基于羟基磷灰石的纳米基因载体的改良及其内耳基因转染的实验研究
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摘要
目的：1)观察新制备的经聚乙烯亚胺(polyethylenimine, PEI)表面修饰的羟基磷灰石纳米载体(hydroxyapatite nanoparticles nHAT)(PEI-nHAT)的生物学性能,评估其体外结合与保护DNA的能力；2)评估携带增强型绿色荧光蛋白报告基因(EGFP)及神经营养素3治疗基因(NT-3)的PEI-nHAT载体-基因复合物(PEI-nHAT-pEGFPC2-NT3)经完整圆窗膜途径向耳蜗转染的安全性及有效性；3)评估PEI-nHAT-pEGFPC2-NT3经完整圆窗膜途径向前庭转染的安全性及有效性。
方法：1)应用化学共沉淀-水热合成法制备nHAT,用PEI对其进行表面修饰后,对PEI-nHAT进行透射电镜观察并行Zeta电位测定；观察PEI-nHAT在体外的分散性及稳定性；并在不同浓度下观察其与DNA结合能力以及保护DNA抗核酸酶消化的能力。2)将南美栗鼠分成转染实验组、空载体转染组及生理盐水对照组三组,将其麻醉后,行枕后径路打开术耳听泡,分别将20μl PEI-nHAT-pEGFPC2-NT3复合物或20μl PEI-nHAT混悬液或20μ1生理盐水经完整圆窗膜途径向耳蜗转染。在手术前及转染48小时后分别测试双耳在1kHz、4kHz8kHz、12kHz及16kHz处短纯音诱发的听性脑干反应(Auditory Brainstem Response, ABR)及畸变产物耳声发射(Distortion Product Otoacoustic Emissions, DPOAE)。听力学检测完后将动物处死,双侧耳蜗固定、取材,行免疫组织化学、免疫荧光染色、常规耳蜗铺片及冰冻切片,应用激光共聚焦荧光显微镜进行耳蜗组织学和形态学观察及绿色荧光蛋白定位表达观察。3)将南美栗鼠分成转染实验组、空载体转染组及生理盐水对照组三组,将其麻醉后,行枕后径路打开术耳听泡,分别将20μl PEI-nHAT-pEGFPC2-NT3复合物或20μ1PEI-nHAT混悬液或20μ1生理盐水经完整圆窗膜途径向内耳转染。转染48小时后将动物处死,双侧内耳前庭器官固定、取材,行免疫组织化学、免疫荧光染色、常规铺片及冰冻切片,应用激光共聚焦荧光显微镜进行前庭器官组织学和形态学观察及绿色荧光蛋白在前庭中的定位表达观察。
结果：1)PEI-nHAT颗粒在电镜下形态为短棒状,粒径均匀,分散性及稳定性好,其平均粒径(X±SD)为73.09±27.32nm；在pH为7、浓度为250μg/ml的条件下,PEI-nHAT的Zeta电位(X±SD)为42.46±10.9mV; PEI-nHAT颗粒在125μg/ml-2000μg/ml浓度范围内都有较好的DNA结合能力,并且PEI-nHAT在上述浓度范围内均能良好地保护DNA不被DNase I消化。2)各组动物在转染前及转染后48小时在1kHz、4kHz、8kHz、12kHz及16kHz处短纯音诱发的ABR阈值无明显变化,各频率DPOAE平均幅值差异也无统计学意义(P>0.05)。基因转染48小时后,各组动物耳蜗内、外毛细胞、血管纹上皮及螺旋神经节细胞形态规则,排列整齐,未见明显破坏现象；转染实验组动物在耳蜗基底膜的外柱细胞区域、部分血管纹上皮细胞、螺旋神经管内的部分细胞中出现绿色荧光表达,但转染率不高。3)各组动物术后48小时内均无偏头、步态不稳、强迫环绕运动等前庭功能障碍表现。各组动物前庭毛细胞形态规则,无缺失,纤毛束排列整齐；周围支持细胞大小形态正常。在转染实验组中,浓密的绿色荧光信号存在于前庭的暗细胞区及其过渡细胞区,在椭圆囊斑、球囊斑及壶腹嵴的毛细胞区域中也出现不同程度的绿色荧光。
结论：1)PEI-nHAT载体表面带正电荷,其体外的分散性和稳定性良好；2)PEI-nHAT载体在体外能有效结合和保护其所携带的重组基因质粒免受酶化学物质的破坏；3)完整圆窗膜途径基因转染对耳蜗的形态学及听力学功能无明显不良影响,该途径是进行耳蜗和前庭局部基因转染的一条安全有效的导入方式；4)前庭暗细胞是PEI-nHAT载体在内耳的一种特异性靶细胞,它可能具有主动摄取PEI-nHAT载体的能力,其原理尚待进一步研究；5)PEI-nHAT是一种具有研究价值的用于内耳基因治疗载体的无机纳米材料。
Objective:1) To investigate the biological properties of hydroxyapatite nanoparticles surface-modified by polyethylenimine (PEI-nHAT), and the DNA-binding and DNA protection capacity of PEI-nHAT in vitro.2) To investigate the safety and effectiveness on transfection of PEI-nHAT carrying enhanced green fluorescent protein with neurotrophin-3(pEGFPC2-NT3) in the cochlea via intact round window membrane (RWM).3) To investigate the safety and effectiveness on transfection of PEI-nHAT-pEGFPC2-NT3in the vestibular system via intact RWM.
Methods:1) nHAT were synthesized by a chemical coprecipitation-hydrothermal synthesis method. Transmission electron microscopy (TEM) observation and Zeta potential detection were carried out after surface modification with PEI. Dispersibility and stability of PEI-nHAT were also observed, the capacity of PEI-nHAT on DNA-binding and DNA protection against nuclease digestion were assessed in different concentration as well.2) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The tone burst-evoked Auditory Brainstem Responses (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) were recorded in1kHz、4kHz、8kHz、12kHz