大鼠胃壁细胞GnRH受体的定位及GnRH类似物对壁细胞内钙的影响  

LOCATION OF GnRH RECEPTOR AND EFFECTS OF GnRH ANALOG ON INTRACELLULAR CALCIUM MOBILIZATION IN CULTURED GASTRIC PARIETAL CELLS OF RATS

作　　者：陈蕾[1] 黄威权[1] 孙绪德[2] 吕宝真[1] 蒲若蕾[1] 

机构地区：[1]第四军医大学组织学胚胎学教研室,西安710032 [2]唐都医院麻醉科,西安710032

出　　处：《解剖学报》2003年第1期79-84,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目 (3 9770 3 88)

摘　　要：目的　观察促性腺激素释放激素 (gonadotropin releasinghormone,GnRH)受体在培养的大鼠胃粘膜壁细胞中的定位 ,并探讨GnRH类似物阿拉瑞林 (alarelin)对壁细胞内游离钙动员的机制。　方法　采用免疫组织化学和原位杂交技术 ;应用Ca2 + 指示剂Fluo 3 AM作为细胞内钙离子的荧光探针 ,对负载培养的胃壁细胞 ,应用激光共聚焦显微镜技术检测单个细胞内钙荧光强度的变化。　结果　大鼠胃壁细胞呈GnRH受体免疫反应阳性 ,阳性物质位于细胞质 ,细胞核为阴性 ;同样壁细胞内可检测到GnRH受体mRNA杂交信号 ,阳性物质位于细胞质 ,细胞核为阴性 ;胞内Ca2 + 浓度变化为 :1 在Hank液中 ,GnRH类似物浓度为 10 - 8、10 - 7、10 - 6 mol L时 ,胃壁细胞内Ca2 + 浓度逐渐升高 ,其峰高 (峰值减去静息值 )分别为 7 1± 1 4、12 1± 1 7、16 8± 2 2。其达峰时间也逐渐增快 ,分别为 34 2± 6 4s、18 9± 1 2s、10 4± 2 3s。相邻两组间其峰高及达峰时间均存在显著性差异 (P <0 0 5 ) ,且呈明显剂量依赖性。 2 在D Hank液 (去除外钙 )中 ,阿拉瑞林可轻度短暂升高胞内Ca2 + ;用内罗啶孵育后再加入阿拉瑞林也可轻度短暂升高胞内Ca2 + ,二者无显著性差异。 3 当用拉西地平孵育后再加入阿拉瑞林 ,可明显抑制胞内Ca2 + 的增加?Objective To observe whether gonadotropin releasing hormone(GnRH)receptor can be expressed in cultured gastric parietal cells of rats and study the effects of GnRH analog (Alarelin)on intracellular Ca 2+ mobilization in gastric parietal cells of rats. Methods Immunohistochemical ABC method and hybridization in situ method were used in the experiment.Cells were loaded with Fluo 3 AM.[Ca 2+ ] i and measured by fluorescent intensity(FI)in each cell with confocal microscope. Results Gastric parietal cells showed GnRH receptor immunoreactivity,positive material was located in cytoplasm with negative nuclei.GnRH receptor mRNA hybridized signals were also detected in cytoplasm with negative nuclei.Effects of intracellular Ca 2+ as follows:1.In Hank solution,10 -8 、10 -7 、10 -6 mol/L alarelin could elevate intracellular Ca 2+ .Their heights of peak were 7 1±1 4,12 1±1 7,16 8±2 2 respectively and their times to peak were 34 2±6 4 s,18 9±1 2s,10 4±2 3s respectively,which indicate that the level of [Ca 2+ ] i acts in dose and time dependancy.2.In D Hank solution (removal the extracellular Ca 2+ )and in the group of ryanodine,alarelin could induce a transient increase[Ca 2+ ] i,and they were no difference( P >0 05).3.When preincubated with lacidipine in Hank solution,alarelin was inhibited and the [Ca 2+ ] i was significantly lower than that of the group which was only added alarelin ( P <0 01).4.When pretreated with the compund 48/80(PLC inhibitor)and sphingosine (PKC inhibitor)respectively,the effects of alarelin were both inhibited which compared with 10 -6 mol/L alarelin,it was significantly lower( P <0 01).Conclusion\ Our data suggest that GnRH receptor could be expressed by gastric parietal cells of rats.Alarelin could increase intracellular Ca 2+ and [Ca 2+ ] i plays an important role in molecular mechanism of intracellular signal transduction pathways by which GnRH analog mediates in gastric parietal cells.T

关 键 词：GNRH类似物 GNRH受体 胃壁细胞 钙 Fluo-3-AM 免疫组织化学 原位杂交 激光共聚焦显微镜 

分 类 号：R392.12[医药卫生—免疫学]