GDF-5联合地塞米松诱导大鼠BMSCs向类髓核样细胞分化  

Differentiation of rat BMSCs into nucleus pulposus-like cells induced by GDF-5 and dexamethasone

作　　者：魏强强[1,2,3] 殷嫦嫦[1] 殷明[2] 何丁文[2] 周荣平[2] 梁广胜[2,3] 

机构地区：[1]九江学院转化医学重点实验室,江西九江332000 [2]南昌大学第二附属医院骨一科 [3]南昌大学研究生院医学部,江西南昌330006

出　　处：《基础医学与临床》2014年第8期1037-1043,共7页Basic and Clinical Medicine

基　　金：国家自然科学基金(81160226)

摘　　要：目的探讨GDF-5联合地塞米松诱导大鼠骨髓间充质干细胞(BMSCs)向类髓核样细胞分化潜能及其表型的改变。方法用全骨髓贴壁法及序贯酶消化法分别分离培养大鼠BMSCs和髓核细胞(NPCs),均取P3代细胞分为1)对照组,2)BMSCs+Dex组3)BMSCs+GDF-5组4)BMSCs+Dex+GDF-5组(联合诱导组)5)NPCs组。诱导培养21 d,镜下观察细胞形态及密度变化,阿辛蓝染色检测蛋白多糖,RT-PCR检测COL1、ACAN、SOX9及COL2和髓核细胞阳性基因KRT19、PAX1和FOXF1的表达。Western blot检测COL2及COL1蛋白表达。结果 BMSCs形态呈长梭型、漩涡样生长;NPCs呈球形、岛状生长的特点;诱导后BMSCs向类圆形、三角形转变,细胞体积缩小,胞质颜色与对照组相比逐渐加深;蛋白多糖分泌增加,以联合诱导效果最佳。ACAN、SOX9、COL2基因的相对表达量逐渐增高,ACAN/COL2的比例呈递增趋势,COL1的表达呈递减趋势。NPCs阳性基因KRT19、PAX1、FOXF1与对照组相比(P<0.05)。含GDF-5的诱导组COL2蛋白表达高于对照组(P<0.05),而联合诱导组COL1蛋白的表达最少。结论GDF-5可以诱导大鼠BMSCs向类髓核样细胞分化,且地塞米松能起到显著促进作用,为BMSCs移植治疗椎间盘退变提供了理论依据。Objective To investigate the NP-like cell differentiation of rat bone marrow mesenchymal stem cells induced by GDF-5 and Dexamethasone. Methods BMSCs were collected from Sprague Dawley rats and the feasibility of isolating and culturing rats nucleus pulposus cells （NPCs） by the sequential enzyme method. Cells at passage 3 were used and divided into control group, group of BMSCs ＋ Dex, group of BMSCs ＋ GDF-5, group of BMSCs ＋ Dex ＋ GDF-5, group of NPCs, then the cellular morphology was observed under microscope and induced for 21 days. A1- cian Blue stain showed the changes of proteoglycan, RT-PCR detected expression COL1, ACAN, SOX9, COL2 and positive genes of nucleus pulposus cells for KRT19, PAX1, COL1. Results BMSCs presented a single long spindle with characteristics and island growth. BMSCs became circles, FOXF1. Western blot was used to detect COL2 and whirlpool-like morphology; NPCs presented spherical triangles, then shrinkaged and the cytoplasmic color of cells gradually deepened as compared with the control group. The content of proteoglycan was increased obviously by Co-induction. The relative expression levels of genes of ACAN, SOX9, COL2 were gradually increased, and the pro- portion of which ACAN/COL2 showed an increasing trend. The mRNA expression of COL1 showed a decreasing trend. The positive genes of NPCs for KRT19, PAX1, FOXF1 were significant different as compared with control group（P 〈0. 05）. The expression of COL2 protein in GDF-5 group was significantly increased as compared with control group （P 〈 0. 05 ）, then the expression of COL1 protein was minimum by Co-induction. Conclusions GDF-5 may induce rat BMSCs to differentiate into NP-like cells and dexamethasone plays a promotion effect in the differentiation of induction, and provides a theoretical basis for BMSCs transplantation in the treatment of interverte- bral disc degeneration.

关 键 词：GDF-5 地塞米松 BMSCS 分化 类髓核样细胞 

分 类 号：R681.5[医药卫生—骨科学]