腺病毒转染新生及成年鼠内耳细胞的研究

Study on adenovirus-infected inner ear cells in newborn and adult mouse

目的探讨腺病毒携带目的基因Cre和Oct4导入新生和成年小鼠转染内耳细胞的可行性,为内耳基因治疗提供可行性研究。方法采用纳升级显微操作系统,经耳蜗中阶显微注射携带基因编辑基因Cre和绿色荧光蛋白(GFP)基因的5型重组腺病毒悬液于新生鼠(P1)和成年鼠,并以腺病毒携带Oct4基因转染入成年鼠内耳,注射4d后取双侧耳蜗标本做基底膜铺片,观察GFP和Oct4表达情况。结果新生鼠组:术后第4d见耳蜗底圈62%和中圈54%支持细胞表达GFP。成年鼠组:术后第4天可见耳蜗底圈96%和中圈51%内毛细胞表达GFP,底圈72%和中圈40%支持细胞表达GFP。31%内毛细胞和43%支持细胞表达Oct4。未注射耳未见GFP和Oct4表达。结论显微注射腺病毒携带目的基因Cre和Oct4可高效导入新生鼠及成年鼠内耳并表达,可用来研究内耳基因功能和基因治疗。

Objective To investigate the feasibility of target gene transfer into the inner ear cells of neonatal and adult mice by adenovirus vectors, so as to provide basis for gene therapy of inner ear. Methods Adenovirus vector 5 carrying GFP linked to a genome editing gene Cre recombinase （Ad-Cre-GFP） was injected into inner ear cells of neonatal （pl） and adult mice through nano-liter grade micro-operating system by cochleostomy.The Adenovirus vectors carrying Oct4 gene（Ad-Oct4） was administered into inner cells of another adult mice group. After 4 days of injection, bilateral cochlea was harvested for whole-mount membrane was sectioned into slices. Immunofluorescence was used to observe the expression of target protein. Results In neonatal（P1） cochleae injected with Ad-Cre-GFP, GFP expression was detected in 62 % of supporting cells in the base circle and 54 % of supporting cells in middle circle after 4 days of injection. In adult mice, GFP was expressed in 72 % and 40 % supporting cells at base and middle circle of cochlear respectively. 96 % and 51% inner hair cells was found expressing GFP at base and middle circles respectively. In addition, Oct4 was detected in 3 1% inner hair cells and 43 % supporting cells after Ad-Oct4 injection. No GFP and Oct4 expression was observed in the control ears. Conclusion Our study indicate that micro-injection of adenovirus into both neonatal and adult mouse inner ears can achieve efficient exogenous gene transfer, which has great potential in inner cell gene therapy and inner ear gene function study.