早期移植生长分化因子11表达沉默的骨髓基质干细胞促进大鼠激素性股骨头坏死区成骨的研究  

Early transplantation of growth differentiation factor 11-silenced bone marrow stromal cells promotes osteogenesis in necrotic femoral head induced by steroid in rats

作　　者：孔令驰 孙兴亮 左荣台 王梦玮 关俊杰[1] 康庆林[1] Kong Lingchi;Sun Xingliang;Zuo Rongtai;Wang Mengwei;Guan Junjie;Kang Qinglin(Department of Orthopaedic Surgery,The Sixth People's Hospital Affiliated to Shanghai Jiao Tong University,Shanghai 200233,China;Department of Orthopaedic Surgery,Wulian People’s Hospital,Wulian 262300,Shandong,China)

机构地区：[1]上海交通大学附属第六人民医院骨科,200233 [2]山东省五莲县人民医院骨二科,262300

出　　处：《中华创伤骨科杂志》2019年第9期802-809,共8页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金面上项目(81572121).

摘　　要：目的探讨早期移植生长分化因子(GDF)11表达沉默的骨髓基质干细胞(BMSCs)对大鼠激素性股骨头坏死区的成骨作用。方法转染小干扰RNA(siRNA)以沉默大鼠BMSCs中的GDF11表达,并检测转染效率和细胞毒性。细胞实验(n=3)和动物实验(n=8)各分4组:空白对照组(未干预)、模型组(糖皮质激素)、实验组(糖皮质激素+siRNA)和阴性对照组(糖皮质激素+siRNA阴性对照物)。采用碱性磷酸酶(ALP)染色与茜素红染色检测BMSCs成骨分化效果;反转录聚合酶链式反应检测成骨标志物的表达水平。利用显微CT、HE染色、免疫组织化学和生物力学等方式评估大鼠股骨头的成骨效果。结果转染siRNA对大鼠BMSCs活性无影响。细胞实验:实验组ALP活性与钙结节数量优于模型组;模型组ALP、核心结合蛋白因子2、骨钙素和Ⅰ型胶原蛋白α1链的mRNA相对表达量分别为0.46±0.11、0.50±0.11、0.35±0.01、0.57±0.02,较空白对照组(1.00±0.09、1.02±0.23、1.03±0.30、1.02±0.25)显著下降,实验组(1.97±0.30、0.94±0.19、1.50±0.18、1.28±0.37)又较模型组显著升高,差异均有统计学意义(P<0.05)。动物实验:实验组较模型组大鼠股骨头密度升高,骨结构更完整,骨小梁参数和生物力学参数比较差异均有统计学意义(P<0.05),且核心结合蛋白因子2和Ⅰ型胶原表达量升高。结论在糖皮质激素作用下,GDF11表达沉默可增强BMSCs的成骨分化。早期移植GDF11表达沉默的BMSCs可有效促进大鼠股骨头坏死区成骨。Objective To study the effect of growth differentiation factor(GDF)11-silenced bone marrow stromal cells(BMSCs)on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats.Methods After GDF11 expression in BMSCs was inhibited by siRNA,the knockdown efficiency and transfection cytotoxicity were detected.The further experiments both in vitro(n=3)and in vivo(n=8)were divided into 4 groups respectively:blank control group(without any intervention),model group(glucocorticoid treatment),experimental group(siRNA transfection and glucocorticoid treatment)and negative control group(negative control transfection and glucocorticoid treatment).The BMSCs were induced into osteogenic differentiation.Alkaline phosphatase(ALP)staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction(qRT-PCR)was employed to detect the relative expression levels of osteogenic markers.The osteogenesis in the necrotic femoral head was evaluated by microCT,H&E staining,immunohistochemistry staining and biomechanical test.Results No transfection cytotoxicity was found(P>0.05).The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group.At the level of mRNA,the relative expression of ALP,runt-related transcription factor(Runx)2,osteocalcin(OCN)and typeⅠcollagen(α1)(COL1A1)in the blank control group(1.00±0.09,1.02±0.23,1.03±0.30 and 1.02±0.25,respectively)were significantly higher than those in the model group(0.46±0.11,0.50±0.11,0.35±0.01 and 0.57±0.02,respectively)but significantly lower than those in the experimental group(1.97±0.30,0.94±0.19,1.50±0.18 and 1.28±0.37)(all P<0.05).MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group(P<0.05).Histological examination showed better bone regeneration and higher exp

关 键 词：股骨头坏死 骨髓细胞 生长分化因子11 糖皮质激素类 成骨分化 

分 类 号：R681.8[医药卫生—骨科学]