% and increased the levels of phospho-Akt by 35.6% and BCL-2 by 81%, respectively. Cardiac myocyte apoptosis after I/R was significantly reduced by 59% in the Ad5-dnMyD88 group. The results demonstrate that both inhibition of the NFkappaB activation pathway and activation of the Akt signaling pathway may be responsible for the protective effect of transfection of dominant negative MyD88.     

AP-4F,antennapedia peptide linked to an amphipathic alpha helical peptide,increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells

Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.     

Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO2 technique

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO(2) (scCO(2)) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 +/- 73 microm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO(2) processing exhibited a slow release and extended gene expression profile.     

The identification of nuclear proteins that bind the homopyrimidine strand of d(GA.TC)n DNA sequences, but not the homopurine strand.

Alternating d(GA.TC)(n)DNA sequences, which are abundant in eukaryotic genomes, can form altered DNA structures. Depending on the environmental conditions, the formation of (GA.GA) hairpins or [C+T(GA.TC)] and [GA(GA.TC)] intramolecular triplexes was observed in vitro. In vivo, the formation of these non-B-DNA structures would likely require the contribution of specific stabilizing factors. Here, we show that Friend's nuclear extracts are rich in proteins which bind the pyrimidine d(TC)(n)strand but not the purine d(GA)n strand (NOGA proteins). Upon chromatographic fractionation, four major proteins were detected (NOGA1-4) that have been purified and characterized. Purified NOGAs bind single-stranded d(TC)n with high affinity and specificity, showing no significant affinity for either d(GA)n or d(GA.TC)nDNA sequences. We also show that NOGA1, -2 and -3, which constitute the three most abundant and specific NOGA proteins, correspond to the single-stranded nucleic acid binding proteins hnRNP-L, -K and -I, respectively. These results are discussed in the context of the possible contribution of the NOGA proteins to the stabilization of the (GA.GA) and [GA(GA.TC)] conformers of the d(GA.TC)n DNA sequences.     

磁性纳米颗粒作为基因转染载体的研究

通过扫描电子显微镜和Zeta电位仪对磁性纳米颗粒的形貌、粒径、表面电位等进行了表征。利用凝胶电泳阻滞试验分析磁性纳米颗粒与DNA的结合情况,研究磁性纳米颗粒对DNA的保护效果,运用MTT和流式细胞术分析磁性纳米颗粒对细胞的毒性。以绿色荧光蛋白基因为报告基因进行293T细胞的转染,研究磁性纳米颗粒与质粒DNA不同比例条件下对293T细胞的转染效率,并与脂质体(Lipofectamine2000)介导的转染进行比较分析。结果表明,磁性纳米颗粒与DNA可以稳定结合,可以保护DNA免受酶的消化作用,当磁性纳米颗粒与DNA比为1 1时,转染效率最高,优于脂质体(Lipotamine2000)介导的转染,且对细胞的毒害作用小于Lipotamine2000。     

Comparison of non-viral transfection methods in melanoma cell primary cultures

Melanoma primary cultures were transiently transfected via electroporation and lipofection for comparison. Transfection efficiency was superior with electroporation (58+/-9%) as compared to lipofection (23+/-9%) as determined by enhanced green fluorescent plasmid (EGFP) transfection. Secretion of IL-2 persisted for up to 3 weeks after electroporation. The increase in sensitivity against immunologic effector cells by transfection with IL-2 was not significant. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction.     

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.     

Ultrasound-guided intrafollicular treatment in mares

A technique for intrafollicular treatment with a transvaginal ultrasound-guided injection needle was developed using equine chorionic gonadotropin (eCG) as the test substance. An injection was made into one growing follicle of a wave when the follicles were 20 to 23 mm. The treated follicles were injected with 1000 iu of eCG in 0.2 ml saline solution and control follicles were injected with 0.2 ml of the saline vehicle (10 mares per group, 1 follicle per mare). The injection system used an inner 25-gauge needle and an outer 20-gauge needle inserted together through the needle-guide channel of a linear-array trans vaginal transducer. The outer needle was pushed through the vaginal wall and the inner needle was then advanced into the follicle during monitoring on the ultrasound screen. The turbulence in the follicular fluid associated with injection was observable on the screen. Seven follicles were successfully injected in each group. The follicular fluid in the control follicles remained anechoic until the follicle was no longer identifiable or ovulated. All 7 follicles in the eCG group showed ultrasonic indications of luteinization, based on the formation of an echogenic, thickened wall or area. Five of the 7 developed a central area that had the ultrasonic appearance of a blood clot similar to the appearance of a corpus hemorrhagicum. Ovulation was not detected in any of the eCG-treated follicles. The maximum post-treatment diameter of follicles was greater (P < 0.05) for the eCG group (32.7 +/- 3.8 mm) than for the control group (23.4 +/- 1.8 mm). The mean diameter for the first 5 days post-treatment (before the occurrence of an ovulation in any mare) was also greater (P < 0.002) in the eCG group (21.6 +/- 0.8 mm vs 19.6 +/- 0.8 mm). Results indicated that this novel research approach is practical and has potential for studies on folliculogenesis. The technique provides a research model between the extremes of an in vitro culture system and treatment of the whole animal.     

