摘要
目的培养骨关节炎患者软骨下骨成骨细胞,探讨硬化区和非硬化区软骨下骨成骨细胞的生物学特性。方法收集本科2009年10月至2010年3月拟行全膝关节置换入院的骨关节炎患者7例,男性5例,女性2例,平均年龄68(55～79)岁,留取术后遗弃的胫骨平台骨组织。应用Ⅰ型胶原酶消化联合贴壁培养方法,培养软骨下骨的成骨细胞,光镜下观察细胞形态,免疫组化检测Ⅰ型胶原,NBT/BCIP染色法检测碱性磷酸酶,茜素红染色检测矿化结节,油红O染色检测脂质成分。应用实时定量RT-PCR技术检测并比较硬化区和非硬化区软骨下骨成骨细胞相关基因[碱性磷酸酶(ALP),骨钙蛋白(OCN),转化生长因子β1(TGF-β1),胰岛素样生长因子1(IGF-1),Ⅰ型胶原(COL1)A1/(COL1)A2,基质金属蛋白酶13(MMP-13),骨桥蛋白(OPN),肿瘤坏死因子(TNF-α),白介素(IL)-6和IL-8]的表达。应用茜素红染色比较两种细胞矿化能力。结果光镜下观察到细胞渐从骨片中爬出的生长过程,免疫组化证实细胞表达Ⅰ型胶原,NBT/BCIP染色显示细胞表达碱性磷酸酶,茜素红染色显示细胞能形成矿化结节,油红O染色显示细胞内无脂质成分,证实Ⅰ型胶原酶消化联合贴壁培养方法能较好的培养出成骨细胞。茜素红染色显示软骨下骨硬化区成骨细胞矿化能力低于非硬化区[(0.92±0.06),(1.32±0.15),P<0.01]。RT-PCR显示,硬化区软骨下骨成骨细胞比非硬化区软骨下骨成骨细胞高表达基因OPN[(2.62±1.47)×10-2,(9.63±3.37)×10-4,P<0.05]、OCN[(6.04±6.27)×10-6,(4.84±2.18)×10-6,P<0.05]、ALP[(73.62±15.75)×10-4,(3.10±0.33)×10-4,P<0.01]、IGF-1[(14.84±1.23)×10-4,(6.33±2.80)×10-4,P<0.01]、IL-6[(8.24±1.05)×10-4,(2.64±0.37)×10-4,P<0.01]、IL-8[(14.68±6.97)×10-6,(2.60±0.89)×10-6,P<0.01]、MMP-13[(1.50±0.11)×10-3,(1.23±0.10)×10-3,P<0.05]、TGF-β1[(4.41±0.42)×10-3,(2.96±0.36)×10-3,P<0.01]、COL1A1[(55.44±21.61)×10-2,(9.17±3.30)×10-2,P<0.01];而TNF-α[(5.62±2.03)×10-6,(5.62±3.146)×10-6]和COL1A2[0.64±0.07,0.46±0.12]表达水平没有明显差异(P>0.05)。结论硬化区软骨下骨成骨细胞的生物学表型变化是骨关节炎患者病变的重要特征之一。
Objective To culture subchondral bone osteoblasts and to study the biological characteristic differences between the osteoblasts in sclerotic area and non-sclerotic area in osteoarthritis（OA） patients.Methods We collected the bone tissue of tibial plateaus resected from 7 OA patients（5 men and 2 women）,with an average age of 68 years（55-79）,who received total knee replacement between October 2009 and March 2010 in our hospital.Subchondral bone osteoblasts were obtained from the sclerotic area and non-sclerotic area of the tibial plateaus through type-Ⅰ collagenase digestion and adherent culture.The appearances of the osteoblasts were observed under a light microscope.Immunohistochemistry,NBT/BCIP staining,alizarin red staining,and oil red O staining were performed on the osteoblasts to detect type-I collagen（COL1）,alkaline phosphate（ALP）,mineralized nodules,and fat,respectively.Real-time RT-PCR was performed to evaluate the mRNA expression levels of ALP,osteocalcin（OCN）,transforming growth factor-β1（TGF-β1）,insulin-like growth factor-1（IGF-1）,COL1A1,COL1A2,matrix metalloproteinase-13（MMP-13）,osteopontin（OPN）,tumor necrosis factor-α（TNF-α）,IL-6,and IL-8 of the osteoblasts in the sclerotic area and non-sclerotic area.Alizarin red staining was performed to compare the mineralization degrees of the osteoblasts in the sclerotic area and non-sclerotic area.Results The growth of subchondral bone osteoblasts could be observed under the light microscope.COL1,ALP,and mineralized nodules were detected in the osteoblasts,while no fat granules were found.The alizarin red staining showed that the subchondral bone osteoblasts in the sclerotic area had a lower mineralization degree than those in the non-sclerotic area[（0.92±0.06）,（1.32±0.15）,P0.01].Compared with those in the non-sclerotic area,the osteoblasts in the sclerotic area had significantly higher mRNA expression levels of OPN[（2.62±1.47）×10-2,（9.63±3.37）×10-4,（P0.05）],OCN[（6.04±6.27）×10-6,（4.84±2.18）×10-6,P0.05],ALP[（73.62±15.75）×10-4,（3.10±0.33）×10-4,P0.01],IGF-1[（14.84±1.23）×10-4,（6.33±2.80）×10-4,P0.01],IL-6[（8.24±1.05）×10-4,（2.64±0.37）×10-4,P0.01],IL-8[（14.68±6.97）×10-6,（2.60±0.89）×10-6,P0.01],MMP-13[（1.50±0.11）×10-3,（1.23±0.10）×10-3,P0.05],TGFβ1[（4.41±0.42）×10-3,（2.96±0.36）×10-3,P0.01],and COL1A1[（55.44±21.61）×10-2,（9.17±3.30）×10-2,P0.01],while no significant differences（P0.05） were found in terms of the mRNA expression levels of TNF-α[（5.62±2.03）×10-6,（5.62±3.146）×10-6] and COL1A2[0.64±0.07,0.46±0.12].Conclusion Type-I collagenase digestion in conjugation with adherent culture is an effective means of culturing subchondral bone osteoblasts.The biological characteristic changes of the subchondral bone osteoblasts in sclerotic area are important pathological characteristics of OA patients.A further research on these changes and their causes will provide a great help for the research and therapy of OA.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第10期1003-1007,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(30772182)~~
