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背景:体内溶血磷脂酸存在3种受体,已有研究表明溶血磷脂酸受体1,2,3广泛分布于小鼠大脑皮质、肾脏的外髓层和内髓层、睾丸、胸腺、心脏、肺、胃、脾脏,而肝脏、小肠和骨骼肌分布较少,小鼠血管平滑肌细胞膜上是否存在多种溶血磷脂酸受体有待研究。目的:观察血管平滑肌细胞中溶血磷脂酸受体mRNA表达含量情况。设计:观察对比实验。单位:华中科技大学同济医学院。材料:选用24只C57BL/6小鼠,雌雄不限,7～8周龄,体质量(25±3)g左右,由华中科技大学同济医学院实验动物学部提供。Trizol试剂购自美国Invitrogen公司;dNTP Mix、RNasin购自TaKaRa公司;M-MLV逆转录酶及缓冲系统购自Promega公司;Taq DNA聚合酶及缓冲系统购自Biostar公司;Oligo(dT)18引物购自上海生工。引物参照文献及核苷酸序列数据库设计,由上海生工合成。方法:实验于2005-08/2006-01在华中科技大学同济医学院综合实验室完成。20g/L氯胺酮(5mL/kg)腹腔麻醉小鼠,无菌操作取出胸主动脉段,采用贴壁法培养血管平滑肌细胞,实验用4～6代细胞,细胞纯度达95%以上。采用RT-PCR技术测定小鼠血管平滑肌细胞溶血磷脂酸3种受体基因的mRNA相对表达量。主要观察指标:小鼠血管平滑肌细胞溶血磷脂酸受体mRNA的相对表达量检测及受体种类的比较。结果:溶血磷脂酸受体1与溶血磷脂酸受体2在小鼠血管平滑肌细胞的表达量相近(P>0.05)。溶血磷脂酸受体3在小鼠血管平滑肌细胞的表达量为(0.53±0.05),少于溶血磷脂酸受体1和溶血磷脂酸受体2(0.79±0.05,0.82±0.06,q=23.78,26.53,P<0.01)。结论:血管平滑肌细胞存在3种溶血磷脂酸受体,溶血磷脂酸受体1与溶血磷脂酸受体2在小鼠血管平滑肌细胞的表达量没有差别,而溶血磷脂酸受体3在小鼠血管平滑肌细胞的表达量少。
BACKGROUND: There are three receptors for lysophosphatidic acid in vivo. Studies have shown that lysophosphatidic acid receptors 1, 2, and 3 are widely distributed in the cerebral cortex, outer and medullary layers of the kidney, testis, thymus, heart, Lung, stomach, spleen, and liver, small intestine and skeletal muscle distribution is less, the existence of a variety of vascular smooth muscle cell membrane lysophosphatidic acid receptor to be studied. Objective: To observe the content of lysophosphatidic acid receptor mRNA in vascular smooth muscle cells. Design: observe the contrast experiment. Unit: Huazhong University of Science and Technology Tongji Medical College. MATERIALS: Twenty-four C57BL / 6 mice were selected. They were male and female, aged 7-8 weeks, and had a body mass of about 25 ± 3 g. They were provided by the Laboratory Animalology Department, Tongji Medical College, Huazhong University of Science and Technology. Trizol reagent was purchased from Invitrogen Corporation, USA; dNTP Mix, RNasin was purchased from TaKaRa; M-MLV reverse transcriptase and buffer system were purchased from Promega; Taq DNA polymerase and buffer system was purchased from Biostar; Oligo (dT) Live from Shanghai. Primer references and nucleotide sequence database design, synthesized by Shanghai Biotechnology. Methods: The experiment was performed at the Comprehensive Laboratory of Tongji Medical College, Huazhong University of Science and Technology from August 2005 to January 2006. 20g / L ketamine (5mL / kg) intraperitoneal anesthesia mice, aseptic removal of the thoracic aorta, the use of adherent method cultured vascular smooth muscle cells, the experiment with 4 to 6 generations of cells, cell purity of 95% or more. RT-PCR was used to determine the relative mRNA expression of lysophosphatidic acid receptors in mouse vascular smooth muscle cells. MAIN OUTCOME MEASURES: Detection of relative expression of lysophosphatidic acid receptor mRNA in mouse vascular smooth muscle cells and comparison of receptor types. Results: The expression of lysophosphatidic acid receptor 1 and lysophosphatidic acid receptor 2 in vascular smooth muscle cells of mice were similar (P> 0.05). The expression of lysophosphatidic acid receptor 3 in mouse vascular smooth muscle cells was (0.53 ± 0.05) less than that of lysophosphatidic acid receptor 1 and lysophosphatidic acid receptor 2 (0.79 ± 0.05, 0.82 ± 0.06, q = 23.78, 26.53, P <0.01). CONCLUSION: There are three lysophosphatidic acid receptors in vascular smooth muscle cells. There is no difference in the expression of lysophosphatidic acid receptor 1 and lysophosphatidic acid receptor 2 in mouse vascular smooth muscle cells. However, lysophosphatidic acid receptor 3 Smooth muscle cell expression is low.