【作者】 郝亚宁；

【导师】 骆文龙；

【作者基本信息】 重庆医科大学 ， 耳鼻咽喉头颈外科， 2013， 博士

【摘要】 由于面神经的解剖结构和功能比较特殊，在受到外伤、手术操作、炎症及肿瘤侵袭时容易损伤，且神经再生是一个极为复杂的过程，所以，目前最紧迫的是要找出一种新的方法以提高面神经的再生速度。通常来讲，神经生长的微环境对于受损神经能否有效的恢复至关重要。神经损伤的基因治疗便是从这一方面着手，通过基因治疗将对神经生长有促进作用的神经生长因子基因导入受损的神经周围，但目前用于基因治疗的有效载体仍存在各种各样的缺陷：病毒载体虽然转染效率很高，但由于其自身较高的毒性，进入人体内会诱导强烈的免疫应答而限制了其应用。非病毒载体虽然毒性比较小，但转染效率较低也受到了限制。那么我们需要探索一种既安全又高效的新的基因载体。近几年的研究表明，超声已经从诊断领域逐渐向治疗领域发展，例如脂质微泡可以在一定强度和频率的超声作用下，顺利的将基因或药物转染到靶组织或靶器官，从而达到靶向治疗的作用，这一发现已经收到越来越多的学者们的注意[1-3]。本课题是以肝细胞生长因子（HGF）作为目的基因，然后在自制超生微泡造影剂的基础上，优化工艺，制备一种能与HGF基因高效结合的脂质体微泡造影剂，然后运用超声靶向破坏微泡(UTMD)介导HGF治疗大鼠面神经损伤，最后采用神经电生理、westernblot以及RT-PCR观察治疗后HGF基因在受损面神经的表达情况及治疗效果，并初步研究超声微泡的作用机理。为研究和评价超声微泡造影剂的性质和作用提供一种新的方法，从而为超声微泡作为一种基因或药物的载体提供更为可靠的实验依据。第一部分携基因的微泡造影剂的制备与性质检测目的制备一种可以携带基因的脂质微泡造影剂，并且研究这种脂质微泡携带基因的能力及其物理和化学的性质方法通过机械振荡的方法制备脂质微泡造影剂，经过60Co射线灭菌后，在显微镜下观察脂质微泡造影剂的浓度、形态、电位改变及其平均大小，最后检测这种脂质微泡造影剂与目的基因结合能力的大小。结果脂质微泡造影剂的平均颗粒直径范围在2.21-6.15μm内，脂质微泡的分布相对均匀。浓度为3.16×109个/ml;脂质微泡制成后经60Co射线消毒灭菌后，通过显微镜能够观察到脂质微泡的形态，颗粒直径大小以及电位等均未见明显的改变；基因量效优化结果显示，脂质微泡的基因结合率最高位23%，基因结合量最大为0.44mg/ml。结论本实验所制备的脂质微泡已经达到了标准微泡造影剂的规格，而且这种脂质微泡造影剂的理化性质稳定，制作过程简单，携带基因的效率非常高，因此是一种理想的微泡造影剂，可以用作一种新型的基因载体。第二部分超声辐照微泡介导HGF促进大鼠损伤面神经的修复目的观察超声辐照微泡促进肝细胞生长因子（HGF）转染大鼠面神经的可行性方法制作大鼠面神经受损模型，将HGF作为目的基因；随机选取48只SD大鼠分成4组：A组：单纯手术组（PBS）；B组：HGF+微泡组（HGF+MB）；C组：HGF+超声组（HGF+US）；D组：HGF+超声+微泡组（HGF+US+MB），每组12只。将含有HGF基因的微泡经静脉注入体内，局部给予超声照射。在第7天、14天、21天、28天后，观察各组大鼠的一般情况，检测面神经的传导速度、潜伏期及神经电位波幅的变化。提取面神经组织，通过western blot及RT-PCR检测HGF的蛋白及mRNA的表达水平。结果经行为学检测，D组（HGF+超声+微泡组）大鼠在触须摆动及鼻尖位置均明显优于其余3组。D组（HGF+超声+微泡组）面神经传导速度、神经电位波幅明显高于其余3组，潜伏期明显低于其余3组(p＜0.05)。D组（HGF+超声+微泡组）中HGF蛋白和mRNA的表达量明显高于其他3组(p＜0.05)。其中C组（HGF+超声组）的HGF蛋白和mRNA的表达量也明显高于A（单纯手术组）、B（HGF+微泡组）两组。结论超声介导携基因微泡促进HGF转染面神经被证明是一种效率较高的基因转染方法，在一定频率和强度的超声作用下，超声微泡可以将目的基因转染损伤后的面神经,从而有利于面神经的修复。由于损伤后的面神经在超声辐照微泡的作用下进行的基因转染并没有损害周围正常的面神经组织，因此超声辐照微泡可以安全、高效的将基因或药物送入损伤后的面神经，为面神经损伤的治疗提供了一条新型、安全、高效的方法。更多还原

