Hepatocyte Growth Factor Loaded Ultrasound Microbubble Contrast Facilitates The Restoration Of Injuried Facial Nerve Of Rats

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.