大鼠耳蜗前体细胞的体外培养与分化鉴定

In vitro culture,differentiation,and identification of cochlear progenitor cells derived from newborn rats

目的 :分离、培养大鼠的耳蜗前体细胞,鉴定其增殖和分化能力,分析内耳发育相关基因在其分化中的表达。方法 :从新生大鼠的耳蜗组织中分离获得前体细胞,进行体外培养,并加入血清诱导分化,通过免疫细胞化学的方法鉴定其增殖和分化为毛细胞的能力,利用荧光定量PCR的方法分析不同分化阶段的耳蜗前体细胞中内耳发育相关基因的表达。结果:原代培养的耳蜗前体细胞经免疫细胞化学鉴定呈nestin、Brd U阳性,经血清诱导分化后呈myosinⅦA阳性。荧光定量PCR的结果表明Sox2、Cyclin A2在耳蜗前体细胞分化的过程中表达逐渐下降,而Jagged1在分化过程中表达不断升高。结论 :分离的耳蜗前体细胞具有增殖能力和分化为毛细胞的潜能,在其分化过程中Sox2、Cyclin A2和Jagged1可能参与调控大鼠前体细胞的分化和耳蜗组织的发育。

Objective：We sought to cuhure and isolate cochlear progenitor cells （CPCs）,identify their proliferation and differentiation capacity,and demonstrate the expression of genes involved with the development of inner ear during the CPCs differentiation. Methods： CPCs were isolated from newborn rats＇ cochlea,in vitro cultured and differentiated by serum. Proliferation and differentiation of hair cells were identified by immunocytochemistry method. The expressions of Sox2,CyclinA2,and Jagged1 genes during CPCs differentiation were analyzed using real time qRT-PCR. Results：The cultured CPCs expressed nestin and BrdU, indicating the proliferation ability of CPCs undergoing mitosis. The expression of myosin VII A in the CPCs-derived differentiated cells indicated the CPCs＇ potential to differentiate into hair cells. It was also noteworthy that the expressions of Sox2 and CyclinA2 were downregulated and the Jaggedl expression was upregulated during the CPCs differentiation. Conclusion：The CPCs in vitro culture showed that it has the ability of proliferation and potential of differentiation into hair ceils. Additionally,it was suggested that Sox2,CyclinA2 and Jagged 1 genes could potentially participate in the modulation of CPCs differentiation and development of cochlear tissues in rats.