培养的大鼠胃壁细胞促性腺激素释放激素的定位、克隆及序列分析  

DISTRIBUTION, CLONING AND AMPLIFICATION SEQUENCE ANALYSIS OF GnRH GENE IN CULTURED GASTRIC PARIETAL CELLS OF RATS

作　　者：陈蕾[1] 孙绪德[2] 赵晶[3] 杨安钢[3] 黄威权[1] 

机构地区：[1]第四军医大学组织学胚胎学教研室 [2]第四军医大学唐都医院麻醉科,西安710032 [3]第四军医大学生物化学教研室,西安710032

出　　处：《解剖学报》2003年第3期284-288,共5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　研究培养的大鼠胃壁细胞GnRH的定位及其基因序列。　方法　应用免疫组织化学和原位杂交的方法进行定位研究 ;从壁细胞中提取总RNA ,应用RT PCR的方法扩增GnRH基因 ,并对产物进行纯化回收 ,连接入pUC19载体中扩增 ,经PCR和酶切鉴定后进行序列分析。　结果　大鼠胃壁细胞呈GnRH免疫反应性阳性物质位于细胞质 ,细胞核为阴性 ;壁细胞内亦可检测到GnRHmRNA杂交信号 ,信号物质位于细胞质 ,细胞核为阴性 ;同样从大鼠胃壁细胞中扩增出GnRH基因的特异性条带 ,经序列分析发现 ,扩增产物的基因序列与文献报道的大鼠下丘脑的完全一致。　结论　大鼠胃粘膜壁细胞能表达GnRH基因 ,其序列和下丘脑的完全一致 ,并能自身合成GnRH ,说明GnRH可能以自分泌或以旁分泌的方式参与胃壁细胞功能的调节。Objective To study the distribution and sequence analysis of gonadotropin releasing hormone (GnRH) gene in cultured gastric parietal cells of rats. Methods The distribution of GnRH molecule and GnRH mRNA were observed out through immunohistochemical ABC methods and in situ hybridization methods in cultured gastric parietal cells of rats. After isolation of the total RNA from the parietal cells, RT PCR was conducted to obtain GnRH cDNA. Then, the products of PCR was purified, digested by the restriction enzyme of Hind Ⅲ and EcoR Ⅰ, and DNA fragments interests were cloned into pUC19 vector. The products of PCR were analyzed by sequenceing with Sanger's method after identified by PCR and digestion of restriction enzyme. Results Gastric parietal cells showed GnRH immunoreactivity, positive material was located in cytoplasm with negative nuclei. GnRH mRNA hybridized signals were also detected in cytoplasm with negative nuclei. The specific amplified band of GnRH mRNA was detected through agarose gel electrophoresis and gene sequence is identical to the GnRH which has been reported in rat hypothalamus.Conclusion Our data suggest that GnRH could be produced by gastric parietal cells of rats and may modulate physiological function of gastric parietal cells of rats.\;[

关 键 词：细胞培养 大鼠 胃壁细胞 促性腺激素释放激素 基因定位 基因克隆 序列分析 基因序列 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R-33[医药卫生—基础医学]