摘要
目的比较大鼠骨髓来源与前交叉韧带(anterior cruciate ligament,ACL)来源的MSCs体外增殖及成骨、成软骨、成脂分化潜能的差异。方法取SPF级6周龄雄性BN大鼠10只,体质量200~220 g,无菌条件下分别取大鼠骨髓及ACL,贴壁法培养获取BMSCs与ACL来源MSCs并进行传代,倒置相差显微镜下观察细胞形态变化。取生长良好的第3代细胞,流式细胞仪检测细胞免疫表型CD34、CD45、CD90和CD29表达;细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞体外增殖能力,克隆形成实验检测细胞体外克隆形成能力;行成骨、成软骨及成脂体外诱导多向分化能力检测;实时荧光定量PCR检测成骨[ALP、骨钙蛋白、RUNX2、BMP-2、分泌性磷蛋白1(secreted phosphoprotein 1,Spp1)]、成软骨[Ⅱ型胶原α1(collagen typeⅡα1,Col2α1)、软骨聚糖蛋白(Aggrecan,Acan)、Sox9]及成脂[过氧化物酶体增殖物激活受体γ2(peroxisome proliferator activated receptorγ2,PPARγ2)、CCAAT/增强子结合蛋白α]相关基因mRNA相对表达量。结果第3代ACL来源MSCs与BMSCs形态相似,均表现为贴壁生长的长梭形细胞。流式细胞仪检测示,两种细胞均表达CD29、CD90,基本不表达CD45及CD34。CCK-8法检测示,ACL来源MSCs的吸光度(A)值(1.11±0.08)显著高于BMSCs(0.78±0.05),差异有统计学意义(t=3.599,P=0.023);ACL来源MSCs的细胞集落数[(53.00±5.51)个/孔]亦明显多于BMSCs[(30.67±4.84)个/孔](t=3.045,P=0.038)。经成骨、成软骨、成脂体外诱导培养21d后,BMSCs及ACL来源MSCs均表现为茜素红、甲苯胺蓝及油红O染色阳性。实时荧光定量PCR检测示,ACL来源MSCs的BMP-2、Spp1、Col2α1、Acan、Sox9及PPARγ2 mRNA相对表达量显著高于BMSCs,差异均有统计学意义(P<0.01)。结论 ACL来源MSCs比BMSCs具有更强的体外增殖能力,在相同体外条件下更易向软骨分化,是一种有潜力的促进腱骨愈合的种子细胞。
Objective To compare the biological characteristics of bone marrow mesenchymal stem cells （BMSCs） and anterior cruciate ligament derived mesenchymal stem cells （ACL-MSCs） from ratsin vitro. Methods Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 （CCK-8）, and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas （ALP）, bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 （BMP-2）, secreted phosphoprotein 1 （Spp1）, collagen type II α1 （Col2α1）, Aggrecan （Acan）, Sox9, peroxisome proliferator activated receptor γ2 （PPARγ2）, and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR. Results BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance （A） value of ACL-MSCs （1.11±0.08） was significantly higher than that of BMSCs （0.78±0.05） （t=3.599,P=0.023）; the number of colonies of ACL-MSCs [（53.00±5.51）/hole] was significantly more than that of BMSCs [（30.67±4.84）/hole] （t=3.045,P=0.038）. The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs （P〈0.01）. Conclusion The proliferation potential of ACL-MSCs is g
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2017年第4期473-480,共8页
Chinese Journal of Reparative and Reconstructive Surgery
基金
南京市卫生局科技基金项目(790000031)
国家自然科学基金资助项目(81672159)~~
关键词
BMSCS
前交叉韧带来源MSCs
增殖分化
大鼠
Bone marrow mesenchymal stem cells
anterior cruciate ligament derived mesenchymal stem cells
proliferation and differentiation
rat
