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超声微泡介导增强型绿色荧光蛋白基因转染视网膜神经节细胞的体内实验 总被引:2,自引:0,他引:2
BACKGROUND: Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal ganglial cells (RGCs). OBJECTIVE: To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING: In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS: Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS: A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls (n = 5) were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group (n = 15) was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group (n = 15), the eyes were irradiated with low-energy ultrasound wave (0.5 W/cm^2) for a total of 60 seconds (irradiated for 5 seconds, at 10-second intervals) immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group (n = 15), the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES: After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS: The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION: Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs. 相似文献
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超声微泡造影剂介导脑源性神经营养因子联合转染视网膜和视皮质对视神经损伤后视网膜神经节细胞的保护作用 总被引:1,自引:1,他引:0
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Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P<0.01). The difference of the number of RGC among the four groups was not significant(F= 0. 877, P>0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats. 相似文献
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Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P<0.01). The difference of the number of RGC among the four groups was not significant(F= 0. 877, P>0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats. 相似文献
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目的评价中国人群let-7基因rs10877887多态性与癌症风险之间的相关性。方法通过检索PubMed、Embase、万方数据库中的相关文献(检索日期截止到2016年12月31日),共纳入4个符合标准的病例-对照研究,包含2 754例病例和3 481例对照。采用优势比(OR)和95%可信区间(95%CI)进行let-7基因rs10877887多态性与癌症风险之间的相关性评价,同时也对敏感性和发表偏倚进行分析。结果 Meta分析结果表明,在Dominant模型(CC+CTvs.TT)下,let-7基因rs10877887多态性与癌症风险之间存在显著相关性(CC+CT vs.TT:OR=0.90,95%CI=0.82~1.00,P=0.048),可降低癌症发生的总体风险。结论基于目前研究的Meta分析结果表明,中国人群中let-7基因rs10877887多态性与癌症风险具有显著相关性,可降低癌症发生的总体风险。 相似文献
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背景选择理想的基因载体是当前基因研究和治疗的前提与关键,超声微泡造影剂作为一种新型的基因载体,可安全、快速、有效地增强目的基因的转染和表达。目的观察超声微泡造影剂介导睫状神经营养因子(CNTF)基因转染视神经损伤大鼠对视功能及视网膜神经节细胞(RGCs)存活的影响。方法SD大鼠60只随机分为正常对照组、假伤组、单纯损伤组、单纯质粒组、质粒+超声组、超声微泡组。采用钳夹大鼠右眼视神经法制作视神经损伤大鼠模型,然后各处理组大鼠分别接受相应的干预处理。质粒采用玻璃体腔注射法注入,超声则采用辐照法进行干预。造模前1d和损伤后第7天检测每组大鼠闪光视觉诱发电位(F—VEP),并于第7天处死各组大鼠。应用荧光金逆行标记法计数各组大鼠RGCs存活数,采用实时定量聚合酶链反应(real—timePCR)检测大鼠视网膜中CNTFmRNA的表达量。结果损伤后第7天,单纯损伤组大鼠F—VEPP.波的隐含时较单纯质粒组、质粒+超声组和超声微泡组明显延长,超声微泡组P,波的隐含时短于单纯质粒组和质粒+超声组,差异均有统计学意义(P〈0.05)。单纯质粒组、质粒+超声组和超声微泡组F—VEPP,波的振幅均高于单纯损伤组,超声微泡组高于单纯质粒组和质粒+超声组,差异均有统计学意义(P〈0.05)。超声微泡组大鼠与正常对照组比较P,波振幅降低,差异有统计学意义(P〈0.05)。单纯质粒组、质粒+超声组和超声微泡组平均RGCs数目均明显高于单纯损伤组,超声微泡组平均RGCs数多于单纯质粒组和质粒+超声组,但少于对照组及假伤组,差异均有统计学意义(P〈0.05)。超声微泡组CNTFmRNA表达量高于正常对照组、假伤组、单纯损伤组、单纯质粒组和质粒+超声组,差异均有统计学意义(P〈0.05)。结论超声微泡能增强CNTF基因在眼内的转染及表达,对视神经损伤大鼠RGCs早期有明显的保护作用,可有效促进视功能的恢氪. 相似文献
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目的 观察超声微泡造影剂提高重组腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因在体内转染视网膜神经节细胞(RGC)的效率.方法 Sprague-Dawley大鼠40只,随机分为A、B、C、D 4组,每组各10只大鼠.A组玻璃体腔注射磷酸盐缓冲液(PBS)5μl;B组玻璃体腔注射rAAV2-EGFP 5 μl;C组玻璃体腔注射rAAV2-EGFP 5 μl后立即用超声波辐照眼球;D组玻璃体腔注射rAAV2-EGFP和微泡造影剂的混悬液5 μl后立即用超声辐照眼球.玻璃体腔注射21 d后,3%荧光金逆行标记RGC.逆行标记7 d后取出眼球,制作视网膜铺片及视网膜冰冻切片,在激光共聚焦显微镜下观察并计算EGFP基因在RGC的转染率及在RGC表达的平均吸光度[A,旧称光密度(OD)]值;用RGC计数判断损伤情况.结果 荧光金标记RGC后,B、C、D 3组均可观察到RGC中有EGFP表达.其中,D组平均A值为95.02±7.25,RGC转染率为(20.10±0.74)%、均明显高于B、C组,差异有统计学意义(F平均A值=25.970,F转染率=25.799;P<0.01);A、B、C、D组RGC计数差异无统计学意义(F=0.877,P>0.05).结论 在低频和一定能量的超声和微泡造影剂作用下,rAAV2介导EGFP基因转染体内RGC的效率能够安全、有效地提高. 相似文献
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