【Abstract】 Facial nerve due to its anatomical structure and function of a special,facial nerve may be injuried due to trauma, inflammation, tumor or surgicaloperation and nerve regeneration is a very complex process, therefore,looking for new repair methods to improve nerve regeneration speed andquality is the current problem. Gene therapy plays a significant role inimproving nerve damage, but the currently available vectors (viruses,liposomes, etc.) may produce potentially harmful to the body, there is aneed to explore more suitable gene carrier. In recent years, the ultrasonicfield appears a new carrier: ultrasound contrast agent targeted therapyachieved gratifying results, making it more and more attention of themedical profession.This research is hepatocyte growth factor (HGF) as the target geneand then manufacture microbubbles with efficient binding to the HGF genefor preparing a liposomal microbubbles. Then use ultrasound-targetedmicrobubble destruction (UTMD) mediated HGF treatment of facial nerveinjury in rats, the last expression HGF mRNA and protein and facial nerve treatment effects observed after treatment by the neurophysiological,westernblot well as RT-PCR.To the study and evaluation of ultrasoundtargeted microbubble destruction mediated gene transfection provides anew ways and means to provide a reliable experimental basis for the genetherapy for facial nerve.PART I EXPERIMENTAL STUDY ON PREPARATIONAND CHARACTERISTICS OF THE GENE-LOADEDLIPID MICROBUBBLESObjective To prepare a lipid ultrasound microbubble contrast agentcan be used as a gene carrier, its physical and chemical properties and itscontained the genetic ability to study.Method Prepared by a method using mechanical oscillation can beused as a gene carrier lipid ultrasound microbubbles observed60Coy raysterilization before and after the appearance of the contrast agent, shape,concentration, average particle size, and the potential change, testing itsability to combine genes.Results The average particle diameter in the range of lipidmicrobubbles in2.21-6.15μm, relatively uniform particle size distribution.Its concentration is about3.16×109/ml; Under the microscope after60Coγ-ray sterilization were no significant changes observed microbubbles form,particle size and potential; Gene combined amount effective optimizationresults show that the maximum DNA binding of the contrast agent efficiency was23%, and the largest amount of DNA binding to0.44mg/ml.Conclusion The prepared lipid microbubbles has reached the standardspecifications of the microbubbles, and this lipid microbubbles stablephysical and chemical properties, the production process is simple,gene-carrying efficiency is very high, and therefore is aideal microbubblecontrast agent, can be used as a novel gene vector.PART II: EXPERIMENTAL RESEARCH ON THERESTORATION OF INJURIED FACIAL NERVEINDUCED BY HGF DIRECTEDULTRASOUND-TARGETED MICROBUBBLEDESTRUCTIONObjective Observation the feasibility and effectiveness of ultrasoundmicrobubble mediated hepatocyte growth factor (HGF) gene to promotethe damaged facial nerve repair.Method Create a model of facial nerve injury in rats, Hepatocytegrowth factor (HGF) as a therapeutic gene.40SD rats were randomlydivided into four groups: Group A: surgery alone group (PBS); B,HGF andmicrobuble (HGF+MB) group; C,HGF and ultrasound (HGF+US) group;D,HGF+ultrasound+microbubble (HGF+MB/US) group. local injection ofmicrobubbles containing hepatocyte growth factor and Ultrasonicirradiation after facial nerve injury in rats, At7,14,21and28days,respectively, to observe the general condition of the rats and detect the facial nerve conduction velocity, latency and nerve potential amplitudechanges in each group. Rats in each group were sacrificed, and take thenerve tissue, HGF expression was detected by Western blot. HGF mRNAexpression was detected by real-timeRT-PCR,ResultsThe behavior alanalyses results showed thatThenosetipposition and vibrissae movement in D group were better thanthat of the other groups. The NCV and potential amplitude of the facialnerve in D group were increased, but the potentiallatency was decreased.HGF expressions was higher than those of other groups in D gouop. Theexpression levels of HGF mRNA in D group was significant lyhigher thanthat of the other groups(p＜0.05).Conclusion Ultrasound-mediated mircobuble could increase thetransfection of HGF ininjury facial nerve safely and facilitatesitsrestoration.更多还原