and16kHz pre-operation and48h post-transfection. Chinchillas were sacrificed after auditory function evaluation. The temporal bones containing cochleae were fixed for surface preparations and cryosections. Immunohistochemistry and immunofluorescence staining were carried out to observe the morphologic changes and localize the EGFP expression in cochlea under confocal microscope.3) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The vestibular samples were fixed and then prepared as surface preparations and cryosections after animals were sacrificed. The morphologic observations of vestibular organs and localized observation of EGFP expression were carried out after immunohistochemistry and immunofluorescence staining.
Results:1) After surface modification with PEI, the shape of PEI-nHAT was similar to a short rod with uniform size. The PEI-nHAT showed good dispersivity and stability in solution. The mean size (Mean±SD) of PEI-nHAT was73.09±27.32(nm). At pH7, the Zeta potential of PEI-nHAT was42.46±10.9mV at the concentration of250μg/ml. PEI-nHAT showed good capacity of DNA-binding and DNA protection against DNase I digestion at concentrations from125μg/ml to2000μg/ml.2) Neither significant threshold shift in tone burst-evoked ABR nor significant amplitude changes in DPOAE occurred at all tested frequencies between pre-operation and48h post-transfection in each group (P>0.05). The cells in cochlear basilar membrane, stria vasularis and cochlear spiral ganglions appeared morphologically normal after48h transfection in each group. EGFP expression was detected in some outer pillar cells in the organ of Corti, a few marginal cells in stria vascularis, some cells in Rosenthal's canal in the PEI-nHAT-pEGFPC2-NT3transfected group, although the transfecton efficiency in cochlea was not high.3) There was no vestibular dysfunction occurred such as step instability, head tilting, rolling or circling after48h transfection in each group. Vestibular hair cells and the supporting cells appeared morphologically normal after48h transfection in each group. Abundant, condensed green fluorescence was found in the zone of dark cells and transitional zone on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells.
Conclusion:1) PEI-nHAT are positive charged and have good dispersivity and stability;2) PEI-nHAT have good capacity of DNA-binding and DNA protection against nuclease digestion at concentrations from125μg/ml to2000μg/ml.3) The approach of gene transfer through intact RWM has no affect on the morphology of cochlea or auditory function, which can serve as an safe and effective way of transferring gene into cochlea and vestibule.4) Vestibular dark cell is the specific targeted cell of PEI-nHAT for gene therapy in the inner ear. It might have the ability of actively uptaking PEI-nHAT-pEGFPC2-NT3complex, although the detailed mechanism is not clear.5) PEI-nHAT are innogarnic materials which could be a potential vector of gene therapy for the inner ear diseases, especially for the vestibular disorders.
