中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2010年
12期
1223-1227
,共5页
王钊%金丹%文君%陀泳华%郭小磊
王釗%金丹%文君%陀泳華%郭小磊
왕쇠%금단%문군%타영화%곽소뢰
降钙素基因相关肽%P物质%骨髓祖代细胞%细胞周期
降鈣素基因相關肽%P物質%骨髓祖代細胞%細胞週期
강개소기인상관태%P물질%골수조대세포%세포주기
Calcitonin gene-related peptide%Substance P%Myeloid progenitor cells%Cell cycle
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.
目的 觀察外源性降鈣素基因相關肽(calcitonin gene-related peptide,CGRP)、P物質(substance P,SP)對大鼠骨髓間質榦細胞(bone marrow mesenchymal stem cells,BMSCs)增殖的影響,併探討其機製.方法 使用全骨髓法分離、培養大鼠BMSCs,採用RT-PCR、Western Blot在傳代培養的不同時間(1、2、3週)檢測BMSCs神經肽受體mRNA、蛋白的錶達情況.在不同時間點(1、3、5、7、9 d)使用濃度為1×10-8 mol/L的CGRP、SP分彆作用于BMSCs,使用MTT比色法檢測BMSCs增殖能力,Western Blot法檢測BMSCs細胞週期相關調控基因cyclin D1、cyclin E、p53的蛋白錶達變化.結果 RT-PCR、Western Blot結果顯示BMSCs穩定錶達CGRP受體、SP受體,同一時間點的CGRP受體錶達高于SP受體錶達.MTT結果顯示,與對照組相比,CGRP在9d時促進BMSCs增殖,SP在7、9 d時促進BMSCs增殖.Western Blot結果顯示,與對照組相比,CGRP促進BMSCs內cyclin D1、cyclin E蛋白錶達,在5 d時達到高峰;SP促進BMSCs內cyclin E蛋白錶達,在5 d時達到高峰;兩種神經肽均降低BMSCs內p53蛋白錶達.結論 CGRP、SP對BMSCs的增殖有直接促進作用,影響細胞週期相關調控基因蛋白的錶達是其作用機製之一.
목적 관찰외원성강개소기인상관태(calcitonin gene-related peptide,CGRP)、P물질(substance P,SP)대대서골수간질간세포(bone marrow mesenchymal stem cells,BMSCs)증식적영향,병탐토기궤제.방법 사용전골수법분리、배양대서BMSCs,채용RT-PCR、Western Blot재전대배양적불동시간(1、2、3주)검측BMSCs신경태수체mRNA、단백적표체정황.재불동시간점(1、3、5、7、9 d)사용농도위1×10-8 mol/L적CGRP、SP분별작용우BMSCs,사용MTT비색법검측BMSCs증식능력,Western Blot법검측BMSCs세포주기상관조공기인cyclin D1、cyclin E、p53적단백표체변화.결과 RT-PCR、Western Blot결과현시BMSCs은정표체CGRP수체、SP수체,동일시간점적CGRP수체표체고우SP수체표체.MTT결과현시,여대조조상비,CGRP재9d시촉진BMSCs증식,SP재7、9 d시촉진BMSCs증식.Western Blot결과현시,여대조조상비,CGRP촉진BMSCs내cyclin D1、cyclin E단백표체,재5 d시체도고봉;SP촉진BMSCs내cyclin E단백표체,재5 d시체도고봉;량충신경태균강저BMSCs내p53단백표체.결론 CGRP、SP대BMSCs적증식유직접촉진작용,영향세포주기상관조공기인단백적표체시기작용궤제지일.
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.
