降钙素基因相关肽对骨质疏松性骨折愈合影响的体外研究  

Effect of calcitonin gene-related peptide on osteoporotic fracture healing in vitro

作　　者：梁伟[1] 吕天成 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,545005

出　　处：《中华骨科杂志》2017年第6期353-359,共7页Chinese Journal of Orthopaedics

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

摘　　要：目的探讨降钙素相关基因肽（calcitonin gene-related peptide，CGRP）对骨质疏松大鼠的骨髓基质干细胞（bone mesenchymal stem cells，BMSCs）成骨分化能力及其血管内皮细胞（vascular endothelial cells，VECs）增殖的影响，及其促进骨质疏松性骨折愈合的细胞分子学作用机制。方法取3月龄SD大鼠10只，予双侧卵巢切除术制作骨质疏松模型，术后6个月予显微CT（Micro-CT）行骨密度检测，确定建模成功；后采用全血贴壁法分离骨质疏松性大鼠的BMSCs及VECs；在细胞培养液中加入不同浓度（1×10^-7~1×10^-10 mol/L）的CGRP，采用细胞增殖检测（CCK-8）法检测细胞增殖能力；CGRP对骨质疏松大鼠BMSCs成骨分化能力的影响：通过碱性磷酸酶活性检测及逆转录-聚合酶链反应（RT-PCR）方法检测碱性磷酸酶、Ⅰ型胶原、重组人骨形态发生蛋白2、骨黏连蛋白、RunX2蛋白等成骨相关细胞基因mRNA的表达。CGRP对骨质疏松大鼠VECs血管化的影响：采用ELISA、RT-PCR法分别检测细胞内皮细胞生长因子（vascular cell endothelial growth factor，VEGF）的蛋白分泌和基因表达水平。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对BMSCs细胞的影响：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OD值为0.58±0.02，CGRP组中10^-7~10^-10 mol/L分别为0.80±0.03，0.79±0.03，0.74±0.03，0.69±0.03，与对照组的差异均有统计学意义；CGRP对VECs细胞的影响：对照组OD值为2.20±0.01，CGRP组中10^-7~10^-10 mol/L分别为4.12±0.04，3.90±0.02，3.56±0.04，3.12±0.04，与对照组的差异均有统计学意义。碱性磷酸酶活性检测及RT-PCR方法检测碱性磷酸酶、Ⅰ型胶原、重组人骨形态发生蛋白2、骨黏连蛋白、RunX2蛋白等成骨相关细胞基因mRNA的表达，对照组与CGRP应用的各浓度组的差异均有统计学意义。结论10^-7~10^-10 mol/L的CGRP可以Objective To explore the effects of calcitonin gene-related peptide （CGRP） on proliferation of vascular endo- thelial cells （VECs） and osteogenic differentiation of bone mesenchymal stem cells （BMSCs） of osteoporotic rats in vitro, and cell/ molecular mechanism of promoting osteoporotic fracture healing. Methods 10 3-month-old SD rats were employed to make osteo- porosis model by taking bilateral ovariectomy. Micro-CT analysis was used to determine the success of modeling at 6 months of postoperation. BMSCs and VECs were isolated from osteoporotic rats by blood adherence method. 10-7-10-l~ mol/L CGRP were added to the culture medium. CCK-8 method was used to examine the cell proliferation. Effect of CGRP on osteogenic differentia- tion of BMSCs： Alkaline phosphatase （ALP） activity, and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the expression of ALP, Collagen I, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs： Vas- cular cell endothelial growth factor （VEGF） protein secretion and gene expression level were tested by ELISA and RT-PCR. Re- suits BMSCs and VECs of osteoporotic rats were isolated successfully.The optical density （OD） values of CCK- 8 method in- creased in a dose-dependent manner, for BMSCs at 7 d, the control group of OD value was 0.58±0.02, the CGRP group 10-7 -10-~~ mol/L was to 0.80±0.03, 0.79±0.03, 0.74±0.03, 0.69±0.03 , difference was significant; for VECs, the control group of OD value was 2.20±0.01, the CGRP group 10^-7 -10^-10 mol/L was to 4.12＋0.04, 3.90±0.02, 3.56±0.04, 3.12±0.04, difference was significant. Effect of CGRP on osteogenic differentiation of BMSCs： Alkaline phosphatase （ALP） activity, and reverse transcription polymerase chain reaction （RT-PCR） were used to detect the expression of ALP, Collagen I, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs： Vascular cell endothelial growth factor （VEGF） protein secretion and gene expressio

关 键 词：降钙素基因相关肽 骨质疏松 骨折 

分 类 号：R580[医药卫生—内分泌] R683[医药卫生—内科学]