Investigation Of RAMP1 And CRLR Enhancing The Inhaibition Effect Of CGRP On The Proliferation Of VSMCs

AIM :1.To Observe the effect of RAMP1 overexpression to CGRP inhibite AngII- induced vascular smooth muscle cell proliferation 2.To explore the role of RAMP1 overexpression on distribution of CRLR.METHODS:1. Construction of pCDNA3.1(+)-RAMP1 eukaryon expression vector:Extract total RNA of A10 cell and run RT-PCR. Double digest the RAMP1 ORF and pCDNA3.1(+) vector by EcoRI/BamHI respectively, then transform into DH5α. Judgments the sequence exactitude of RAMP1 gene by Sequencing, then extract the recombination for a standby. 2. Stable cell line:G418 solution was prepared with different concentration to screen cell, as a basic concentration for kill all cells for 14 days. To optimize the ratio of transfection reagent to plasmid then transfect cell by standard procedure. Screen positive cell with G418 since 48 hour later by transfection. The expressions of RAMP1 mRNA and protein were detected by RT PCR and western blot, respectively. 3.Synchronize cell for 24h by 0.1%FBS; The VSMCs were treated with CGRP or (and) AngII, the viability of cultured VSMCs were estimated by MTT assay;the expression of CRLR were detected by RT-PCR and Western Blot; the distribution of RAMP1 and CRLR in membrane were detected by Immunofluorescence.RESULTS:1. Success in construction of RAMP1 eukaryon expression vector by identification of EcoRI/BamHI double digate and sequencing. 2. The OD570 Values of RAMP1 overexpression was higher than No plasmid and pCDNA3.1(+) by MTT assay; The mRNA levels of CRLR in each group have no significance by RT PCR; Treating cell with CGRP and AngII,we found that the CRLR proteins expression were lower in the RAMP1 overexpression group, but higher under only stimulus of CGRP, meanwhile, the CRLR proteins level have on change between no plasmid and pCDNA3.1(+)-RAMP1 treated with 0.1% FBS or AngII; The membrane distribution of RAMP1 and CRLR in pCDNA3.1(+)-RAMP1 were more than No plasmid and pCDNA3.1(+) by Immunofluorescence .CONCLUSION 1.Success in construction of RAMP1 eukaryon expression vector 2.Overexpression of RAMP1 significantly enhance effect of CGRP on antiprolifer- ation of VSMC induced by AngII , which maybe increase the distribution of CRLR in the membrane,and enhance the sensitivity of CGRP receptor to CGRP.