CAJ | 학술논문

目的：探讨一种适宜耳蜗组织贴壁培养的方法。方法 PET滤膜预先置入层粘连蛋白、鸟氨酸、胎牛血清按2：2：1比例混合均匀的液体中在4℃预孵过夜，选取出生四天的C57BL/6J小鼠作为研究对象。剥取基底膜以PET滤膜作为承载介质培养在加入含青霉素的培液中，48小时后收取样本，进行免疫荧光（一抗goat anti-otoancorin、mouse anti-beta tubu?linⅢ及对应二抗采用cy3 donkey anti goat，647 donkey anti mouse，染核试剂DAPI，phalloidin)观察不同部位细胞，扫描电镜及制备原位超薄切片透射电镜观察体外培养新生小鼠基底膜内外毛细胞、螺旋缘内细胞、螺旋神经元细胞显微结构的改变。结果耳蜗基底膜在PET膜上贴附生长良好。各种细胞的微小结构和超微结构保持正常状态。结论PET滤膜能作为一种新颖的承载基底膜培养的介质。
목적：탐토일충괄의이와조직첩벽배양적방법。방법 PET려막예선치입층점련단백、조안산、태우혈청안2：2：1비례혼합균균적액체중재4℃예부과야，선취출생사천적C57BL/6J소서작위연구대상。박취기저막이PET려막작위승재개질배양재가입함청매소적배액중，48소시후수취양본，진행면역형광（일항goat anti-otoancorin、mouse anti-beta tubu?linⅢ급대응이항채용cy3 donkey anti goat，647 donkey anti mouse，염핵시제DAPI，phalloidin)관찰불동부위세포，소묘전경급제비원위초박절편투사전경관찰체외배양신생소서기저막내외모세포、라선연내세포、라선신경원세포현미결구적개변。결과이와기저막재PET막상첩부생장량호。각충세포적미소결구화초미결구보지정상상태。결론PET려막능작위일충신영적승재기저막배양적개질。
Objective To report a practical method for tissue adhere in cochlear organotypic cultures. Methods The polyethylene terephthalate (PET) membrane was precoated with laminin, poly-L-ornithine, and fetal bovine serum at a ratio of 2:1:1 overnight. The cochlear basilar membrane was micro-dissected from C57BL/6J mouse pups at postnatal day 4, and placed on the PET membrane in a culture medium containing DMEM, 10%fetal bovine serum, and 30U/ml penicillin. After 48 hours, the cochlear specimens were fixed with 10%formalin in PBS. Anti-otoancorin and anti-tubulinⅢantibodies were dou?ble stained for labeling of nonsensory cells, interdental cells in the inner ear and auditory nerve fibers respectively. Fluores?cence conjugated phalloidin was also used to label stereocilia and cuticular plate of hair cells, and DAPI was also applied for nuclear staining. In addition, cochlear explants were also examined using scanning and transmission electron microscopy. Re?sults The cochlear basilar membrane firmly attached to the surface of PET. The microstructure and ultrastructure of cochlear cells appeared normal. Conclusion PET membrane can be used as an ideal holder for cochlear organotypic cultures.