方法：1)应用化学共沉淀-水热合成法制备nHAT,用PEI对其进行表面修饰后,对PEI-nHAT进行透射电镜观察并行Zeta电位测定；观察PEI-nHAT在体外的分散性及稳定性；并在不同浓度下观察其与DNA结合能力以及保护DNA抗核酸酶消化的能力。2)将南美栗鼠分成转染实验组、空载体转染组及生理盐水对照组三组,将其麻醉后,行枕后径路打开术耳听泡,分别将20μl PEI-nHAT-pEGFPC2-NT3复合物或20μl PEI-nHAT混悬液或20μ1生理盐水经完整圆窗膜途径向耳蜗转染。在手术前及转染48小时后分别测试双耳在1kHz、4kHz8kHz、12kHz及16kHz处短纯音诱发的听性脑干反应(Auditory Brainstem Response, ABR)及畸变产物耳声发射(Distortion Product Otoacoustic Emissions, DPOAE)。听力学检测完后将动物处死,双侧耳蜗固定、取材,行免疫组织化学、免疫荧光染色、常规耳蜗铺片及冰冻切片,应用激光共聚焦荧光显微镜进行耳蜗组织学和形态学观察及绿色荧光蛋白定位表达观察。3)将南美栗鼠分成转染实验组、空载体转染组及生理盐水对照组三组,将其麻醉后,行枕后径路打开术耳听泡,分别将20μl PEI-nHAT-pEGFPC2-NT3复合物或20μ1PEI-nHAT混悬液或20μ1生理盐水经完整圆窗膜途径向内耳转染。转染48小时后将动物处死,双侧内耳前庭器官固定、取材,行免疫组织化学、免疫荧光染色、常规铺片及冰冻切片,应用激光共聚焦荧光显微镜进行前庭器官组织学和形态学观察及绿色荧光蛋白在前庭中的定位表达观察。
结果：1)PEI-nHAT颗粒在电镜下形态为短棒状,粒径均匀,分散性及稳定性好,其平均粒径(X±SD)为73.09±27.32nm；在pH为7、浓度为250μg/ml的条件下,PEI-nHAT的Zeta电位(X±SD)为42.46±10.9mV; PEI-nHAT颗粒在125μg/ml-2000μg/ml浓度范围内都有较好的DNA结合能力,并且PEI-nHAT在上述浓度范围内均能良好地保护DNA不被DNase I消化。2)各组动物在转染前及转染后48小时在1kHz、4kHz、8kHz、12kHz及16kHz处短纯音诱发的ABR阈值无明显变化,各频率DPOAE平均幅值差异也无统计学意义(P>0.05)。基因转染48小时后,各组动物耳蜗内、外毛细胞、血管纹上皮及螺旋神经节细胞形态规则,排列整齐,未见明显破坏现象；转染实验组动物在耳蜗基底膜的外柱细胞区域、部分血管纹上皮细胞、螺旋神经管内的部分细胞中出现绿色荧光表达,但转染率不高。3)各组动物术后48小时内均无偏头、步态不稳、强迫环绕运动等前庭功能障碍表现。各组动物前庭毛细胞形态规则,无缺失,纤毛束排列整齐；周围支持细胞大小形态正常。在转染实验组中,浓密的绿色荧光信号存在于前庭的暗细胞区及其过渡细胞区,在椭圆囊斑、球囊斑及壶腹嵴的毛细胞区域中也出现不同程度的绿色荧光。
结论：1)PEI-nHAT载体表面带正电荷,其体外的分散性和稳定性良好；2)PEI-nHAT载体在体外能有效结合和保护其所携带的重组基因质粒免受酶化学物质的破坏；3)完整圆窗膜途径基因转染对耳蜗的形态学及听力学功能无明显不良影响,该途径是进行耳蜗和前庭局部基因转染的一条安全有效的导入方式；4)前庭暗细胞是PEI-nHAT载体在内耳的一种特异性靶细胞,它可能具有主动摄取PEI-nHAT载体的能力,其原理尚待进一步研究；5)PEI-nHAT是一种具有研究价值的用于内耳基因治疗载体的无机纳米材料。
Objective:1) To investigate the biological properties of hydroxyapatite nanoparticles surface-modified by polyethylenimine (PEI-nHAT), and the DNA-binding and DNA protection capacity of PEI-nHAT in vitro.2) To investigate the safety and effectiveness on transfection of PEI-nHAT carrying enhanced green fluorescent protein with neurotrophin-3(pEGFPC2-NT3) in the cochlea via intact round window membrane (RWM).3) To investigate the safety and effectiveness on transfection of PEI-nHAT-pEGFPC2-NT3in the vestibular system via intact RWM.
