软骨前体细胞的分离鉴定及IL-1β对其成软骨分化的影响  

ISOLATION AND IDENTIFICATION OF CARTILAGE PROGENITOR CELLS AND INFLUENCE OF INTERLEUKIN 1β ON ITS CHONDROGENESIS

作　　者：周建新 杨晓斐[2] 李阳[2] 丁朋[2] 蒋逸秋[2] 桂鉴超[2] 

机构地区：[1]苏州市吴江区第一人民医院骨科,江苏苏州215200 [2]南京医科大学附属南京医院骨科

出　　处：《中国修复重建外科杂志》2015年第7期863-869,共7页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的对正常软骨中的软骨前体细胞进行分离、鉴定,并对不同浓度IL-1β对软骨前体细胞的成软骨分化影响进行研究。方法取正常成年新西兰大白兔的软骨细胞,通过纤连蛋白粘连分离出软骨前体细胞,采用流式细胞仪对其细胞表型进行鉴定,倒置相差显微镜观察其克隆增殖,并行成骨、成脂、成软骨三系分化观察。培养软骨前体细胞团并分为4组,分别加入普通H-DMEM培养基(A组)、成软骨诱导分化培养基(B组)、成软骨诱导分化培养基+0.1 ng/mL IL-1β(C组)、成软骨诱导分化培养基+1.0 ng/mL IL-1β(D组),培养3周行组织学、生物化学、实时荧光定量PCR等检测,观察IL-1β的影响。结果在正常软骨细胞中存在软骨前体细胞,经鉴定有干细胞表型阳性表达,有与干细胞相似的克隆增殖能力及分化能力。HE染色示,C、D组中细胞团块较B组明显减小,细胞呈肥大样改变。番红O、Ⅱ型胶原及Ⅹ型胶原染色示,B组较A组染色深,C、D组均浅于B组,D组浅于C组。生物化学成分测定示,C、D组的总胶原、糖胺聚糖(glycosaminoglycan,GAG)相对含量及GAG/DNA比值均显著低于B组,D组显著低于C组,差异均有统计学意义(P<0.05);C、D组DNA相对含量均显著高于B组(P<0.05),但C、D组间差异无统计学意义(P>0.05)。实时荧光定量PCR检测示,C、D组Ⅱ型胶原、Ⅹ型胶原、Sox-9 mRNA相对表达量均显著低于B组,D组显著低于C组,差异均有统计学意义(P<0.05);而C、D组Runx-2和MMP-13mRNA相对表达量均显著高于B组,D组显著高于C组,差异均有统计学意义(P<0.05)。结论在正常软骨组织中存在一种有干细胞特性的软骨前体细胞,其有克隆和潜在分化的能力。IL-1β对软骨前体细胞成软骨分化有抑制作用,并有促进成骨分化的可能。Objective To isolate and identify the cartilage progenitor cells（CPCs） from normal cartilage, and to explore the influence of interleukin 1β（IL-1β） in different concentrations on its chondrogenesis. Methods CPCs were isolated from normal cartilage of adult New Zealand white rabbit with the fibronectin adhesion assay; the cell phenotype was identified; and the cloning and differentiation of CPCs were observed. CPCs were incubated with H-DMEM in group A, with chondrogenic induced medium in group B, with chondrogenic induced medium＋0.1 ng/mL IL-1β in group C and chondrogenic induced medium＋1.0 ng/mL IL-1β in group D for 3 weeks. The histology, biochemistry, and realtime fluorescence quantitative PCR were performed to observe the effect of IL-1β on the chondrgenic differentiation.Results The CPCs from normal cartilage expressed positively stem cell phenotype, which have similar ability of cloning and differentiation to stem cells. The cell pellets in groups C and D were significantly smaller than those in group B, and cell showed hypertrophic morphology change. There were more expressions of collagen type Ⅱ and collagen type X in group B than in group A, in group B than in groups C and D, and in group C than group D with Safranin O staining. The biochemistry results showed that collagen type Ⅱ content, glycosaminoglycan（GAG） content, and the ratio of GAG/DNA were significantly lower in groups C and D than in group B（P〈0.05）, and in group D than in group C（P〈0.05）; but the DNA content was significantly higher in groups C and D than in group B（P〈0.05）, and no significant difference between groups C and D（P〉0.05）. The real-time fluorescence quantitative PCR results showed that the relative mRNA expressions of collagen type Ⅱ, collagen type X, and Sox-9 were significantly lower in groups C and D than in group B（P〈0.05）, and in group D than in group C（P〈0.05）, but the relative mRNA expressions of Runx-2 and matrix metalloproteinase 13 were significantly higher

关 键 词：软骨前体细胞 炎性因子 IL-1Β 诱导分化 软骨修复 

分 类 号：R687[医药卫生—骨科学] R318.08[医药卫生—外科学]