摘要
目的:探讨去整合素金属蛋白酶(a disintegrin and metalloproteinase,ADAM) 9、15、17在骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨分化中的作用。方法:分离ADAM9、ADAM15、ADAM17的条件基因敲除小鼠及野生型(wild type,WT)小鼠的BMMSCs,培养刺激其分化为成骨细胞。比色法测定成骨标志碱性磷酸酶(alkaline phosphatase,ALP)活性,实时荧光定量PCR检测成骨早期转录因子Runx、Osterix,茜素红染色分析成骨矿物质形成情况。结果:在无刺激的对照培养下,ADAM9组(8. 08±0. 34)、ADAM15组(6. 46±3. 40)、ADAM17(9. 30±2. 30)组的ALP活性与WT组(9. 44±2. 50)相比差异无统计学意义(P均> 0. 05)。用骨形态发生蛋白质2(bone morphogenic protein 2,BMP2)刺激培养,ADAM9组(14. 22±3. 25)、ADAM15组(10. 14±2. 40)的ALP均小于WT组(20. 89±3. 40),差异有统计学意义(P均<0. 05),而ADAM17组(23. 56±2. 50)虽大于WT组(20. 89±3. 40),但差异无统计学意义(P> 0. 05)。用成骨培养基(osteogenic induction medium,OST)刺激培养,ADAM9组(9. 63±1. 00)、ADAM15组(7. 75±1. 30)的ALP均小于WT组(12. 97±1. 30),而ADAM17组(20. 09±1. 68)大于WT组(12. 97±1. 30),差异均有统计学意义(P均<0. 05)。同样,BMP2与OST联合刺激培养后检测ALP,ADAM9组(15. 75±1. 30)、ADAM15组(12. 43±1. 30)均小于WT组(26. 15±1. 50),而ADAM17组(29. 55±2. 10)大于WT组,差异均有统计学意义(P均<0. 05)。Runx2表达,在无刺激的对照培养下,ADAM9组(2. 02±0. 24)、ADAM15组(3. 09±0. 19)、ADAM17组(3. 89±0. 91)分别与WT组(2. 02±0. 21)相比差异均无统计学意义(P均> 0. 05); BMP2刺激培养后ADAM9组(7. 00±0. 23)、ADAM15组(6. 04±0. 23)均小于WT组(12. 6±0. 23),而ADAM17组(18. 52±1. 39)大于WT组,差异均有统计学意义(P均<0. 05)。Osterix表达,在无刺激的对照培养下,ADAM9组(9. 60±3. 87)、ADAM17组(12. 4±3. 00)与WT组(10. 9±1. 10)比较差异均无统计学意义(P均> 0. 05),但ADAM15组(6. 50±1. 51)低于WT组(10. 9±1. 10),差异有统计学意义(P <0. 05); BMP2刺激培养后ADAM9组(39. 20±3. 23)、ADAM15组(20. 50±4. 80)均小于WT组(60. 3±5. 93),而ADAM17组(80. 2±3. 30)大于WT组,差异均有统计学意义(P均<0. 05)。茜素红染色,无刺激组未见明显橘红色团块,BMP2、OST、OST+BMP2刺激培养条件下各实验组均可见局部钙化结节形成,但量化分析差异无统计学意义(P> 0. 05)。结论:ADAM9、15、17参与了BMMSCs的成骨分化,为调节BMMSCs成骨分化提供了新靶点。
Objective： To study the effects of disintegrin and metalloproteinase（ ADAM） 9,15 and 17 on the osteogenic differentiation of bone marrow mesenchymal stem cells（ BMMSCs）. Methods：BMMSCs of ADAM9,ADAM15,ADAM17 conditional knockout mice and wild type mice（ WT） were induced and cultured. Alkaline phosphatase（ ALP） activity was measured by colorimetry,early osteogenic transcription factors Runx and Osterix were detected by Real-time PCR,and mineral formation was analyzed by alizarin red staining. Results： ALP activity was lower in ADAM9 group（ 8. 08 ± 0. 34）,ADAM15 group（ 6. 46 ± 3. 40）,ADAM17 group（ 9. 30 ± 2. 30） than that in WT group（ 9. 44 ± 2. 50）,but there was no significant difference（ P〈0. 05）. Stimulated with bone morphogenetic protein2（ BMP2）,there was significant difference（ P〈0. 05） between ADAM9 group（ 14. 22 ± 3. 25）,ADAM15 group（ 10. 14 ± 2. 40） and WT group（ 20. 89 ± 3. 40）,and ADAM 17 group（ 23. 