摘要
目的 确定人晶状体上皮细胞 (HLEC)系LEC B3细胞内两种钙池调控系统肌醇1,4,5 三磷酸 (IP3 )受体和兰尼碱受体的表达 ,并鉴定其亚型 ,初步探讨两系统对HLEC内Ca2 + 的调控作用。方法 传代培养HLEC系LEC B3细胞 ,选用第 3代融合状态良好的细胞 ,提取细胞总RNA ,反转录获取cDNA ,分别用IP3 受体的Ⅰ、Ⅱ、Ⅲ亚型和兰尼碱受体的Ⅰ、Ⅱ、Ⅲ亚型特异引物进行逆转录聚合酶链反应 ,判定分析mRNA的表达。并在激光扫描共聚焦显微镜下分别采用一定浓度的乙酰胆碱、阿托品、咖啡因、兰尼碱及普鲁卡因作用于细胞外液含Ca2 + 、Mg2 + 和不含Ca2 + 、Mg2 +的同代细胞 ,观察分析Ca2 + 信号的变化。结果 HLEC系LEC B3细胞中分别表达IP3 受体和兰尼碱受体的Ⅰ和Ⅲ型mRNA ,且均Ⅲ型表达强于Ⅰ型 (P <0 0 5 )。10 μmol/L乙酰胆碱、10mmol/L咖啡因及 5 0 μmol/L兰尼碱均可使细胞内Ca2 + 浓度增高 ,而波动的开始时间、幅度及持续时间因细胞外液成分和药物不同而异 ;1μmol/L阿托品可阻断乙酰胆碱的作用 ,5 0 μmol/L普鲁卡因可抑制兰尼碱的作用。结论 HLEC系LEC B3细胞内同时存在IP3 受体和兰尼碱受体基因的转录 ,且存在亚型特异性。两种受体的激动剂和抑制剂均可引起细胞内Ca2 + 浓度的变化 ,提示IP3
Objective To determinate the existence of the inositol 1,4,5-triphosphate receptor (IP 3R) and the ryanodine receptor (RyR), identify their isoforms and assess their functions during calcium ion concentration modulation in a human epithelial cell line (LEC-B3). Methods LEC-B3 cell line was defrosted and subcultured , the 3rd passage cells were chosen as object when they became confluent. Then the total cellular RNA was extracted from the cells, the expression of mRNA of IP 3R and RyR isoforms was analyzed by polymerase chain reaction.Ca 2+ ion changes in the same passage cells were observed and analyzed with laser scanning confocal microscope when aceylcholine (Ach), atropine, caffeine, ryanodine,and procaine were added to the extracellular Ca 2+, Mg 2+ free solutions with ethyleneglycol bis ether tetracetic acid or Ca 2+, Mg 2+ involved solutions. Results The IP 3R isoform Ⅰ ,Ⅲ and RyR isoform Ⅰ,Ⅲ mRNA were expressed,the expression of isoform IP 3R Ⅲ>IP 3RⅠ(P<0.05); RyR Ⅲ>RyRⅠ(P <0.01). 10 μmol/L Ach, 10 mmol/L caffeine and 50 μmol/L ryanodine all could induce the intracellular free Ca 2+ concentration increase, but the incubation periods, ranges and lasting time of the increases are different: when Ach acted, the duration of calcium increase in calcium involved solutions was a little longer than in calcium free solutions; the amplitude of calcium oscillations induced by Ach was much higher than caffein and ryanodine , while the onset of the later is slower than the former. Besides, 1 μmol/L atropine could block the Ach effects, 50 μmol/L procaine could inhibit the ryanodine effects. Conclusions (1) The IP 3R and RyR gene transcription coexist in the human lens epithelial cell line: LEC-B3, and their isoforms have specificity. (2) Different agonists can induce different calcium changes. (3)The increase of Ca 2+ concentration induced by caffeine or ryanodine suggested the involvement of both the cyclic adenosine diphosphate ribose and the inositol 1,4,5-triphosphate systems in the rugulation of intracellular calcium in LEC-B3, and IP 3R is more sensitive to free Ca 2+ modulation than RyR.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2003年第7期389-394,共6页
Chinese Journal of Ophthalmology
关键词
晶状体
上皮细胞
内肌醇
三磷酸受体
HLEC
兰尼碱受体
钙通道
Lens, crystalline
Epithelial cells
Receptors, cytoplasmic and nucleur
Calcium channels
Ryanodine receptor calcium release channel
