不同浓度浓缩生长因子对脂肪间充质干细胞成骨能力的影响  

Effect of different concentrations of concentrated growth factor on osteogenic potential of adipose-derived mesenchymal stem cells

作　　者：黄昕[1] 郭正健 李家宁 张雅婧 张婷婷[1] Huang Xin;Guo Zhengjian;Li Jianing;Zhang Yajing;Zhang Tingting(Department of Oral and Maxillofacial Surgery,Stomatological Hospital,Tianjin Medical University,Tianjin Key Laboratory of Oral Soft and Hard Tissues Restoration and Regeneration,Tianjin Medical University Institute of Stomatology,Tianjin 300070,China)

机构地区：[1]天津医科大学口腔医院口腔颌面外科、天津市口腔软硬组织修复再生重点实验室、天津医科大学口腔研究所,天津300070

出　　处：《国际生物医学工程杂志》2024年第6期537-545,共9页International Journal of Biomedical Engineering

基　　金：天津市教委科研计划项目(2022KJ207);天津市卫健委中医中西医结合科研项目(2023155)。

摘　　要：目的研究不同浓度浓缩生长因子(CGF)对脂肪间充质干细胞(ADSCs)成骨能力的影响。方法差速离心法从大鼠血液中制备CGF, 通过酶联免疫吸附试验(ELISA)测定CGF内源性生长因子水平;从雄性SD大鼠腹股沟脂肪组织中提取ADSCs, 采用含不同浓度(5%、20%、50%、80%)CGF的培养基进行培养, 运用免疫荧光染色检测表面标志物CD44表达和流式细胞术检测CD29和CD90表达来鉴定ADSCs;通过细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性;通过碱性磷酸酶(ALP)染色法检测成骨分化效果, 茜素红染色法检测成骨钙化效果;通过实时荧光定量逆转录PCR检测成骨相关基因相对表达水平;并通过免疫印迹法检测成骨相关蛋白相对表达水平。结果 CGF中骨形态发生蛋白-2(BMP-2)、转化生长因子β1(TGF-β1)和血管内皮生长因子-A(VEGF-A)的检测浓度分别为(33.15±0.72)pg/ml、(95.60±2.45)ng/ml、(351.58±22.28)pg/ml。ADSCs形态呈梭形, 伸展良好, 免疫荧光染色显示细胞质呈均匀的绿色荧光。流式细胞术检测CD29和CD90表达阳性率分别为99.65%、92.72%。低浓度(5%、20%)CGF组ADSCs整体呈稳定的增殖活性, 培养7 d后, 吸光度(A)值分别为3.90±0.25、4.04±0.22, 均高于对照组, 且差异均有统计学意义(均P<0.01)。当CGF浓度为5%时, ALP染色最深, 且5% CGF组ALP的累积A值(2.299×107±7.156×106)高于对照组(4.426×106±1.839×105), 差异具有统计学意义(t=?4.95, P<0.01)。不同浓度CGF组的茜素红染色均比对照组较深, 且5% CGF组钙结节的累积A值(3.599×107±4.094×106)高于对照组(1.413×107±1.298×106), 差异具有统计学意义(t=?5.46, P<0.01)。5% CGF组BMP-2、β-连环蛋白(β-catenin)、TGF-β1 mRNA相对表达水平分别为50.97±1.75、1.84±0.53、1.86±0.24, 均分别高于对照组的26.03±1.94、1.13±0.12、1.14±0.16, 且差异均具有统计学意义(均P<0.01)。5% CGF组BMP-2、β-catenin、TGF-β1蛋白表达水平分别为1 174.33±70.02、1Objective To study the effect of different concentrations of concentrated growth factor(CGF)on the osteogenic potential of adipose-derived mesenchymal stem cells(ADSCs).Methods Preparation of CGF from rat blood by differential centrifugation method,the levels of endogenous growth factors in CGF were quantified by enzyme-linked immunosorbent assay(ELISA).ADSCs were extracted from the inguinal adipose tissue of male SD rats and cultured in media containing different concentrations(5%,20%,50%,80%)of CGF.ADSCs were identified by the expression of the surface marker CD44 detected by immunofluorescence staining and by the expression of CD29 and CD90 detected by flow cytometry.Cell proliferation effects was evaluated by the cell counting kit-8(CCK-8)assay.The effect of osteogenic differentiation was detected by alkaline phosphatase(ALP)staining,and the effect of osteogenic calcification was detected by alizarin red staining.Real-time reverse transcription-PCR was used to detect the expression levels of osteogenic genes,and Western blotting was performed to detect the expression of osteogenesis-related proteins.Results The detected level of bone morphogenetic protein 2(BMP-2),transforming growth factor-β1(TGF-β1),and vascular endothelial growth factor-A(VEGF-A)were(33.15±0.72)pg/ml,(95.60±2.45)ng/ml,and(351.58±22.28)pg/ml in CGF,respectively.ADSCs were spindle-shaped and well-extended.Immunofluorescence staining results showed uniform green fluorescence in the cytoplasm.Flow cytometry analysis revealed that the positive rates for CD29 and CD90 were 99.65%and 92.72%,respectively.The low-concentration(5%and 20%)CGF groups showed stable proliferative activity in ADSCs,with absorbance(A)value reaching 3.90±0.25 and 4.04±0.22 after 7 days of culture,both of them were higher than that of control group,and the differences were statistically significant(both P<0.01).The 5%CGF group showed the most intense ALP staining,with a cumulative A value of ALP(2.299×10^(7)±7.156×10^(6)),which was significantly higher than that i

关 键 词：生长因子 浓缩生长因子 脂肪间充质干细胞 成骨 碱性磷酸酶 骨形态发生蛋白-2 转化生长因子Β1 血管内皮生长因子 

分 类 号：R78[医药卫生—口腔医学]