【摘要】 目的对比不同浓度碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对国产多孔钽-软骨细胞复合物维持软骨细胞表型和去分化作用的影响。方法分离培养3周龄新西兰幼兔膝关节软骨细胞,Ⅱ型胶原免疫细胞化学染色、番红O(Safranin O)染色鉴定软骨细胞;取第3代软骨细胞分为5组:A组(1ng/mL bFGF-多孔钽-软骨细胞),B组(10ng/mL bFGF-多孔钽-软骨细胞),C组(50ng/mL bFGF-多孔钽-软骨细胞),D组(多孔钽-软骨细胞)及E组(软骨细胞);MTT法检测不同浓度bFGF对多孔钽-软骨细胞生长及增殖状态的影响;扫描电镜观察bFGF-多孔钽-软骨细胞复合物细胞形态及生长;Ⅰ、Ⅱ、Ⅸ、Ⅹ型胶原免疫细胞化学染色检测软骨细胞表型变化及去分化状态;real time-PCR检测软骨细胞Ⅱ、Ⅹ型胶原mRNA表达。结果Ⅱ型胶原免疫细胞化学染色及Safranin O染色均呈阳性反应,证实所分离培养的细胞为软骨细胞;1、10、50ng/mL bFGF均可促进软骨细胞增殖,各实验组(A~D组)软骨细胞增殖与对照组(E组)相比,差异有统计学意义,其中10ng/mL bFGF-多孔钽-软骨细胞增殖最为明显(P<0.05);组间比较差异有统计学意义(P<0.05);扫描电镜观察:各组软骨细胞在多孔钽表面及孔隙内生长、黏附,早期呈不等的球形,24h后胞质延展并向周围伸出多条突起,相互连接、延展向孔隙内部生长,细胞间相互融合并覆盖支架材料;Ⅰ、Ⅱ、Ⅸ和Ⅹ型胶原免疫细胞化学染色显示10ng/mL bFGF-多孔钽-软骨细胞组(B组)Ⅱ和Ⅸ型胶原表达高于对照组(E组,P<0.05),组间差异也有统计学意义(P<0.05);而Ⅰ和Ⅹ型胶原表达则低于对照组(E组,P<0.05);real time-PCR检测软骨细胞Ⅱ、Ⅹ型胶原mRNA表达显示:各实验组(A~D组)Ⅱ型胶原mRNA的表达高于对照组(E组,P<0.05),而Ⅹ型胶原mRNA表达量则低于对照组(E组,P<0.05)。结论 bFGF能维持多孔钽-软骨细胞复合物软骨细胞表型,抑制去分化,加强软骨细胞分泌功能。更多还原

【Abstract】 Objective To study the effect of basic fibroblast growth factor(bFGF)of different concentrations on phenotypes and dedifferentiation of rabbit chondrocytes in porous tantalum-chondrocyte composites in vitro,so as to provide theoretic basis for cartilage defect repair.Methods The articular chondrocytes from 3-week-old rabbit were cultured and identified by typeⅡ collagen immunocytochemistry and Safranin O staining.The 3rd generation chondrocytes were implanted in the porous tantalum and was treated with bFGF of various concentrations.The bFGF-chondrocyte-porous tantalum composites(bFGF compostes)were then divided into 5groups:group A(1ng/mL bFGF composites),group B(10ng/mL bFGF composites),group C(50 ng/mL bFGF composites),group D(chondrocyte-porous tantalum),and group E(pure chondrocyte).The proliferation of chondrocytes was measured by MTT and the cell morphology and growth were observed by scanning electron microscopy(SEM).Phenotypes and dedifferentiation(typeⅠ,Ⅱ,Ⅸ,andⅩcollagen)of the chondrocytes were detected by immunocytochemical method.TypeⅡandⅩcollagen mRNA was tested by real-time PCR.Results TypeⅡ collagen immunocytochemistry and Safranin O staining were positive,confirming that the cultured cells were chondrocytes.MTT results showed that chondrocyte proliferation in groups A,B,C,and D were significantly greater than that in group E(P<0.05),with the most significant proliferation promoting effect seen at 10ng/mL;significant difference was also found between different groups(P<0.05).SEM observation:the chondrocytes grew adherently on the surface of the scaffolds and within scaffold pores,in the early stage the chondrocytes were spherical,after 24 hthe chondrocytes stretched,lengthened,and extended pseudopodia gradually over time,and the chondrocytes connected with each other across the pores and covering the scaffold.Immunocytochemical staining showed that collagen Ⅱ and Ⅸ expressions were significantly stronger in 10ng/mL bFGF-tantalum-chondrocytes group(group B)compared with the control group(group E,P<0.05),and there were significant differences between different groups(P<0.05);while collagen Ⅰ and Ⅹ expressions were significantly weaker compared with the control group(P<0.05).Expression of collagen type Ⅱ mRNA in the chondrocytes was significantly up-regulated in group A,B,C,and D compared with group E(P<0.05),and collagen typeⅩ gene was significantly downregulated(P<0.05).Conclusion bFGF can maintain chondrocyte phenotype in tantalum-chondrocyte composites,inhibit dedifferentiation and promote the secretion function of chondrocytes.更多还原