ADSCs和β-TCP复合支架制备及其对猪膝关节软骨缺损组织CollagenⅡ、Sox-9表达的影响  

Preparation of ADSCs composite scaffold and its effect on CollagenⅡ and Sox-9 expression in pigs with knee articular cartilage defects

作　　者：王宇[1] 王琪[1] 杜明昌[1] 刘铭[1] 刘欣伟[1] 张玉彪[1] 刘松波[1] 刘宪民[1] 项良碧[1] 

机构地区：[1]沈阳军区总医院,沈阳110016

出　　处：《山东医药》2016年第32期1-4,I0001,共5页Shandong Medical Journal

基　　金：辽宁省自然科学基金资助项目(2013020175);辽宁省科技攻关项目(2013225089)

摘　　要：目的制备猪脂肪干细胞(ADSCs)和β-磷酸三钙(β-TCP)复合支架,观察其对猪膝关节软骨缺损组织CollagenⅡ和Sox-9表达的影响。方法取1只巴马小型仔猪的颈部皮下脂肪组织,分离并鉴定ADSCs。以β-TCP作为支架材料,将其制成12个圆柱体支架,随机选择其中6个进行成骨诱导。将12个支架与ADSCs复合培养,制备复合支架,对经成骨诱导后复合支架上的ADSCs进行茜素红和碱性磷酸酶染色。将18只巴马小型猪随机分为空白对照组、支架组、成骨诱导支架组,每组6只。支架组、成骨诱导支架组制备与支架大小相符的膝关节软骨缺损模型,并分别植入未经过、经过成骨诱导的复合支架;空白对照组切开膝关节后直接缝合,不进行其他处理。各组常规饲养4周后处死,取缺损处软骨组织,采用荧光定量PCR法检测CollagenⅡmRNA和Sox-9 mRNA相对表达量,Western blotting法检测CollagenⅡ蛋白和Sox-9蛋白相对表达量。结果分离提取的ADSCs在分离初期镜下显示为圆形单个细胞,细胞的边缘比较清楚,细胞贴壁并形成均匀分布的细胞。免疫组化染色显示,分离的细胞CD44和CD105表达均为阳性,证明其为ADSCs。与空白对照组比较,支架组和成骨诱导支架组软骨组织CollagenⅡ和Sox-9 mRNA及蛋白相对表达量均降低,但支架组降低更明显(P均<0.05)。结论成功制备了ADSCs和β-TCP的复合支架;该支架经成骨诱导后可提高猪膝关节软骨缺损组织CollagenⅡ和Sox-9的表达。Objective To prepare the composite scaffold of porcine adipose stem cells（ ADSCs） and β-tricalcium phosphate（ β-TCP）,and then to observe its effect on CollagenⅡ and Sox-9 expression in pigs with articular cartilage defects. Methods The ADSCs were extracted from subcutaneous adipose tissues of one Bama miniature pig,cells were observed under microscopy,and the expression of CD44,CD105 was detected by immunohistochemical staining. We took β-TCP as scaffolds to make 12 cylindrical stents,then 6 of which were randomly selected for osteogenic induction. The 12 stents and ADSCs were co-cultured to prepare the ADSCs composite scaffolds. ADSCs after osteogenic induction received the Alizarin red and alkaline phosphatase staining. Eighteen Bama miniature pigs were randomly divided into the control group,the stent group and osteogenic stent group,6 in each. The knee cartilage defect models matched to stent size were prepared in the stent group and osteogenic stent group,then were implanted into the ADSCs composite scaffolds with or without osteogenic induction. In the control group,we directly sutured the incision after knee cut without any other treatment. The pigs were sacrificed after four-week regular feeding in each group,and we took the cartilage lesions. The fluorogenic quantitative PCR and Western blotting was used to detect the mRNA and protein expression of CollagenⅡ and Sox-9in the cartilage tissues. Results Under microscope,ADSCs in the early separation were circular single cells,and the cell edge was more clear,and adherent cells formed uniformly. Immunohistochemical staining showed that CD44 and CD105 expression was positive in the isolated cells,which demonstrated they were ADSCs. Compared with the control group,the mRNA and protein expression levels of CollagenⅡ and Sox-9 were decreased in the stent group and osteogenic stent group,but the decrease was more significant in the stent group（ all P 〈 0. 05）. Conclusion ADSCs and β-TCP material composite scaffold was successfully prepared

关 键 词：软骨缺损 膝关节 软骨下骨 Β-磷酸三钙 脂肪干细胞 复合支架 猪 

分 类 号：R687.3[医药卫生—骨科学]