Methods:1) nHAT were synthesized by a chemical coprecipitation-hydrothermal synthesis method. Transmission electron microscopy (TEM) observation and Zeta potential detection were carried out after surface modification with PEI. Dispersibility and stability of PEI-nHAT were also observed, the capacity of PEI-nHAT on DNA-binding and DNA protection against nuclease digestion were assessed in different concentration as well.2) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The tone burst-evoked Auditory Brainstem Responses (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) were recorded in1kHz、4kHz、8kHz、12kHz and16kHz pre-operation and48h post-transfection. Chinchillas were sacrificed after auditory function evaluation. The temporal bones containing cochleae were fixed for surface preparations and cryosections. Immunohistochemistry and immunofluorescence staining were carried out to observe the morphologic changes and localize the EGFP expression in cochlea under confocal microscope.3) Eight normal adult chinchillas of both sexes were used in this study. The right ears from six animals were used as the experimental group for local application of PEI-nHAT-pEGFPC2-NT3onto the intact RWM, and the left were treated with the same volume of empty vector (PEI-nHAT) as self control. Both ears from another two animals were treated with the same volume of normal saline as blank control. The vestibular samples were fixed and then prepared as surface preparations and cryosections after animals were sacrificed. The morphologic observations of vestibular organs and localized observation of EGFP expression were carried out after immunohistochemistry and immunofluorescence staining.
Results:1) After surface modification with PEI, the shape of PEI-nHAT was similar to a short rod with uniform size. The PEI-nHAT showed good dispersivity and stability in solution. The mean size (Mean±SD) of PEI-nHAT was73.09±27.32(nm). At pH7, the Zeta potential of PEI-nHAT was42.46±10.9mV at the concentration of250μg/ml. PEI-nHAT showed good capacity of DNA-binding and DNA protection against DNase I digestion at concentrations from125μg/ml to2000μg/ml.2) Neither significant threshold shift in tone burst-evoked ABR nor significant amplitude changes in DPOAE occurred at all tested frequencies between pre-operation and48h post-transfection in each group (P>0.05). The cells in cochlear basilar membrane, stria vasularis and cochlear spiral ganglions appeared morphologically normal after48h transfection in each group. EGFP expression was detected in some outer pillar cells in the organ of Corti, a few marginal cells in stria vascularis, some cells in Rosenthal's canal in the PEI-nHAT-pEGFPC2-NT3transfected group, although the transfecton efficiency in cochlea was not high.3) There was no vestibular dysfunction occurred such as step instability, head tilting, rolling or circling after48h transfection in each group. Vestibular hair cells and the supporting cells appeared morphologically normal after48h transfection in each group. Abundant, condensed green fluorescence was found in the zone of dark cells and transitional zone on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells.
Conclusion:1) PEI-nHAT are positive charged and have good dispersivity and stability;2) PEI-nHAT have good capacity of DNA-binding and DNA protection against nuclease digestion at concentrations from125μg/ml to2000μg/ml.3) The approach of gene transfer through intact RWM has no affect on the morphology of cochlea or auditory function, which can serve as an safe and effective way of transferring gene into cochlea and vestibule.4) Vestibular dark cell is the specific targeted cell of PEI-nHAT for gene therapy in the inner ear. It might have the ability of actively uptaking PEI-nHAT-pEGFPC2-NT3complex, although the detailed mechanism is not clear.5) PEI-nHAT are innogarnic materials which could be a potential vector of gene therapy for the inner ear diseases, especially for the vestibular disorders.
引文
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