豚鼠耳蜗内基因治疗的安全性研究

Safety of gene therapy within cochlea, experimental study with guinea pig

目的 观察携带人神经营养素 3基因的腺病毒 (Ad NT3)导入豚鼠耳蜗内的安全性和NT3基因的表达以及导入手术对听觉功能的影响。方法 将 2 0只豚鼠随机分为 2组 ,经耳蜗底周鼓阶钻孔向外淋巴腔内导入目的基因 ,其中 10只导入Ad NT3,10只导入人工外淋巴液。导入前、导入后即刻及导入后 7d分别检测听觉脑干诱发电位。导入后 7d应用免疫组化方法观察Ad NT3在蜗内的转染。结果 Ad NT3导入前、导入后即刻、导入后 7dABR III波反应阈分别为 35dBSPL± 3 7dBSPL、4 0dBSPL± 2 5dBSPL和 35dBSPL± 3 0dBSPL ,10 0dBSPL短声下的ABR III波潜伏期分别为2 812ms± 0 0 87ms、2 793ms± 0 0 93ms和 2 82 2ms± 0 10 2ms。导入手术操作以及腺病毒导入对于耳蜗结构和听力无明显影响。应用免疫组化方法可以检测到耳蜗内NT3反应产物。结论 应用微量注射方法经鼓阶造孔将目的基因导入外淋巴腔对耳蜗形态及听觉功能无明显影响 ,耳蜗内局部实施基因治疗是安全可行的。

Objective To observe the safety of inoculation of recombinant adenovirus with human neurotrophin 3 (Ad NT3) into cochlea and its effect on hearing function. Methods Twenty healthy guinea pigs were randomly divided into two groups: ten animals were inoculated with Ad NT3 into the perilymphatic space after a small hole was drilled in the bony wall near tympanic scala of the base turn, while the other ten were introduced with artificial perilymphatic fluid (APF) as controls. Auditory brainstem response (ABR) were recorded before, immediately after and seven days after the inoculation. Seven days after the surgery, the animals were killed. Frozen sections and colloidin sections of cochlea were made and observed under microscope. Immunohistochemistry was used to detect the expression of NT3 protein in cochlea. Results The mean ABR threshold values were (35±3 7) dB SPL and (35±3 2) dB SPL before the inoculation, (40±2 5) dB SPL and (40±3 0) dB SPL immediately after the inoculation, and (35±3 0) dB SPL and (35±3 0) dB SPL seven days after inoculation in the Ad NT3 group and AFP group respectively (all P >0 05). The ABR III latency of 100dB SPL click was (2 812±0 087) ms and (2 824±0 121) ms before inoculation, (2 793±0 093) ms and (2 796±0 095) ms immediately after inoculation, and (2 822±0 102) ms and (2 834±0 135) ms seven days after inoculation in Ad NT3 and AFP groups respectively ( P >0 05). No difference of ABR threshold and latency was observed at different time points in both groups. Ad NT3 protein was expressed within each turn of the cochlea, including spiral limbus, spiral ligament and ganglion cells. The cochlear structure remained intact with no sign of infection or cell degeneration. Conclusion Gene therapy by means of introduction of trace AdNT3 into the perilymphatic space through pore in tympanic scala is safe, causing no structure change or hearing loss.