摘要
目的 :观察天然碱性脂 (Stearylamine,SA)脂质体介导绿色荧光蛋白 /碱性成纤维细胞生长因子(GFP/bFGF)基因于不同时间段豚鼠耳蜗中的表达 ,为进一步研究耳聋的基因治疗提供实验基础。方法 :取豚鼠 1 6只 ,分成 4组 ,每组 4只。其中 3只右耳圆窗内注入SA -GFP/bFGF复合物 ,1只同法注入生理盐水作为对照。分别于术后第 2、7、1 4、2 1天取材。在荧光显微镜下观察GFP的表达 ,用免疫组化法检测bFGF的转导情况。结果 :荧光显微镜下见双侧耳蜗于术后第 2天开始部分细胞发出绿色荧光 ,第 7天达到高峰 ,支持细胞及内外毛细胞均显荧光 ,细胞轮廓清晰 ;第 1 4天开始减弱 ,第 2 1天消失。免疫组化染色显示 ,除血管纹外 ,耳蜗各回Corti器、螺旋韧带、螺旋缘及螺旋神经节细胞均有高浓度的表达产物 ,对照动物呈阴性表达。结论 :SA脂质体介导的GFP/bFGF基因单耳给药双侧耳蜗均有高效表达 ,为进一步研究基因治疗耳聋提供了可能。
Objective:To observe the expression of stearylamine (SA) liposome mediated GFP/bFGF gene in guinea pig cochlea in different period of administration,and to provide the groundwork for further studies of the gene therapy in deafness.Method:16 guinea pigs were divided randomly into 4 groups.3 of each group were infused with SA-GFP/bFGF complexes through round window of the right ear,another one with intracochlear saline infusion served as a negative control.The animals of 4 groups then sacrificed on the 2nd,7th,14th and 21st day,respectively.The expression of GFP in cochlea was observed by fluorescent microscope and the transduction of bFGF gene was assayed via immunohistochemistry (ABC method).Result:There were expression of GFP in both cochleae of the guinea pig.It began to express in the supportine cells,outer and inner hair cells at 2nd day after the administration,reached to the highest level at 7th day and reduced thereafter.At the 21st day,it disappeared finally.Animals infused with SA-GFP/bFGF complexes showed intense immunohistochemical reactivity in the spiral limbus,spiral ligament,spiral ganglion cells and the organ of Corti in both infused cochlea and contralateral ear.Cochleae from saline-infused animals were devoid of the DAB stain.The changes of the expressive intensity were similar to the result which observed by fluorescent microscope.Conclusion:SA liposome mediated GFP/bFGF gene that is injected in one ear can express highly in both cochleae of the guinea pig,which suggest the feasibility of gene therapy in deafness.
