神经肽对大鼠骨髓间质干细胞增殖影响的研究

摘要

Objective To investigate the effects and mechanism of calcitonin gene-related peptide (CGRP) and substance P (SP) on proliferation of rat bone marrow mesenchymal stem cells. Methods The rBMSCs were isolated using whole bone marrow adherence method. In the different periods of culturing (1, 2,and 3 weeks), expressions of the neuropeptide receptors were detected by Western Blot and reserve transcriptase-polymerase chain reaction (RT-PCR). The BMSCs were treated with CGRP and SP at concentration 10-8 mol/L at different time (1,3,5,7,9 days), cell proliferation was detected with MTT assay, the protein expressions of cyclin D1 ,cyclin E and p53 were examined using Western Blot. Results The CGRP receptor and SP receptor were expressed in BMSCs. The expression of CGRP receptor was statistically higher than that of SP receptorat the same time point. The growth curves of BMSCs cultured by both neuropeptides had similar appearance. CGRP and SP stimulated the proliferation of BMSCs significantly at 9 days and 7 and 9 days. In this process, the expressions of cyclinDl and cyclinE were up-regulated by CGRP, SP only enhanced the expression of cyclinE; these effects all reached a peak at 5 days. The expression of p53 was down-regulated by both neuropeptides. Conclusion CGRP and SP had direct effects on the proliferation of BMSCs, the regulation of cell cycle proteins is one of the mechanisms.%目的 观察外源性降钙素基因相关肽(calcitonin gene-related peptide,CGRP)、P物质(substance P,SP)对大鼠骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影响,并探讨其机制.方法 使用全骨髓法分离、培养大鼠BMSCs,采用RT-PCR、Western Blot在传代培养的不同时间(1、2、3周)检测BMSCs神经肽受体mRNA、蛋白的表达情况.在不同时间点(1、3、5、7、9 d)使用浓度为1×10-8 mol/L的CGRP、SP分别作用于BMSCs,使用MTT比色法检测BMSCs增殖能力,Western Blot法检测BMSCs细胞周期相关调控基因cyclin D1、cyclin E、p53的蛋白表达变化.结果 RT-PCR、Western Blot结果显示BMSCs稳定表达CGRP受体、SP受体,同一时间点的CGRP受体表达高于SP受体表达.MTT结果显示,与对照组相比,CGRP在9d时促进BMSCs增殖,SP在7、9 d时促进BMSCs增殖.Western Blot结果显示,与对照组相比,CGRP促进BMSCs内cyclin D1、cyclin E蛋白表达,在5 d时达到高峰;SP促进BMSCs内cyclin E蛋白表达,在5 d时达到高峰;两种神经肽均降低BMSCs内p53蛋白表达.结论 CGRP、SP对BMSCs的增殖有直接促进作用,影响细胞周期相关调控基因蛋白的表达是其作用机制之一.