Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (7): 7-13    DOI: 10.13523/j.cb.20180702
研究报告     
SFRP5抑制BMP9诱导人脐带间充质干细胞成骨分化的实验研究 *
郑妍,姚欢,杨珂()
重庆医科大学附属儿童儿科研究所 儿童发育疾病研究教育部重点实验室 儿童发育重大疾病国家国际科技合作基地 儿科学重庆市重点实验室 重庆市干细胞治疗工程技术研究中心 重庆 400014
SFRP5 Inhibites Osteogenic Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Induced by BMP9
Yan ZHENG,Huan YAO,Ke YANG()
Department of Pediatric Research Institute, Children’s Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, China International Science and Technology Cooperation Base of Child development and Critical Disorders,Chongqing Key Laboratory of Pediatrics, Chongqing Engineering Research Center of Stem Cell Therapy,Chongqing 400014, China
 全文: PDF(1375 KB)   HTML
摘要：

目的:探讨脂肪因子分泌型卷曲相关蛋白(secreted frizzled-related protein 5,SFRP5)对骨形态发生蛋白9 (bone morphogenetic protein 9,BMP9)诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的影响。方法:将人脐带间充质干细胞根据不同的处理因素分为4 组:对照组、BMP9组、BMP9+SFRP5组和SFRP5 组;分别在3天、5天和7天进行碱性磷酸酶(alkaline phosphatase,ALP)活性读数,7天进行ALP染色,21天进行茜素红染色检测钙盐沉积 及油红O染色;收集不同组的细胞用于裸鼠皮下注射成骨模型的建立,4周后取出离体骨进行Micro-CT 扫描和分析,获取的标本进行HE、Masson染色,Alcian blue染色及油红O染色检测。Western blot检测成骨分化相关蛋白Runx2和OPN的表达。结果:BMP9 组的ALP活性读数和染色结果和茜素红染色结果均较对照组增加,而BMP9+SFRP5组则较BMP9组降低;BMP9处理后出现少量脂滴,而BMP9+SFRP5组脂滴明显增加,SFRP5组脂滴最多;裸鼠皮下注射成骨模型的观察结果显示,对照组和SFRP5组没有形成肉眼可见的皮下包块,BMP9组和BMP9+SFRP5组能生成异位骨;4周后观测大体标本以及进行MicroCT检测,发现BMP9+SFRP5组的骨密度值小于BMP9组(P<0.05)。HE、Masson染色,Alcian blue染色结果显示,BMP9组的骨分化程度大于BMP9+SFRP5,油红O染色结果示BMP9+SFRP5组有较多的成脂分化;SFRP5能抑制BMP9诱导的Runx2、OPN的蛋白质表达。结论:SFRP5抑制BMP9诱导的人脐带间充质干细胞成骨分化。
关键词： 间充质干细胞;  分泌型卷曲相关蛋白5;  骨形态发生蛋白9    
Abstract:

Objectives: To determine the role of SFRP5 in bone morphogenetic proteins 9 (BMP9)—mediated osteogenesis in human umbilical cord-derived mesenchymal stem cells(hUC-MSCs). Methods: The hUC-MSCs are divided into control group,BMP9 group,BMP9+SFRP5 group and SFRP5 group. Alkaline phosphatase (ALP) activity was carried out on 3, 5 and 7 days respectively; while ALP staining on 7 days. Aizarin red staining and oil red O staining were detected on 21 days. Different groups of cells were collected for subcutaneous injection in nude mice. After 4 weeks, the ectopic bone formation was analyzed by micro-CT scanning. The specimens were stained with H.E. staining, Masson staining, Alcian blue staining and oil red O staining. Osteogenesis differentiation related proteins expression of Runx2 and OPN were detected by Western blot. Results: Compared with control, the ALP activity and alizarin red staining were higher than BMP9 group; and BMP9+SFRP5 group was lower than that of BMP9 group. A small quantity of lipid droplets was detected in BMP9 group; and lipid droplets increased significantly in BMP9+SFRP5 group and SFRP5 group. Ectopic bone formation subcutaneously in nude mice was observed in BMP9 group and BMP9+SFRP5 group. The bone density of BMP9+SFRP5 group was lower than BMP9 group(P<0.05). The osteogenetic differentiation in BMP9 group was greater than that in BMP9+SFRP5 group by H.E. staining, Masson staining and Alcian blue staining, and more adipogenesis in BMP9+SFRP5 group by oil red O staining. SFRP5 inhibited the protein expression of Runx2 and OPN induced by BMP9. Conclusion: SFRP5 can inhibit the osteogenic differentiation of human umbilical cord mesenchymal stem cells induced by BMP9.
Key words: Mesenchymal stem cells    Secreted frizzled-related protein 5    Bone morphogenetic protein 9
收稿日期: 2018-01-19 出版日期: 2018-08-13
ZTFLH:  R394.2R68  
基金资助: 重庆市科委自然科学研究(cstc2017jcyjAX1071);重庆市科委自然科学研究(cstc2014jcyjA10090)
通讯作者: 杨珂     E-mail: 361885986@qq.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
郑妍
姚欢
杨珂