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。     

Electrically induced DNA uptake by cells is a fast process involving DNA electrophoresis.

Simian Cos-1 cells were transfected electrically with the plasmid pCH110 carrying the beta-galactosidase gene. The efficiency of transfection was determined by a transient expression of this gene. When the plasmid was introduced into a cell suspension 2 s after pulse application, the transfection efficiency was shown to be less than 1% as compared with a prepulse addition of DNA. Addition of DNAase to suspension immediately after a pulse did not decrease transfection efficiency, thus the time of DNA translocation was estimated to be less than 3 s. The use of electric treatment medium, in which the postpulse colloid-osmotic cell swelling was prevented, did not affect the transfection efficiency. These results contradict both assumptions of free DNA diffusion into cell through the long-lived pores and of involvement of osmotic effects in DNA translocation. Transfection of cells in monolayer on a porous film allowed creation of the spatial asymmetry of cell-plasmid interaction along the direction of electric field applied. A pulse with a polarity inducing DNA electrophoresis toward the cells resulted in the 10-fold excess of transfection efficiency compared with a pulse with reverse polarity. Ficoll (10%) which increases medium viscosity or Mg2+ ions (10 mM) which decrease the effective charge of DNA, both reduced transfection efficiency 2-3-fold. These results prove a significant role of DNA electrophoresis in the phenomenon considered. The permeability of cell membranes for an indifferent dye was shown to increase noticeably if the cells were pulsed in the presence of DNA. This indicates a possible interaction of DNA translocated with the pores in an electric field, that results in pore expansion.     

Characterization of cationic lipid DNA transfection complexes differing in susceptability to serum inhibition

Background

Cationic lipid DNA complexes based on DOTAP (1,2-dioleoyl-3-(trimethyammonium) propane) and mixtures of DOTAP and cholesterol (DC) have been previously optimized for transfection efficiency in the absence of serum and used as a non-viral gene delivery system. To determine whether DOTAP and DC lipid DNA complexes could be obtained with increased transfection effciency in the presence of high serum concentrations, the composition of the complexes was varied systematically and a total of 162 different complexes were analyzed for transfection efficiency in the presence and absence of high serum concentrations.

Results

Increasing the ratio of DOTAP or DC to DNA led to a dose dependent enhancement of transfection efficiency in the presence of high serum concentrations up to a ratio of approximately 128 nmol lipid/μg DNA. Transfection efficiency could be further increased for all ratios of DOTAP and DC to DNA by addition of the DNA condensing agent protamine sulfate (PS). For DOTAP DNA complexes with ratios of ≤ 32 nmol/μg DNA, peak transfection efficiencies were obtained with 4 μg PS/μg DNA. In contrast, increasing the amount of PS of DC complexes above 0.5 μg PS /μg DNA did not lead to significant further increases in transfection efficiency in the presence of high serum concentrations. Four complexes, which had a similar high transfection efficiency in cell culture in the presence of low serum concentrations but which differed largely in the lipid to DNA ratio and the amount of PS were selected for further analysis. Intravenous injection of the selected complexes led to 22-fold differences in transduction efficiency, which correlated with transfection efficiency in the presence of high serum concentrations. The complex with the highest transfection efficiency in vivo consisted of 64 nmol DC/ 16 μg PS/ μg DNA. Physical analysis revealed a predicted size of 440 nm and the highest zeta potential of the complexes analyzed.

Conclusions

Optimization of cationic lipid DNA complexes for transfection efficiency in the presence of high concentrations of serum led to the identification of a DC complex with high transduction efficiency in mice. This complex differs from previously described ones by higher lipid to DNA and PS to DNA ratios. The stability of this complex in the presence of high concentrations of serum and its high transduction efficiency in mice suggests that it is a promising candidate vehicle for in vivo gene delivery.     

Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography-tandem mass spectrometry and oral bioavailability in rats

We developed a highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS-MS) for a glycopeptide antibacterial drug, vancomycin (VCM), in rat plasma. After precipitating 100 micro l of plasma with 300 micro l of 10% trifluoroacetic acid-methanol (2:1, v/v), the supernatant was diluted with 300 micro l of distilled water and was passed through a filter. LC-MS-MS equipped with electrospray ionization in the positive ion mode used a pair of ions at 725/144 m/z for VCM in the multiple reaction-monitoring mode with a sample injection volume of 20 micro l. The calibration curve had a linear range from 0.01 to 20 micro g/ml when linear least square regression was applied to the concentration versus peak area plot. The drug in the sample was detected within 5 min. Precision, accuracy and limit of quantitation indicated that this method was suitable for the quantitative determination of VCM in rat plasma. Using this method, we defined for the first time that the oral bioavailability of VCM in rats was 0.069%. This method can be applied to basic pharmacokinetic and pharmaceutical studies in rats.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell.

Neumann and coworkers (Neumann, E., M. Schaefer-Ridder, Y. Wang, and P. H. Hofschneider. 1982. EMBO J. 1:841-845) have shown that the efficiency of pulsed electric field (PEF)-induced DNA transfection of mouse L-cells by the thymidine kinase gene is several times higher for the linear DNA than for the closed circular DNA. Transfection of Escherichia coli bacteria by several plasmids indicates that the transfection efficiency was much higher for the closed circular/supercoiled (sc-) and circular/relaxed (cr-) DNA than for the linearized (In-) DNA (Xie, T. D., L. Sun, H. G. Zhao, J. A. Fuchs, and T. Y. Tsong. 1992. Biophys. J. 63:1026-1031). To resolve these conflicting observations, we have systematically examined electrotransfection of NIH3T3 mouse fibroblast by the plasmids, pRSVcat, pRSVneo, and pRSVgpt. Mg(2+)-facilitated surface binding of DNA before, and DNA uptake by 3T3 cells after treatment with PEF, were monitored by 3H-labeled plasmids. Transfection efficiency was evaluated by both the transient expression of chloramphenicol acetyltransferase (cat) activity 2-3 days after, and the permanent expression of neomycin phosphotransferase (neo) and xanthine-guanine phosphoribosyltransferase (gpt) genes in the transformants 2 weeks after the PEF treatment. Our results indicate that cell surface binding and PEF-induced cell uptake of DNA did not depend on the topology of DNA. However, both the transient and the permanent expression of the plasmids were three to five times more efficient for the cr-DNA and the sc-DNA than for the in-DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Effect of xylazine on interovulatory interval and serum progesterone concentrations in the horse mare

The objective of this study was to determine the effect of the alpha(2)-adrenergic agonist, xylazine, on interovulatory interval and progesterone concentrations in the horse mare. Mares were assigned to one of four treatments: Group 1 (controls) received an intramuscular injection (i.m.) of 5 cc saline (n=6), Group 2 received 10 mg prostaglandin F(2alpha) (PGF(2alpha)) i.m. (n=5), Group 3 received 500 mg xylazine i.m. (n=6) and group 4 received an intravenous injection (i.v) of 350 mg xylazine (n=6). Treatment was administered on Day 10 of the estrous cycle (Day 0 = Day of detected ovulation). There was no difference in length of interovulatory interval between PGF(2alpha)-treated mares and control mares (mean +/- SEM; 18.8 +/- 1.0 versus 21.7 +/- 1.6 d). When compared with either xylazine-treated group, PGF(2alpha)-treated mares had a shorter interovulatory interval (18.3 +/- 1.0 d versus 22.2 +/- 0.6 and 22.8 +/- 1.3 d, respectively; P < 0.05). There was no difference in the length of interovulatory interval between control mares and either xylazine-treated group. At the time of treatment all mares had progesterone concentrations > 10 ng/ml, therefore the onset of luteolysis was defined as the day of the estrous cycle when progesterone concentrations decreased below 10 ng/ml. In PGF(2alpha)-treated mares, this event occurred earlier than in any other group (Day 11.2 +/- 0.2 of the estrous cycle versus 16.0 +/- 1.3 for control, Day 15.7 +/- 0.2 for Group 3 and Day 15.2 +/- 0.6 for Group 4; P < 0.002). It was concluded that a single treatment with xylazine, either by an intramuscular or intravenous route, had no significant effect on interovulatory interval or progesterone concentrations in horse mares.     

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56 U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6 µg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79–84%) transfected muscle fibers with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle.