中华耳鼻咽喉头颈外科杂志
中華耳鼻嚥喉頭頸外科雜誌
중화이비인후두경외과잡지
CHINESE JOURNAL OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY
2015年
1期
43-49
,共7页
耳蜗%迷路支持细胞%腺苷三磷酸%大鼠
耳蝸%迷路支持細胞%腺苷三燐痠%大鼠
이와%미로지지세포%선감삼린산%대서
Cochlea%Labyrinth supporting cells%Adenosine triphosphate%Rats
目的 观察体外培养的新生大鼠耳蜗K(o)lliker器支持细胞是否存在并释放ATP,初步探讨其释放机制.方法 选取出生后ld的Sprague-Dawley大鼠,分离耳蜗膜迷路,采用机械分离与酶消化相结合的方法获得单离的K(o)lliker器支持细胞.观察膜迷路和K(o)lliker器支持细胞的喹丫因染色情况.采用生物发光法,通过影响K(o)lliker器支持细胞ATP代谢、改变细胞内外Ca2浓度、抑制细胞内磷脂酶信号通路及添加缝隙连接半通道阻断剂,观察K(o)lliker器支持细胞释放ATP浓度的变化.结果 用喹丫因染色体外培养的K(o)lliker器支持细胞,发现胞质中存在大量绿色星点状染色.采用生物发光法检测的ATP标准曲线呈明显的对数线性关系.随着巴佛洛霉素A1浓度增加,K(o)lliker 器支持细胞培养液中ATP浓度逐渐降低,而随着己二酸二癸酯浓度的增加,培养液中ATP浓度逐渐升高;在一定浓度范围内,随着细胞外Ca2浓度增加,K(o)lliker器支持细胞ATP的释放减少,而随着细胞内游离Ca2浓度增加,K(o)lliker器支持细胞释放ATP量增加;培养液中加入甘珀酸钠或乌热酸抑制半通道后可以显著的降低ATP释放.此外,抑制细胞内磷脂酶信号通路也可以减少ATP的释放.结论 体外培养的新生大鼠耳蜗K(o)lliker器支持细胞存在并释放ATP,细胞内、外液中Ca2+浓度的变化可能通过调节半通道的开放而影响其ATP的释放.
目的 觀察體外培養的新生大鼠耳蝸K(o)lliker器支持細胞是否存在併釋放ATP,初步探討其釋放機製.方法 選取齣生後ld的Sprague-Dawley大鼠,分離耳蝸膜迷路,採用機械分離與酶消化相結閤的方法穫得單離的K(o)lliker器支持細胞.觀察膜迷路和K(o)lliker器支持細胞的喹丫因染色情況.採用生物髮光法,通過影響K(o)lliker器支持細胞ATP代謝、改變細胞內外Ca2濃度、抑製細胞內燐脂酶信號通路及添加縫隙連接半通道阻斷劑,觀察K(o)lliker器支持細胞釋放ATP濃度的變化.結果 用喹丫因染色體外培養的K(o)lliker器支持細胞,髮現胞質中存在大量綠色星點狀染色.採用生物髮光法檢測的ATP標準麯線呈明顯的對數線性關繫.隨著巴彿洛黴素A1濃度增加,K(o)lliker 器支持細胞培養液中ATP濃度逐漸降低,而隨著己二痠二癸酯濃度的增加,培養液中ATP濃度逐漸升高;在一定濃度範圍內,隨著細胞外Ca2濃度增加,K(o)lliker器支持細胞ATP的釋放減少,而隨著細胞內遊離Ca2濃度增加,K(o)lliker器支持細胞釋放ATP量增加;培養液中加入甘珀痠鈉或烏熱痠抑製半通道後可以顯著的降低ATP釋放.此外,抑製細胞內燐脂酶信號通路也可以減少ATP的釋放.結論 體外培養的新生大鼠耳蝸K(o)lliker器支持細胞存在併釋放ATP,細胞內、外液中Ca2+濃度的變化可能通過調節半通道的開放而影響其ATP的釋放.
목적 관찰체외배양적신생대서이와K(o)lliker기지지세포시부존재병석방ATP,초보탐토기석방궤제.방법 선취출생후ld적Sprague-Dawley대서,분리이와막미로,채용궤계분리여매소화상결합적방법획득단리적K(o)lliker기지지세포.관찰막미로화K(o)lliker기지지세포적규아인염색정황.채용생물발광법,통과영향K(o)lliker기지지세포ATP대사、개변세포내외Ca2농도、억제세포내린지매신호통로급첨가봉극련접반통도조단제,관찰K(o)lliker기지지세포석방ATP농도적변화.결과 용규아인염색체외배양적K(o)lliker기지지세포,발현포질중존재대량록색성점상염색.채용생물발광법검측적ATP표준곡선정명현적대수선성관계.수착파불락매소A1농도증가,K(o)lliker 기지지세포배양액중ATP농도축점강저,이수착기이산이계지농도적증가,배양액중ATP농도축점승고;재일정농도범위내,수착세포외Ca2농도증가,K(o)lliker기지지세포ATP적석방감소,이수착세포내유리Ca2농도증가,K(o)lliker기지지세포석방ATP량증가;배양액중가입감박산납혹오열산억제반통도후가이현저적강저ATP석방.차외,억제세포내린지매신호통로야가이감소ATP적석방.결론 체외배양적신생대서이와K(o)lliker기지지세포존재병석방ATP,세포내、외액중Ca2+농도적변화가능통과조절반통도적개방이영향기ATP적석방.
Objective The specific mechanism underlying in the Adenosine triphosphate (ATP) release from the K(o)lliker's organ is still unknown.The present study was designed to investigate whether the supporting cells in the K(o)lliker organ in vitro release ATP and to explore the mechanism of ATP releasing from these cells.Methods Supporting cells in the K(o)lliker organ from P1 rats were isolated,purified and cultured with a combinatorial approach of enzymatic digestion and mechanical separation.Quinacrine staining was used to observe the cochlear membranous labyrinth and supporting cells.the bioluminescence assay was chosen to explore the release ATP from supporting cells in the K(o)lliker organ,when the ATP metabolism of the cells was influenced,the intracellular or extracellular Ca2 + concentration changed,the hemichannels blocked,and the phospholipase signaling pathways inhibited.Results There were intensely numerous starlike green spots of quinacrine staining in the cytoplasm of supporting cells.There was a strong log-linear relationship in the ATP standard curve generated by the bioluminescence assay.With increasing concentrations of bafilomycin Al,the ATP concentration in the culture medium of the supporting cells in the K(o)lliker organ decreased,while with adipic acid didecyl,it increased.In a certain concentration range,with increasing extracellular Ca2+ concentration,the supporting cells in the K(o)lliker organ releasing ATP decreased,while the intracellular Ca2+ concentration increased,the results showed the elevation of the amount of ATP release.Adding chelerythrine chloride or aristolochid acid into the culture medium of the supporting cells in the K(o)lliker organ could decrease the ATP release significantly via inhibiting the hemichannels.In addition,by reducing intracellular Ca2+ concentration,inhibition of intracellular signaling pathways phospholipase also decreased ATP release.Conclusions This study demonstrated the presence and release of ATP from the supporting cells cultured in vitro.It showed that the changes of the intracellular and extracellular Ca2+ concentration could affect on the ATP release from the supporting cells in the K(o)lliker organ by regulating the hemichannels openings.
