小鼠内淋巴囊原代上皮细胞L型钙离子通道定位表达

Localization and expression of L-type calcium channel in mice primary endolymph sac epithelium

目的探讨C57BL/6小鼠内淋巴囊原代上皮细胞中电压门控钙离子通道定位表达。方法选取2日龄C57BL/6(P2B6)小鼠60只,P2B6小鼠8只麻醉处死取得颞骨标本,体视显微镜下定位内淋巴囊位置,提取内淋巴囊原代细胞并在低血清状态下培养。P2B6小鼠52只处理同上,颞骨标本用4%甲醛固定后脱钙包埋,采用免疫组织荧光技术和免疫细胞荧光技术定性原代内淋巴囊上皮细胞并确定L型钙离子通道表达。结果内淋巴囊上皮细胞在P2B6小鼠内淋巴囊中排列紧密,可见细胞聚集呈嵴状突起,胞体较小,细胞呈椭圆状,以扁平上皮为主,细胞培养光镜观察为不规则铺路石样,细胞形态大小不一,胞浆丰富,核椭圆形且大。免疫荧光检测显示L型Ca^(2+)通道在内淋巴囊上皮细胞膜上均呈均匀表达,细胞核上高表达。结论小鼠原代内淋巴囊上皮细胞中L-型电压依赖钙离子通道α1C亚基(CACNA1C)、L-型电压依赖钙离子通道α1S亚基(CACNA1S)、L-型电压依赖钙离子通道α1D亚基(Cav1.3/CACNA1D)表达为阳性,为细胞功能学实验提供依据,有利于进一步研究内淋巴囊维持稳态的机制。

Objective To investigate the localization and expression of voltage-gated calcium channels in primary epithelial cells of the endolymphatic sac in C57 BlL/6 mice.Methods Eight of all 60 C57 BL/6 mices,2 days post portum(P2 B6),were sacrificed under anesthesia to obtain temporal bone specimens.The endolymphatic sac was located and primary cells were extracted to culture in low serum.The other 52 mice were treated as above.The temporal bone specimens were fixed with 4%formaldehyde and decalcified and embedded.The primary endolymphatic sac epithelial cells were characterized with immunohistofluorescence and immunocytofluorescence,and the expression of L-type calcium channels were determined.Results The endolymphatic sac epithelial cells were tightly arranged in P2 B6 mice.The cells could clustered as ridge-like protrusions,which were mainly flat epithelium with small and elliptical cell bodies.The cell culture showed stone-like irregular paving,with different cell shapes and sizes,rich cytoplasm,and oval,large nucleuses.Immunofluorescence showed that the L-type calcium channel protein was uniformly expressed on the endolymphatic sac epithelial cell membrane,and highly expressed in the nucleuses.Conclusion The expression of L-type voltage-dependent calcium channel alpha 1 C subunit(CACNA1 C),L-type voltage-dependent calcium channel alpha 1 S subunit(CACNA1 S),and L-type voltage-dependent calcium channel alpha 1 D subunit(Cav1.3/CACNA1 D)are positive in primary endolymphatic sac epithelial cells of C57 BL/6 mice,which provides a basis for cell function experiments and helps to explore the mechanism of endolymphatic sac in maintaining homeostasis.