引用本文:

郑妍,姚欢,杨珂. SFRP5抑制BMP9诱导人脐带间充质干细胞成骨分化的实验研究 *[J]. 中国生物工程杂志, 2018, 38(7): 7-13.

Yan ZHENG,Huan YAO,Ke YANG. SFRP5 Inhibites Osteogenic Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Induced by BMP9. China Biotechnology, 2018, 38(7): 7-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180702    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I7/7

图1  SFRP5对BMP9诱导的hUC-MSCs细胞的成骨分化早期指标ALP活性的影响
图2  SFRP5对BMP9诱导的hUC-MSCs细胞的成骨分化晚期指标茜素红S染色的影响(200×)
图3  SFRP5对BMP9诱导的hUC-MSCs细胞的油红O染色(200×)
图4  SFRP5对BMP9诱导的hUC-MSCs细胞的裸鼠异位成骨MicroCT 扫描、重建及分析
图5  SFRP5对BMP9诱导的hUC-MSCs裸鼠异位成骨组织的染色
图6  SFRP5对BMP9诱导的成骨相关蛋白的影响
[1] Sadie-Van G H, Crowther N J, Hough F S, et al. The interrelationship between bone and fat: from cellular see-saw to endocrine reciprocity. Cell Mol Life Sci, 2013,70(13):2331-2349.
doi: 10.1007/s00018-012-1211-2 pmid: 23178849
[2] Sheu Y, Cauley J A . The role of bone marrow and visceral fat on bone metabolism. Curr Osteoporos Rep, 2011,9(2):67-75.
doi: 10.1007/s11914-011-0051-6 pmid: 4188476
[3] Thomas H A, Antonio S, Antonia G, et al. Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration. Stem Cell, 2017,20(6):771-784.
[4] Hu W, Li L, Yang M, et al. Circulating SFRP5 is a signature of obesity-related. J Clin Endocrinol Metab, 2013,98(1):290-298.
doi: 10.1210/jc.2012-2466
[5] Chen H, He Y, Wu D, et al. Bone marrow SFRP5 level is negatively associated with bone formation markers. Osteoporos Int, 2017,28(4):1305-1311.
doi: 10.1007/s00198-016-3873-3 pmid: 27986984
[6] Li T, Xia M, Gao Y, et al. Human umbilical cord mesenchymal stem cells: an overview of their potential in cell-based therapy. Expert Opin Biol Ther, 2015,15(9):1293-1306.
doi: 10.1517/14712598.2015.1051528
[7] Kang Q, Sun M H, Cheng H, et al. Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther, 2004,11(17):1312-1320.
doi: 10.1038/sj.gt.3302298
[8] 李娅莎, 刘星, 毕杨, 等. BMP9诱导人脐带间充质干细胞体内外成骨分化的作用研究. 中国生物工程杂志, 2016,36(5):20-26.
doi: 10.13523/j.cb.20160503
Li Y S, Liu X, Bi Y, et al. Effect of BMP9 on osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro and in vivo. China Biotechnology, 2016,36(5):20-26.
doi: 10.13523/j.cb.20160503
[9] Abdallah B M . Marrow adipocytes inhibit the differentiation of mesenchymal stem cells into osteoblasts via suppressing BMP-signaling. J Biomed Sci, 2017,24(1):11.
doi: 10.1186/s12929-017-0321-4
[10] Robert A K, Larissa N, Jessica H, et al. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice. PLoS Genet, 2006,2(5):e81.
