超声微泡介导CNTF基因眼内转染对视神经损伤大鼠作用的研究

Effects of ciliary neurotrophic factor mediated by ultrasound microbubbles intraocular transfer after optic nerve injury in rat

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．

Background The key premise of genetic research and treatment is to select the desired gene vector,ultrasound microbubbles as a new type of gene vector can safe, fast and effectively enhance the gene transfection and expression by reversibility increasing the permeability of cells. Objective This study was to observe the effects of ciliary neurotrophic factor （CNTF） gene mediated by ultrasound microbubbles intraocular transfer on visual function and retinal ganglion cell（RGCs） after optic nerve injury. Methods Fifty-five adult Sprague-Dawley（SD） rats were divided into 6 groups randomly, including normal control group （ n = 5 ） , sham injury group （ n = 10 ） , simple injury group （ n = 10 ） , naked plasmid group （ n = 10 ） , plasmid ＋ultrasonic irradiation group （ n = 10） and ultrasound microbubbles group（n= 10）. The model of optic nerve injury was made by forceps clip on the right optic nerve,and the corresponding intervene was performed in different groups. Flash visual evoked potential（ F-VEP） was recorded in all rats before injury and 7 days after injury to evaluate the change of visual function, and survival of RGCs of rats was assessed by retrogradely label of FluoroGold （FG）. Expression of CNTF mRNA in rat retina was determined by real time-PCR. Results Latency of F-VEP P~ wave in naked plasmid group,plasmid＋ ultrasonic irradiation group and ultrasound microbubbles group were shorter than simple injury group, and that of ultrasound microbubbles group were shorter than naked plasmid group and plasmid ＋ ultrasonic irradiation group, showing significant difference among them （P〈0.05）. The amplitude of PI wave in naked plasmid group, plasmid ＋ ultrasonic irradiation group and ultrasound microbubbles group were elevated in comparison with simple injury group, and that of ultrasound microbubbles group was higher than naked plasmid group and plasmid＋ultrasonic irradiation group（P〈0.05）. Significant difference in Pl amplitude was also seen between ultrasound microbubbles group and control group（P〈0.05）. The RGCs in simple injury group was least among various groups （P〈0.05）. In ultrasound microbubbles group,the RGCs were more than naked plasmid group and plasmid ＋ultrasonic irradiation group, but less than control group and sham injury group （P〈0.05）. RT-PCR revealed that the relative expression values of CNTF mRNA in ultrasound microbubbles group were higher than those of control group, sham injury group, simple injury group,naked plasmid group and plasmid ＋ultrasonic irradiation group with the statistically significant difference among them （ P 〈 0. 05 ）. Conclusion Under irradiation of low-frequency ultrasound wave, ultrasound-mediated microbubbles destruction can increase intraocular transfection and expression of CNTF genc and promote the recovery of visual function.