title: [Effects of CGRP and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cells].
abstract: OBJECTIVE
  To explore the effects and mechanism of secretory calcitonin gene-related peptide (CGRP) and CGRP receptor modified mesenchymal stem cells on proliferation and phenotypic transformation of vascular smooth muscle cell.
  
  
  METHODS
  Firstly (Lenti-GFP-CGRP, referred to CGRP (+/+)) MSCs were transfected with high expression lentivirus vector of CGRP (MSCs(CGRP+/+)). Protein secretion in the above-mentioned MSCs(CGRP+/+) supernatant was detected with enzyme-linked immunosorbent assay (ELISA). And then MSCs(CGRP+/+) was co-cultured with VSMCs(RAMP1)(+/+) and VSMCs(RAMP1-/-) respectively. Experimental groups were as follows: MSCs +VSMCs MSCs(CGRP+/+) +VSMCs, MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+) and MSCs(CGRP+/+) + VSMCs (RAMP1-/-). Flow cytometry was applied to detect the cycle variation of smooth muscle cells, thiazolyl blue tetrazolium bromide method for detecting the proliferation of smooth muscle cells and Western blot for examining the expression changes of spectrin α-SM-actin and synthetic protein OPN in each group respectively.
  
  
  RESULTS
  After the transfection of CGRP(+/+), MSCs(CGRP+/+) secreted and expressed CGRP protein, the secretory volume of CGRP protein in MSCs(CGRP+/+) increased significantly compared with the control group (19.530 ± 0.498 vs 3.133 ± 0.160 and 3.120 ± 0.001, P < 0.05) . After a 72 h co-culturing with VSMCs, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)(+/+)group declined significantly (0.270 ± 0.263 vs 0.413 ± 0.070, P < 0.05) and the number of cells staying in G0 phase significantly increased (93.51% ± 0.38% vs 84.48% ± 0.31%, P < 0.05) , the expression of contractile phenotype protein α-SM-actin increased and intermediate phenotype OPN declined significantly as compared with MSCsCGRP(+/+) +VSMCs group { (α-SM-actin 102 946 ± 3847 vs 51 759 ± 635, P < 0.05), OPN (26 026 ± 2595 vs 44 201 ± 2811, P < 0.05) }; but compared with MSCs(CGRP+/+)+VSMCs(RAMP1)(+/+)group, the proliferation of VSMCs in MSCs(CGRP+/+) +VSMCs(RAMP1)-/-group significantly increased (0.601 ± 0.04 vs 0.270 ± 0.263, P < 0.05) while the number of cells staying in G0 phase significantly declined (78.57% ± 0.68% vs 93.51% ± 0.38%, P < 0.05) . The expression of contractile phenotype protein α-SM-actin declined while intermediate phenotype OPN increased significantly in MSCs(CGRP+/+)+VSMCs(RAMP1)-/-group {α-SM-actin (34 400 ± 2179 vs 102 946 ± 3847, P < 0.05), OPN (53 933 ± 1192 vs 26 026 ± 2595, P < 0.05)}.
  
  
  CONCLUSIONS
  CGRP gene-modified MSCs may secrete target protein stably. And CGRP-modified MSCs inhibits VSMCs phenotypic transformation and cell proliferation. It is probably associated with enhanced CGRP effect due to an up-regulation of CGRP receptor.
authors.affiliation: Deparment of Cardiology, First Affiliated Hospital, Zunyi Medical College, Zunyi 563003, China. Email: shi-bei2147@yahoo.com.cn.
authors.forename: Bei
authors.initials: B
authors.lastname: Shi
authors.forename: Can
authors.initials: C
authors.lastname: Cui
authors.forename: Xian-ping
authors.initials: XP
authors.lastname: Long
authors.forename: Wen-ming
authors.initials: WM
authors.lastname: Chen
authors.forename: Ran-zun
authors.initials: RZ
authors.lastname: Zhao
authors.forename: Zhi-jiang
authors.initials: ZJ
authors.lastname: Liu
authors.forename: Zhu-jun
authors.initials: ZJ
authors.lastname: Ao
mesh_terms: D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics*; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000235:genetics*; D014162:Transfection
chemical_list: D018015:Receptors, Calcitonin Gene-Related Peptide; D015740:Calcitonin Gene-Related Peptide