碱性成纤维细胞生长因子对豚鼠耳蜗外毛细胞钙调控的影响

The effects of bFGF on the calicum influx in OHCs

目的 观察碱性成纤维细胞生长因子 (Basicfibroblastgrowthfactor ,bFGF)对豚鼠耳蜗外毛细胞 (Outerhaircells,OHCs)钙调控的影响以及这种影响与硫酸链霉素的拮抗效应 ,旨在深入探讨硫酸链霉素急性耳毒性和bFGF防治作用的分子生物学机制。方法 豚鼠全麻后断头活杀 ,酶 -机械法分离获得单离的OHCs。 10 μmol·L-1的Fluo 3 /AM负载 ,室温下放置 60min后用ACASUltima型激光扫描共聚焦显微镜 (LSCM ,MeridianUSA)观察各种条件下OHCs[Ca2 + ]i的变化。结果 ① 10 0mmol·L-1的高钾细胞外液和含 1nmol·L-1bFGF的正常细胞外液灌流分别导致 10 /11和 9/9个细胞的 [Ca2 + ]i升高 ,而高钾无钙细胞外液和含 1nmol·L-1bFGF的无钙细胞外液灌流则分别为 0 /7和 0 /8个细胞 [Ca2 + ]i 升高。②经 1 0mmol·L-1的硫酸链霉素处理后 ,灌流高钾细胞外液则 0 /12个细胞 [Ca2 + ]i 升高 ,灌流bFGF则为 4/8个细胞 [Ca2 + ]i 升高。③ 1nmol·L-1的bFGF作用后 ,灌流高钾细胞外液 ,OHCs[Ca2 + ]i升高的幅度较高 ,持续时间较长。④ 3组细胞均提前 1h加入 1mmol·L-1的硫酸链霉素 ,实验前 5min分别加入 0 1、1 0和 10nmol·L-1的bFGF ,灌流高钾细胞外液则分别导致 4/11、6/9和 12 /12个细胞 [Ca2 + ]i的升高。结论 bFGF和?

AIM To examined the effect of bFGF on the calcium influx in outer hair cells (OHCs) and its antagonistical effects with streptomycin. The aim of the study is to explore the mechanism of acute ototoxicity of streptomycin and antagonism of bFGF. METHODSE The OHCs of guinea pig were isolated using an enzyme machine methods and loaded with 10 μmol·L -1 Fluo 3/AM for 30 min at 37℃,then cultured 60 min at room temperature. Individual Fluo 3 loaded OHCs were examined with a confocal microscope (ACAS Ultima, USA) using a 20× objective lens and linear scan mean. The fluorescent images, collected every 5 sec for 300 s, were stored in a computer. The fluorescent intensity of the images were analyzed by the software cooperated with the confocal microscope, and a function curve showing the change trend of fluorescent intensity with time was obtained. RESULTS The OHCs [Ca 2+ ] i hold steady within the process of normal extracellular liquid perfusion. OHCs [Ca 2+ ] i increased when perfused 100 mmol·L -1 high potassium media(10/11) and normal media containing 1 nmol·L -1 bFGF(9/9), but the OHCs [Ca 2+ ] i don't changes in high potassium free calcium media(7/7) and free calcium media containing 1 nmol·L -1 bFGF(8/8). After treated by 1 mmol·L -1 streptomycin, the OHCs [Ca 2+ ] i increased 0/12, 4/8 respectively when perfused high potassium media and bFGF media. And after treated by 1 nmol·L -1 bFGF, high potassium media make OHCs [Ca 2+ ] i increased more obviously and keeping longer time. After treated by 1 mmol·L -1 streptomycin, respectively, 0 1,1 and 10 nmol·L -1 bFGF make 4/11,6/9 and 12/12 OHCs [Ca 2+ ] i increasing when perfused high potassium media. CONCLUSION High potassium media and bFGF perfusion can result in increasing of OHCs [Ca 2+ ] i ,and OHCs [Ca 2+ ] i increase rooted in the calicum influx, there is synergic effects between high potassium and bFGF. The streptomycin can block the process of calcium influx induced by high potassium media and the block effects can be antagonized by bFGF, and the antagonistical effects have bFGF concentration dependence.