不同时期bFGF或副甲状腺激素相关肽对TGF-β_1诱导的兔BMSCs向软骨细胞分化的影响  

EFFECT OF BASIC FIBROBLAST GROWTH FACTOR AND PARATHYROID HORMONE-RELATED PROTEIN ON EARLY AND LATE CHONDROGENIC DIFFERENTIATION OF RABBIT BONE MARROW MESENCHYMAL STEM CELLS INDUCED BY TRANSFORMING GROWTH FACTOR β_1

作　　者：刘印[1] 贺利平[1] 田京[1] 

机构地区：[1]南方医科大学珠江医院骨科,广州510282

出　　处：《中国修复重建外科杂志》2013年第2期199-206,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：广东省科技计划资助项目(2011B031800147)~~

摘　　要：目的探讨bFGF和副甲状腺激素相关肽(parathyroid hormone-related protein,PTHrP)对TGF-β1诱导兔BMSCs向软骨细胞分化的影响。方法 2月龄健康日本大耳白兔3只,雌雄不限,体重1.6～2.1 kg;取兔胫骨骨髓分离培养BMSCs。取第3代细胞行团块状立体培养,并按照不同诱导条件分为TGF-β1组(A组)、TGF-β1/bFGF组(B组)、TGF-β1/21 d bFGF组(C组)、TGF-β1/PTHrP组(D组)、TGF-β1/21d PTHrP组(E组)。于团块状立体培养开始时,各组均加入10 ng/mL TGF-β1;B、D组同时加入10 ng/mL bFGF或10 ng/mL PTHrP;C、E组于培养21 d时对应加入10 ng/mLbFGF或10 ng/mL PTHrP。培养后1、2、3、4、5、6周检测各组BMSCs向软骨细胞分化的标志性基因Ⅰ型胶原(collagentypeⅠ,ColⅠ)、ColⅡ、ColⅩ和基质金属蛋白酶(matrix metalloproteinases,MMP)13的表达,1、2、3、4、6周检测ALP活性,6周时行1,9二甲基亚甲蓝(1,9-dimethylmethylene blue,DMMB)染色观察细胞外基质分泌情况。结果 RT-PCR检测示,3周后C、E组ColⅠ基因表达呈显著下降趋势,4、5周时A组显著高于C、E组(P<0.05),3～6周A组显著高于B、D组(P<0.05);B、C组3、4周时及D、E组3周时比较,差异有统计学意义(P<0.05)。C、E组3周后ColⅡ、ColⅩ基因表达均逐渐下降,4～6周时显著低于A组(P<0.05);B、D组各时间点两基因表达均未见显著升高,与A组比较差异均有统计学意义(P<0.05)。A组各时间点MMP-13均未见明显表达,3周时B组与A、C组比较差异有统计学意义(P<0.05),D组显著高于A、E组(P<0.05)。随着时间延长A组ALP活性逐渐升高,4周后C、E组显著下降,均低于A组(P<0.05);B、C组间及D、E组间比较,仅在2、3周时差异有统计学意义(P<0.05)。DMMB染色显示6周时A组软骨陷窝明显,其余各组软骨陷窝明显较少。结论 bFGF和PHTrP能通过改变软骨细胞外基质的合成和分解,抑制TGF-β1诱导的兔BMSCs向软骨细胞分化。这种抑制作用不仅通过抑制ColⅩ基因表达而实现,可能还通�Objective To explore the impact of basic fibroblast growth factor （bFGF） and parathyroid hormone- related protein （PTHrP） on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells （BMSCs） induced by transforming growth factor β1 （TGF-β1）. Methods BMSCs were isolated from 3 healthy Japanese rabbits （2-month-old, weighing 1.6-2.1 kg, male or female）, and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions： TGF-β1 group （group A）, TGF-β1/bFGF group （group B）, TGF-β1/21 days bFGF group （group C）, TGF-β1/PTHrP group （group D）, and TGF-β1/21 days PTHrP group （group E）. At the beginning, TGF-β1 （10 ng/mL） was added to all groups, then bFGF and PTHrP （10 ng/mL） were added to groups B and D respectively; bFGF and PTHrP （10 ng/mL） were added to groups C and E at 21 days respectively. The gene expressions of collagen type I （Col I）, Col II, Col X, matrix metalloproteinases （MMP）-13, and alkaline phosphatase （ALP） activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue （DMMB） staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks （P 〈 0.05）, and than that in groups B and D at 3-6 weeks （P 〈 0.05）; and significant differences were found between groups B and C at 3 and 4 weeks, andbetween groups D and E at 3 weeks （P 〈 0.05）. After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks （P 〈 0.05）. Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A （

关 键 词：软骨组织工程 BMSCS BFGF 副甲状腺激素相关肽 成软骨分化 兔 

分 类 号：R687.3[医药卫生—骨科学]