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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 investigate the localization of gonadotropin releasing hormone (GnRH) receptor in cultured rat gastric mucosa parietal cells and to explore the mechanism of GnRH analog Alalalin on intracellular free calcium mobilization in parietal cells. Methods Immunofluorescence staining and in situ hybridization were used to detect the intracellular calcium fluorescence of cultured gastric parietal cells using Fluo 3 AM, a Ca2 + indicator, as a fluorescent probe for intracellular calcium. Variety. Results The GnRH receptor immunoreactivity was positive in the rat gastric parietal cells. The positive cells were located in the cytoplasm and the nuclei were negative. In the same parietal cells, the GnRH receptor mRNA was detected. The positive cells were located in the cytoplasm and the nuclei were negative. The intracellular Ca2 + The changes were as follows: 1 In Hank’s solution, the concentration of Ca2 + in gastric parietal cells increased gradually with the concentration of GnRH analogues being 10 - 8,10 - 7,10 - 6 mol L, the peak height (peak minus the resting value) Respectively, 7 1 ± 1 4,12 1 ± 1 7,16 8 ± 2 2. The peak time also gradually increased, respectively, 34 2 ± 6 4s, 18 9 ± 1 2s, 104 ± 23s. There was a significant difference (P <0 05) between peak height and peak time in adjacent two groups, and showed a dose-dependent manner. 2 in D Hank solution (to remove extra-calcium), Alarelin slightly elevated intracellular Ca2 + transiently; incubated with ethidium and then added to Alarelin also mildly elevated intracellular Ca2 +, two No significant difference. 3 when incubated with lacidipine and then added Alarelin, can significantly inhibit the increase of intracellular Ca2 +?