移形细胞在庆大霉素由前庭外淋巴转运至内淋巴的作用及机制研究

Transtympanic administration of gentamicin(GM) has been considered as an effective approach for the treatment of intractable Meniere’s Disease. Several studies revealed that GM-induced toxicity for vestibular sensory hair cells partially ablated vestibular function, thus resolving vertigo spells. However, few researches focused on pathway for GM trafficking from vestibular perilymph into endolymph. Some research reported that trans-strial vascularis way was very important for GM to enter the endolymph, during which marginal cell is critical. In our study, we observed intense, punctate gentamicin fluorescence in the apical region of transitional cell from 12 hours to 28 days after the administration. We also observed that GM fluorescence in transitional cells didn’t increase with increasing GTTR doses, suggestting rapid cellular saturation with the drug. We find that transitional cell have almost similar structure, function and ion channels with marginal cell. The ototoxic effects of GM can be greatly enhanced by co-administration with loop inhibiting diuretics. The massive hair-cell loss that occurs shortly after GM/diurtics treatment because diuretics affect the ion channel, NKCC1 and Pendrin protein, to enhance the permeability of stria.vascularis, which accelerates the influx of GM into endolymph followed by entry through the apical surface of the hair cell into the cytoplasm. Some research also showed that NKCC1 and Pendrin proteins were also expressed in vestibular transitional cells. We deduce that transitional cells may act as a pathway for GM trafficking from vestibular perilymph into endolymph and NKCC1 and Pendrin proteins probably play a pivotal role. In this research we used GM conjugated toTexas Red (GTTR) as a tracer to explore GM and diuretics, separately and corporately, which was administrated in vivo or in vitro to study the morphology, ultrastructure；Electrophysiology；molecular biology change to to clarify the mechanism of GM trafficking from vestibular perilymph into endolymph。

鼓室内庆大霉素注射是治疗难治性眩晕的有效方法,它通过部分损伤前庭毛细胞降低前庭敏感性而控制眩晕的发作。前庭毛细胞位于内淋巴顶端的离子通道是庆大霉素进入细胞的主要方式，但目前对其如何由前庭外淋巴转运至内淋巴研究甚少。既往研究认为血管纹是庆大霉素在耳蜗由外淋巴快速转运至内淋巴的通道，其中边缘细胞起到了关键作用,其表达的离子通道NKCC1和Pendrin蛋白起了重要作用。本课题组实验中发现庆大霉素在前庭移形细胞分布较多，且有出现早、饱和性和持续时间长的特点；同时其与边缘细胞在形态、主要离子通道表达及对庆大霉素的吸收分布具有类似性。因此本课题推测移形细胞可能是庆大霉素在前庭由外淋巴转运至内淋巴的关键细胞，NKCC1和Pendrin蛋白可能为其转运的关键分子。本课题将研究庆大霉素作用下移形细胞在体和离体组织培养状态下的形态学、分子生物学、电生理及超微结构的变化，从而阐明其在庆大霉素转运中的作用机制

本课题通过庆大霉素得克萨斯红进行小剂量庆大霉素在移形细胞的时间和空间分布的研究，发现和明确庆大霉素早期在移形细胞分布较感觉上皮较高，随着时间的延长和浓度的增高，移形细胞分布略有增高，但成饱和状态，但其分布可维持较长时间，因此移形细胞可能是庆大霉素内外淋巴转运的通路。通过庆大霉素损伤性电镜研究发现，移形细胞损伤较轻，细胞间连接无明显破坏，因此庆大霉素的转运并非通过改变细胞间的紧密连接结构的使得庆大霉素通过“内外淋巴leak”的转运。庆大霉素的转运可能是通过受体介导的特异性或者非特异性转运， Pendrin蛋白作为候选蛋白在移行细胞基底面表达，且免疫蛋白印记显示内淋巴积水模型中蛋白含量较对照组下降。本课题通过内淋巴囊切除手术制作内淋巴积水模型,进一步研究梅尼埃病病理性内淋巴积水状态下庆大霉素在前庭的分布及超微结构的改变,实验发现在内淋巴积水模型庆大霉素得克萨斯红鼓室内注射后,庆大霉素在积水模型内淋巴在感觉上皮和移行细胞分布均较对照组有明显减少，与免疫蛋白印记显示的Pendrin的呈现减少一致，因此我们认为该蛋白可能是介导庆大霉素在内外淋巴转运的受体。但进一步需要进行Pendrin蛋白耳蜗特异性敲除小鼠的研究。

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