慢病毒载体介导IL-4基因修饰BMSCs内耳局部应用治疗免疫性感音神经性聋的实验研究

Therapeutic effect of locally injection of bone-marrow mesenchymal stem cells decorated by recombinant vector of lentivirus with IL-4 gene into inner ear on immune-mediated immune sensorineural hearing loss in guinea pigs

目的:探讨白细胞介素4(IL-4)基因修饰骨髓间充质干细胞(BMSCs)内耳局部植入对免疫性感音神经性聋动物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BMSCs组(A组)、空载慢病毒感染的BMSCs对照组(B组,即BMSCs空载对照组)和模拟手术对照组(C组),均将BMSCs细胞悬液经鼓阶开窗植入内耳。提取豚鼠BMSCs,重组IL-4基因的慢病毒载体体外转染BMSCs,成功后鼓阶开窗植入内耳。采用免疫荧光组织化学法和免疫酶组织化学法分别观察导入内耳的BMSCs及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,并行内耳石蜡切片光镜观察。结果:局部KLH免疫后与内耳局部BMSCs植入后2周,特异性抗KLH抗体水平差异无统计学意义;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(P<0.05)。免疫组织化学结果显示BMSCs(荧光反应阳性细胞)主要分布在鼓阶、前庭阶,酶反应阳性(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BMSCs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基因的慢病毒载体植入内耳后,可在内耳迁移并产生基因产物IL-4。经IL-4基因修饰的BMSCs和空载在体外成功地转染BMSCs。经鼓阶途径BMSCs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症反应和听觉功能损伤,前者作用更为显著。从而提示BMSCs(包括经IL-4基因修饰的BMSCs)局部应用可对免疫性内耳病的免疫炎症损伤产生一定的调节和治疗作用,并有向病变部位迁移、聚集的倾向。

Objective： To evaluate the therapeutic effect of local implanting with bone-marrow mesenchymal stem cells （BMSCs） decorated with recombinant lentivirus vocters carrying interleukin-4 （IL-4） gene into inner ear, on guinea pigs with immune-mediated sensorineural hearing loss （IMSNHL）. Methods： Guinea pigs were immunized with keyhole limpet hemocyanin （KLH） and 33 animal models were established, which were divided into three groups on refering to matched-pairs design. Recombinant lentiviral vector integrated IL-4 gene to infect the BMSCs in vitro, the green fluorescence report and the result of RT-PCR to confirm the successful infection of BMSCs. BM- SCs decorated with interleukin-4 （IL-4） gene （group A）and without gene （group B） were implanted separately in- to scala tymani. Group C as control group were injected phosphate buffer （PBS）. Auditory functions and the KLH level of Guinea pigs＇ blood were monitored respectively by auditor brain stern response （ABR） and ELISA test ; Fluorography immunohistochemistry test was performed for tracing BMSCs and enzyme immunohistochemistry test for the situation of IL-4 gene express and productive protein distribution in inner ear. HE stain and light microscopy were carried out. Results： There were no significant differences in levels of KLH in blood serum 2 weeks after im- munization （pretransplantation） and l week after the transplantation of the BMSCs in the same guinea pigs from each group. The threshold of ABR m wave decreased in group A and group B, the result was more significant in group A and group B than that in group C （P 〈 0.05 ）. The results of immunohistochemistry test showed that fluo- rescence positive BMSCs mainly scattered in scala tymani and scala vestibule. Enzyme immune-reaction positive product （i. e. IL-4 gene product） mainly located at spiral ganglion, Corti organ, stria vascularis, around areas of BMSCs in cochlear duct and etc. After the transplantation of cells, the degree of immunity inflammatory reaction and level of hearing impairment were relieved. These situation was more significant in group A than that in group B. Conclusion： These results suggest that BMSCs and BMSCs decorated with IL-4 gene can be locally implanted into inner ear and move forward to pathologic site, importantly develop therapeutical effect to immune-mediated sensorineural hearing loss.