瑞香素联合IGF-1对大鼠脂肪间充质干细胞成软骨分化的影响  

Effects of daphnetin combined with insulin-like growth factor 1 on chondrogenic differentiation of adipose-derived mesenchymal stem cells in rats

作　　者：申宁 席晓云 李飞 吕超亮[3] 张普晟[1] SHEN Ning;XI Xiaoyun;LI Fei;LU Chaoliang;ZHANG Pusheng(Department of Orthopedics,Jinxiang Hospital of Jining Medical University,Jining Shandong,272200,P.R.China;Department of Radiology,Jinxiang Hospital of Jining Medical University,Jining Shandong,272200,P.R.China;Department of Orthopedics,Jining First People's Hospital,Jining Shandong,272200,P.R.China)

机构地区：[1]济宁医学院附属金乡医院骨科,山东济宁272200 [2]济宁医学院附属金乡医院放射科,山东济宁272200 [3]济宁市第一人民医院骨科,山东济宁272200

出　　处：《中国修复重建外科杂志》2019年第6期743-749,共7页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的初步研究瑞香素(dephnetin,DAP)联合IGF-1基因转染对大鼠脂肪间充质干细胞(adiposederived mesenchymal stem cells,ADSCs)成软骨分化的影响。方法采用酶消化法分离培养大鼠ADSCs。取第3代ADSCs以IGF-1基因转染作为实验组,未转染的ADSCs作为对照组。两组细胞分别用不同浓度DAP(0、30、60、90μg/mL)处理,培养72 h后采用细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞增殖活性;培养14 d分别采用实时荧光定量PCR和Western blot检测Ⅱ型胶原和蛋白聚糖(Aggrecan)mRNA和蛋白表达,并行甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色观察。结果 CCK-8法检测示,随DAP作用浓度增加,对照组和实验组细胞吸光度(A)值均逐渐增加(P<0.05);相同DAP浓度下,实验组细胞A值显著高于对照组(P<0.05)。实时荧光定量PCR和Western blot检测示,随DAP作用浓度增加,对照组细胞Ⅱ型胶原和Aggrecan的mRNA和蛋白相对表达量无明显变化,各浓度组间差异无统计学意义(P>0.05);实验组细胞Ⅱ型胶原和Aggrecan的mRNA和蛋白相对表达量逐渐增加,其中60、90μg/mL DAP浓度组显著高于0μg/mL DAP浓度组(P<0.05)。相同DAP浓度下,实验组细胞Ⅱ型胶原和Aggrecan的mRNA和蛋白相对表达量显著高于对照组(P<0.05)。甲苯胺蓝染色示,随DAP作用浓度增加,对照组内和实验组内细胞着色无明显差异;相同DAP浓度下,实验组比对照组细胞着色略深。Ⅱ型胶原免疫组织化学染色示,随DAP作用浓度增加,对照组细胞的细胞质内着色无明显差异;实验组细胞的细胞质内着色逐渐加深,其中60、90μg/mL DAP浓度组明显深于0μg/mL DAP浓度组。相同DAP浓度下,实验组比对照组细胞着色明显加深。结论 DAP对大鼠ADSCs增殖有一定促进作用;单独使用DAP诱导大鼠ADSCs向软骨细胞分化作用极微弱,但DAP联合IGF-1基因转染有明显协同作用,促进ADSCs成软骨分化。Objective To investigate the effect of daphnetin(DAP) combined with insulin-like growth factor 1(IGF-1) gene transfection on chondrogenic differentiation of adipose-derived mesenchymal stem cells(ADSCs) in rats.Methods Rat ADSCs were isolated and amplified by enzymatic digestion. The third generation ADSCs were treated with IGF-1 gene transfection as experimental group and normal ADSCs as control group. The cells of the two groups were treated with different concentrations of DAP(0, 30, 60, 90 μg/mL), respectively. Cell proliferation was detected by cell counting kit 8(CCK-8) after cultured for 72 hours. After 14 days, real-time fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of chondrocyte markers(collagen type Ⅱ and Aggrecan) in each group;and toluidine blue staining and collagen type Ⅱ immunohistochemical staining were performed.Results CCK-8 assay showed that with the increase of DAP concentration, the cell absorbance(A) value of the control group and the experimental group increased gradually(P<0.05). At the same DAP concentration, the cell A value of the experimental group was significantly higher than that of the control group(P<0.05). Real-time fluorescence quantitative PCR and Western blot showed that with the increase of DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the control group did not change significantly, and there was no significant difference among the different concentration groups(P>0.05). But the mRNA and protein expressions of collagen type Ⅱ and Aggrecan in the experimental group increased gradually, and the 60 and 90 μg/mL DAP concentration groups were significantly higher than 0 μg/mL DAP concentration group(P<0.05). At the same DAP concentration, the relative mRNA and protein expressions of collagen type Ⅱ and Aggrecan were significantly higher in the experimental group than in the control group(P<0.05). Toluidine blue staining showed that with the increase of DAP concentr

关 键 词：脂肪间充质干细胞 瑞香素 IGF-1 成软骨分化 大鼠 

分 类 号：R68[医药卫生—骨科学]