Determining the mRNA expression of lysophosphatidic acid receptors in vascular smooth muscle cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Lysophosphatidic acid (LPA) had 3 kinds of receptors in vitro. Some researches had showed that LPA1, LPA2 and LPA3 receptors distributed widely in mouse cerebral cortex, nephritic external medulla layer and internal medulla layer, spermary, thymus, heart, lung, stomach, spleen, whereas less in liver, small intestine and skeletal muscles. Whether there are various LPA receptors in mouse VSMC membrane deserves further study.OBJECTIVE: To observe the mRNA expression of LPA receptors in vascular smooth muscle cells (VSMC).DESIGN: Observational comparative experiment.SETTING: Tongji Medical College, Huazhong University of Science and Technology.MATERIALS A total of 24 C57BL/6 mice aged 7-8 weeks, of either sex, with the body mass of approximately (25±3) g,were purchased from the Animal Department of Tongji Medical College, Huazhong University of Science and Technology. Trizol was purchased from America Invitrogen; dNTP Mix, Rnasin were obtained from TaKaRa; M-MLV reverse transcriptase and buffer system were from Promega; Taq DNA-polymerase and buffer system were from Biostar.Oligo(dT)18 primer were from Sangon, Shanghai. Primer sequences were designed referring to literature and nucleotide sequence database and synthesized by Sangon, Shanghai.METHODS: The experiment was conducted at the Comprehensive Laboratory, Tongji Medical College, Huazhong University of Science and Technology between August 2005 and January 2006. Mice were anaesthetized by abdominal cavity with 20 g/L ketamine (5 mL/kg). Thoracic aorta was obtained sterilely, and VSMC was cultured with adherence method. The 4th-6th passage cells were used in the trial. Cell purity was over 95%. Reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to determine the mRNA expression of LPA receptors gene in mouse VSMC.MAIN OUTCOME MEASURES: Detection of mRNA expression of LPA receptors in mouse VSMC and comparison of receptor types.RESULTS: There were no significant differences between the expressions of LPA1 receptors and LPA2 receptors (P ＞ 0.05). Compared with LPA1 receptors and LPA2 receptors (0.79±0.05,0.82±0.06), the LPA3 receptor expression was lower (0.53±0.05, q =23.78,26.53, P＜ 0.01 ).CONCLUSION: There are 3 kinds of LPA receptors in VSMC, and their molecular masses are 600 bp, 463 bp and 899 bp,respectively. There are no differences for the expressions between LPA1 receptors and LPA2 receptors, while the LPA3 receptor expression is less.