应用酶消化耳蜗基底膜上皮细胞的研究

摘要

Objective To establish a new method for isolating progenitor cells from cochlear basilar membrane epi-thelium by combinatorial enzymatic digestion. Methods Basilar membranes were separated from the cochlea of P1-3 neo-natal SD rats. The isolated basilar membrane sheets were treated by trypsin digestion (Group A), thermolysin digestion (Group B), type I collagenase digestion (Group C), or combined thermolysin and type I collagenase digestion (Group D). Cells isolated by enzymatic digestion were collected and cultured under suspension and adherent conditions. The number of otospheres, dominance by epithelial cells and proliferation and differentiation of progenitor cells were evaluated. Results Cells collected in all four groups gave rise to otospheres, with positive nestin and BrdU expression. Under the differentia-tion condition, cells from basilar membrane epithelium differentiated into hair cell-like cells. The epithelial origin was con-firmed by cytokeratin-18 and E-cadherin staining. Epithelial cells dominance was the greatest in Group D and significantly lower than other three groups in Group A. Conclusions Combinatorial enzymatic digestion with thermolysin and type I col-lagenase can significantly improve isolation of epithelial cells. This method provides a new tool for cochlear progenitor cells research.%目的：比较联合应用嗜热菌蛋白酶和I型胶原酶消化对耳蜗基底膜上皮细胞的分离效果。方法分离P0-3天SD大鼠基底膜，并将其分为四组，分别为：A组胰酶消化组；B组嗜热菌蛋白酶消化组；C组I型胶原酶消化组；D组嗜热菌蛋白酶和I型胶原酶消化组。收集各组消化的细胞进行悬浮培养和诱导分化，分别计数各组形成的细胞球数目，应用免疫荧光对获得的细胞来源进行鉴定，并比较各组Cytokeratin-18阳性细胞的百分率。结果从4种方法分离得到的细胞经培养后均可形成细胞球，表达干细胞标记物Nestin和细胞分裂标记物BrdU。获得细胞大部分均表达上皮来标记物E-cadherin和cytokeratin-18，而不表达间质细胞标志物Vimentin，经过诱导分化后可以分化成毛细胞样细胞。应用嗜热菌蛋白酶和I型胶原酶消化获得的细胞球数目显著高于其它组(P<0.05)；而采用胰酶消化获得的细胞cytokeratin-18阳性率显著低于其它组(P<0.05)。结论联合应用嗜热菌蛋白酶和I型胶原酶消化耳蜗基底膜上皮细胞可以显著提高基底膜上皮细胞的分离效果，从而耳蜗前体细胞的研究提供有力研究基础。