doi: 10.1371/journal.pgen.0020081 pmid: 1464831
[11] Taipalesnmaki H, Abdalla B M, Aidahrnash A, et al. Wnt signaling mediates the crosstalk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations. Exp Cell Res, 2011,317(6):745-756.
doi: 10.1016/j.yexcr.2010.12.015
[12] Cruciat C M, Niehrs C . Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb Perspect Biol, 2013,5(3):a015081.
[13] Miyazono K, Maeda S, Imamura T . BMP receptor signaling: transcriptional targets, regulation of signals, and signaling crosstalk. Cytokine Growth Factor Rev, 2005,16(3):251-263.
doi: 10.1016/j.cytogfr.2005.01.009
[14] Long L, Ormiston M L, Yang X , et al. Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension. Nat Med, 2015,21(7) : 777-785.
doi: 10.1038/nm.3877 pmid: 4496295
[15] Alfaro M P, Vincent A, Saraswati S , et al. SFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair. J Biol Chem, 2010,285(46):35645-35653.
doi: 10.1074/jbc.M110.135335
[1] 王宇轩,陈婷,张永亮. MiR-148生物学功能研究进展*[J]. 中国生物工程杂志, 2021, 41(7): 74-80.
[2] 李开秀,司维. 间充质干细胞来源的外泌体治疗炎症性肠病研究进展*[J]. 中国生物工程杂志, 2021, 41(7): 66-73.
[3] 赵久梅,王哲,李学英. 调控软骨形成的信号通路及相关因子在骨髓间充质干细胞骨向分化中的作用*[J]. 中国生物工程杂志, 2021, 41(10): 62-72.
[4] 苑亚坤,刘广洋,刘拥军,谢亚芳,吴昊. 间充质干细胞基础研究与临床转化的中美比较[J]. 中国生物工程杂志, 2020, 40(4): 97-107.
[5] 陈利军,屈晶晶,项春生. 间充质干细胞在2019新型冠状病毒肺炎(COVID-19)中的治疗潜能、临床研究与应用前景*[J]. 中国生物工程杂志, 2020, 40(11): 43-55.
[6] 朱永朝,陶金,任萌萌,熊燃,何亚琴,周瑜,卢震辉,杜勇,杨芝红. 自噬抑制肿瘤坏死因子α诱导人胎盘胎儿来源间充质干细胞发生凋亡 *[J]. 中国生物工程杂志, 2019, 39(9): 62-67.
[7] 施文雯,张蕾. 力学微环境影响间充质干细胞分化的研究现状 *[J]. 中国生物工程杂志, 2018, 38(8): 76-83.
[8] 袁雅红, 赵珊珊, 王小莉, 腾智平, 李东升, 曾毅. HIV-1 Tat蛋白抑制骨髓间充质干细胞的造血支持功能[J]. 中国生物工程杂志, 2017, 37(6): 1-8.
[9] 刘红霞, 施琼, 周一青, 安利钦, 严树涓, 张汝益, 翁亚光. 过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化[J]. 中国生物工程杂志, 2017, 37(5): 9-18.
[10] 曹俊杰, 李爱芳, 卫亚琳, 廉静, 唐敏. Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨[J]. 中国生物工程杂志, 2017, 37(4): 48-55.
[11] 吉彩霞, 刘晓骅, 徐丽, 董超群, 罗进勇. Runx1促进BMP9诱导的间充质干细胞MEFs的成骨分化[J]. 中国生物工程杂志, 2017, 37(3): 10-17.
[12] 陈文杰, 汪建样, 殷明, 殷嫦嫦. 人脐带间充质干细胞抗肿瘤机制的研究进展[J]. 中国生物工程杂志, 2017, 37(3): 78-82.
[13] 赵正德, 陈振银, 张慧楠, 龚剑萍, 许少丹, 罗忠礼. 自组装短肽水凝胶支架三维培养环境对骨髓间充质干细胞生物学特性及心肌方向分化的影响[J]. 中国生物工程杂志, 2017, 37(11): 45-51.
[14] 徐丽, 吉彩霞, 刘晓骅, 喻婷婷, 罗进勇. DLX1对BMP9诱导的间充质干细胞C3H10T1/2成骨分化的影响[J]. 中国生物工程杂志, 2017, 37(10): 8-15.
[15] 毛开云, 范月蕾, 王跃, 陆娇, 陈大明. 间充质干细胞治疗产品开发现状与趋势[J]. 中国生物工程杂志, 2017, 37(10): 126-135.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会