56 ± 2. 50）was higher than WT group（ 20. 89 ± 3. 40）,but no significant difference（ P〈0. 05）. Similarly,cultured by osteogenic induction medium（ OST）,compared with WT group（ 12. 97 ± 1. 30）,ADAM9 group（ 9. 63 ± 1. 00） and ADAM15 group（ 7. 75 ± 1. 30） were lower,ADAM17 group（ 20. 09 ± 1. 68） was higher,and the difference was statistically significant（ P〈0. 05）. Using stimulated culture by BMP2 and OST combined,ADAM9 group（ 15. 75 ± 1. 30）,ADAM 15 group（ 12. 43 ± 1. 30） were less than WT group（ 26. 15 ± 1. 50）,while ADAM17 group（ 29. 55 ± 2. 10） was higher than WT group were statistically significant（ P〈0. 05）. The expression of Runx2 in ADAM9 group（ 2. 02 ± 0. 24）,ADAM15 group（ 3. 09 ± 0. 19）,ADAM17 group（ 3. 89 ± 0. 91） had no significant difference compared with WT（ 2. 02 ± 0. 21） group（ P〈0. 05）. ADAM9 group stimulated by BMP2（ 7. 00 ± 0. 23）,ADAM15 group（ 6. 04 ± 0. 23） were lower than WT group（ 12. 6 ± 0. 23）,ADAM17 group（ 18. 52 ± 1. 39） was higher than WT group（ 12. 6 ± 0. 23）,and the difference was statistically significant（ P〈0. 05）. In non-stimulating culture,there was no significant difference in Osterix expression between ADAM9 group（ 9. 60 ±3. 87）,ADAM17 group（ 12. 40 ± 3. 00） and WT group（ 10. 9 ± 1. 10,P〈0. 05）,but in ADAM15 group（ 6. 50 ± 1. 51） it was slightly lower than that in WT group（ P〈0. 05）. After BMP2 stimulation,ADAM9 group（ 39. 20 ± 3. 23） and ADAM15 group（ 20. 50 ± 4. 80） were less than WT group（ 60. 30 ±5. 93）,while ADAM17 group（ 80. 20 ± 3. 30） was higher than WT group（ P〈0. 05）. Alizarin red staining showed no obvious orange-red mass in the non-induction group. Local calcified nodules could be seen in the BMP2,OST,OST ＋ BMP2 induction culture conditions in all the experimental groups,but there was no significant difference in quantitative analysis（ P〈0. 05）. Conclusion： ADAM9,15,17 took part in the osteogenic differentiation of BMMSCs,and provided new targets for its regulation.
作者
刘霞
李英妮
孙晓麟
彭清林
卢昕
王国春
LIU Xia;LI Ying-ni;SUN Xiao-lin;PENG Qing-lin;LU Xin;WANG Guo-ehun(Department of Rheumatology and hnmunology,China-Japan Friendship Hospital,Beijing 100029,China;Depart-ment of Rheumatology and hnmunology,Peking University People＇s Hospital,Beijing 100044,China)
出处
《北京大学学报（医学版）》
CAS
CSCD
北大核心
2018年第6期962-967,共6页
Journal of Peking University:Health Sciences
基金
国家自然科学基金(81302586)~~
关键词
去整合素金属蛋白酶
骨髓间充质干细胞
成骨分化
A disintegrin and metalloproteinase
Bone marrow mesenchymal stem cells
Osteogenic differentiation
