降钙素基因相关肽对骨质疏松大鼠骨髓基质干细胞增殖及分化的影响  

Effect of calcitonin gene - related peptide on proliferation and osteogenic differentiation of bone mesenchymal stem cells in osteoporotic rats

作　　者：梁伟[1] 李理[1] 李兵[1] 

机构地区：[1]广西医科大学第四附属医院柳州市工人医院骨科,柳州545005

出　　处：《中华实验外科杂志》2018年第1期41-45,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81260273）;广西自然科学基金（2013GXNSFAA019269）

摘　　要：目的观察降钙素相关基因肽（CGRP）对骨质疏松大鼠骨髓基质干细胞（BMSCs）增殖及成骨分化能力的影响，探讨其促进骨质疏松性骨折愈合的细胞/分子作用机制。方法3个月龄SD大鼠，行双侧卵巢切除术，术后常规喂养，术后6个月用显微CT（Micro-CT）行骨密度检测，确定骨质疏松模型建立成功；处死骨质疏松大鼠，采用全血贴壁法分离BMSCs，用流式细胞仪进行鉴定；以培养基中加入不同浓度（10－7～10－10 mol/L）的CGRP建立实验组，用细胞计数试剂盒（CCK-8）法检测细胞增殖能力；碱性磷酸酶（ALP）活性检测及茜素红染色法观察CGRP对骨质疏松大鼠BMSCs成骨分化及细胞外基质矿化能力的影响；采用反转录酶-聚合酶链反应（RT-PCR）方法检测ALP、Ⅰ型胶原蛋白（COLL-1）、骨形态发生蛋白-2（BMP-2）、骨粘连蛋白（Osteonectin）、Run家族转录因子（RunX2）等成骨相关细胞基因mRNA的表达；使用Western blot分析来检测RunX2及Osteonectin的蛋白含量。 结果成功分离培养出骨质疏松大鼠BMSCs；CCK-8法测定细胞增殖能力，CGRP各浓度组较对照组均增加，且呈剂量依赖关系，7 d时对照组吸光度（A）值为0.580 4±0.015 0，CGRP组中10－7 mol/L为0.804 1±0.029 9（P＝0.000），10－8 mol/L为0.788 6±0.028 0（P＝0.000），10－9 mol/L为0.743 6±0.028 8（P＝0.001），10－10 mol/L为0.693 3±0.029 2（P＝0.004），差异有统计学意义；ALP活性测定结果显示，各CGRP组较对照组ALP活性均增高，14 d时对照组为4.633±0.423，CGRP组中10－7 mol/L为44.047±2.446（P＝0.000），10－8 mol/L为42.030±2.626（P＝0.000），10－9 mol/L为39.333±0.527（P＝0.000），10－10 mol/L为24.647±1.135（P＝0.000）[单位：10－3 μmol/（min·mg）]，差异有统计学意义；钙结节染色均呈阳性，而对照组无显色；基因检测结果提示，各CGRP组的基因表达较对照组升高，7 d时ALP空�ObjectiveTo research the effects of calcitonin gene-related peptide （CGRP） on the proliferation and osteogenic differentiation of bone mesenchymal stem cells （BMSCs） of osteoporotic rats in vitro.MethodsThree-month-old SD rats were used, taking bilateral ovariectomy through the abdomen. Micro-computed tomography （Micro-CT） analysis was used to determine the success of modeling at 6 months after surgery. BMSCs were isolated from osteoporotic rats by blood adherence method. The biological characteristics of BMSCs were examined by flow cytometry. Different concentrations of CGRP （10-7-10-10 mol/L） were added to the culture medium of third generation BMSCs for examination of proliferation according CCK-8 method, alkaline phosphatase （ALP） activity and alizarin red staining were used to evaluate the ability of cells differentiation and mineralization. And reverse transcription polymerase chain reaction （RT-PCR） was used to detect the expression of ALP, Collagen I, BMP-2, Osteonectin and RunX2 gene. Western blotting was used to detect the expression of RunX2 and Osteonectin protein.ResultsThe bone marrow stromal cells of osteoporotic rats were isolated successfully. The optical density （A） values of CCK-8 method increased in a dose-dependent manner, at 7 days, the control group of A value was 0.580 4±0.015 0, the CGRP group 10-7 mol/L was to 0.804 1±0.029 9（P=0.000）, 10-8 mol/L to 0.788 6±0.028 0（P=0.000）, 10-9 mol/L to 0.743 6±0.028 8 （P=0.001） and 10-10 mol/L to 0.693 3±0.029 2（P=0.004）, difference was significant; The ALP activity was positive, at 14 days, the control group of A value was 4.633±0.423, the CGRP group 10-7 mol/L was to 44.047±2.446 （P=0.000）, 10-8 mol/L to 42.030±2.626 （P=0.000）, 10-9 mol/L to 39.333±0.527 （P=0.000）, 10-10 mol/L to 24.647±1.135 （P=0.000） [unit： 10-3 μmol/（min·mg）}, difference was significant （P〈0.05）; The formation of calcium nodules were proved by alizarin red staining; The target genes expression wer

关 键 词：降钙素基因相关肽 骨质疏松 骨髓基质干细胞 成骨分化 

分 类 号：R580[医药卫生—内分泌]