慢病毒载体介导IL-4基因修饰骨髓间充质干细胞内耳局部应用治疗免疫性感音神经性聋实验研究
发布时间：2018-10-19 15:18
【摘要】：目的:探讨白细胞介素4(IL-4)基因修饰骨髓间充质干细胞(bone-marrow mesenchymal stem cells, BMMSCs)内耳局部植入对免疫性感音神经性聋动物的内耳病理损伤和生理功能障碍的调节与治疗作用。 方法:采用钥孔嘁血蓝蛋白(KLH)抗原在已致敏的豚鼠圆窗龛局部免疫,造成免疫性感音神经性聋动物模型33只。分为IL-4基因修饰BMMSCs组(A组)、空载慢病毒感染的BMMSCs对照组(B组,即BMMSCs空载对照组)和模拟手术对照组(C组),均将各组细胞悬液经鼓阶开窗植入内耳。提取豚鼠BMMSCs,重组IL-4基因的慢病毒载体体外转染BMMSCs,成功后鼓阶开窗植入内耳。采用免疫荧光组织化学试验法观察导入内耳的BMMSCs和免疫酶组织化学试验法观察IL-4基因产物在内耳组织结构中的分布和表达情况。应用酶联免疫吸附试验(ELISA)和听性脑干诱发电位(ABR)测试观察血清抗KLH特异性抗体水平和听觉功能变化。并行内耳石蜡切片和光镜观察。 结果:局部KLH免疫后与内耳局部BMMSCs植入后2周对比,特异性抗KLH抗体水平无显著性差异;A组和B组ABRⅢ波阈值不同程度降低,但前者阈值降低更为明显,两组间比较差异有统计学意义(P0.05);免疫组织化学结果显示BMMSCs(荧光反应阳性细胞)主要分布在鼓阶、前庭阶。酶反应阳性物(IL-4基因产物)部位主要集中于螺旋神经节、骨螺旋板唇部、Corti器、血管纹、耳蜗骨壁、以及蜗管中的BMMSCs的细胞和其周围。内耳光镜观察结果显示:A组和B组仅在鼓阶内有絮状物、注射部位有少量红细胞和白细胞;C组对照组可见螺旋神经节和蜗轴小血管周围有单个核细胞浸润,部分听损耳还可见螺旋神经节细胞数目减少,不同程度的膜迷路积水,蜗管内有絮状物和漂浮细胞。 结论:重组IL-4基因和空载慢病毒在体外可成功地转染BMMSCs。经鼓阶途径行IL-4基因修饰BMMSCs和空载慢病毒转染BMMSCs内耳移植,均可明显减轻免疫性感音神经性聋动物的内耳免疫炎性反应和听觉功能损伤,前者作用更为显著。从而提示BMMSCs(包括经IL-4基因修饰的BMMSCs)局部应用可对免疫性内耳病的免疫炎性损伤产生一定程度的调节和治疗作用,并有向病变部位迁移、聚集的倾向。
[Abstract]:Aim: to investigate the effects of interleukin-4 (IL-4) gene modified bone marrow mesenchymal stem cells (bone-marrow mesenchymal stem cells, BMMSCs) implanted locally on the pathological injury and physiological dysfunction of the inner ear in rats with immune sensorineural hearing loss. Methods: 33 animal models of immune sensorineural deafness were established by local immunization of keyhole chirin (KLH) antigen in the sensitized rounding window niche of guinea pigs. They were divided into IL-4 gene modified BMMSCs group (group A), BMMSCs control group (group B) and simulated operation control group (group C). The lentivirus vector containing recombinant IL-4 gene from guinea pig BMMSCs, was successfully transfected into BMMSCs, in vitro. The distribution and expression of IL-4 gene products in the inner ear were observed by immunofluorescence histochemical assay and immunohistochemical method respectively. The changes of serum anti KLH specific antibody and auditory function were observed by enzyme linked immunosorbent assay (ELISA) and auditory brainstem evoked potential (ABR). Paraffin sections of inner ear and light microscope were observed. Results: there was no significant difference in the level of specific anti KLH antibody between two weeks after local KLH immunization and 2 weeks after BMMSCs implantation in the inner ear, but the threshold of ABR 鈪，
本文编号：2281526
[Abstract]:Aim: to investigate the effects of interleukin-4 (IL-4) gene modified bone marrow mesenchymal stem cells (bone-marrow mesenchymal stem cells, BMMSCs) implanted locally on the pathological injury and physiological dysfunction of the inner ear in rats with immune sensorineural hearing loss. Methods: 33 animal models of immune sensorineural deafness were established by local immunization of keyhole chirin (KLH) antigen in the sensitized rounding window niche of guinea pigs. They were divided into IL-4 gene modified BMMSCs group (group A), BMMSCs control group (group B) and simulated operation control group (group C). The lentivirus vector containing recombinant IL-4 gene from guinea pig BMMSCs, was successfully transfected into BMMSCs, in vitro. The distribution and expression of IL-4 gene products in the inner ear were observed by immunofluorescence histochemical assay and immunohistochemical method respectively. The changes of serum anti KLH specific antibody and auditory function were observed by enzyme linked immunosorbent assay (ELISA) and auditory brainstem evoked potential (ABR). Paraffin sections of inner ear and light microscope were observed. Results: there was no significant difference in the level of specific anti KLH antibody between two weeks after local KLH immunization and 2 weeks after BMMSCs implantation in the inner ear, but the threshold of ABR 鈪，
本文编号：2281526
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