中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (1): 7-13.doi: 10.3969/j.issn.2095-4344.1874

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

脂肪间充质干细胞成骨分化条件培养基联合骨形态发生蛋白2治疗去卵巢大鼠骨质疏松症

杨九杰1,赵  伟2,王  楠2,陈晓春2,李  治2,马  骥2,申德伟2,王  勇2,牛庆飞2,王  涛2,周禹伯2,张  洋2   

  1. 1沈阳医学院,辽宁省沈阳市  110024;2沈阳医学院附属中心医院,辽宁省沈阳市  110000
  • 收稿日期:2019-04-29 修回日期:2019-05-06 接受日期:2019-06-22 出版日期:2020-01-08 发布日期:2019-12-11
  • 通讯作者: 张洋,博士,副主任医师,沈阳医学院附属中心医院,辽宁省沈阳市 110000
  • 作者简介:杨九杰,女,1992年生,辽宁省沈阳市人,汉族,沈阳医学院在读硕士,医师,主要从事骨科疾病诊断与治疗研究。
  • 基金资助:
    沈阳医学院青年基金(20152033)

Conditioned medium of adipose-derived mesenchymal stem cells combined with bone morphogenetic protein 2 effectively mitigates ovariectomy induced osteoporosis in rats

Yang Jiujie1, Zhao Wei2, Wang Nan2, Chen Xiaochun2, Li Zhi2, Ma Ji2, Shen Dewei2, Wang Yong2, Niu Qingfei2, Wang Tao2, Zhou Yubo2,  Zhang Yang2   

  1. 1Shenyang Medical College, Shenyang 1100247, Liaoning Province, China; 2Central Hospital Affiliated to Shenyang Medical College, Shenyang 110000, Liaoning Province, China
  • Received:2019-04-29 Revised:2019-05-06 Accepted:2019-06-22 Online:2020-01-08 Published:2019-12-11
  • Contact: Zhang Yang, MD, Associate chief physician, Central Hospital Affiliated to Shenyang Medical College, Shenyang 110000, Liaoning Province, China
  • About author:Yang Jiujie, Master candidate, Physician, Shenyang Medical College, Shenyang 1100247, Liaoning Province, China
  • Supported by:
    the Youth Foundation of Shenyang Medical College, No. 20152033

摘要:


文题释义:

骨质疏松症:是由多种原因引起的老年性骨病。通常由于骨量下降、骨的细微结构发生改变,导致骨脆性增加,易发生骨折,好发于胸、腰椎椎体、桡骨远端及股骨上端等。骨质疏松可发生在任何年龄和任何性别,但通常多发生于绝经后妇女和老年男性。

RANKL/OPG:骨重建需要骨形成和骨吸收之间的精确平衡,RANKL/OPG在骨重建中起着至关重要的作用。RANKL是一种细胞因子,可诱导祖细胞分化为成熟的破骨细胞。OPG通过与RANKL结合而成为诱骗受体。因此,RANKL/OPG的比例是维持骨形成和骨吸收之间平衡的关键。



背景:脂肪间充质干细胞分泌各种骨重塑需要的细胞因子和生长因子,被认为是骨再生的优良候选细胞。骨形态发生蛋白2与脂肪间充质干细胞对骨再生有协同作用,并能显著增强脂肪间充质干细胞的成骨分化作用。

目的:探讨脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用对大鼠绝经后骨质疏松症的影响。

方法:8-10月龄雌性SD大鼠75只,随机取60只采用卵巢切除方法建立绝经后骨质疏松症大鼠模型,余15只进行假手术,未切除卵巢。将60只造模成功大鼠随机分为4组:骨质疏松症组、条件培养基组、骨形态发生蛋白2组、联合治疗组,通过尾静脉分别注射DMEM培养基、脂肪间充质干细胞成骨分化条件培养基、骨形态发生蛋白2、脂肪间充质干细胞成骨分化条件培养基联合骨形态发生蛋白2。治疗12周,取各组大鼠股骨和血清,组织学观察骨小梁数量和结构以及骨小梁间距,ELISA检测血清P1NP、ALP、TRAP、OPG、RANKL水平,Western blot、Realtime PCR检测RANKL、OPG蛋白和mRNA水平,细胞因子芯片分析脂肪间充质干细胞成骨分化条件培养基中细胞因子水平。

结果与结论:①与假手术组相比,骨质疏松症组大鼠的骨小梁间距扩大,骨小梁数量明显减少,骨小梁失去正常结构并且不连续。与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠骨小梁间距扩大较少,骨小梁结构更完整、更连续;②与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组大鼠血清P1NP和ALP水平显著升高(P < 0.05),血清TRAP水平显著降低(P < 0.05);③与骨质疏松症组、条件培养基组、骨形态发生蛋白2组比较,联合治疗组RANKL/OPG比值显著降低(P < 0.01),从而促进更多的骨形成;④脂肪间充质干细胞成骨分化条件培养基含有多种与骨形成密不可分的细胞因子,包括骨形态发生蛋白4和7、白血病抑制因子、脑源性神经营养因子、骨保护素、胰岛素样生长因子1等;⑤结果表明,脂肪间充质干细胞成骨分化条件培养基与骨形态发生蛋白2联合应用可减轻卵巢切除大鼠骨质疏松,可能成为治疗绝经后骨质疏松症的新方案。

ORCID: 0000-0003-0552-4818(杨九杰)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程


关键词: 绝经后骨质疏松症, 脂肪间充质干细胞, 条件培养基, 骨形态发生蛋白2, 成骨分化, 骨再生

Abstract:

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) secrete various cytokines and growth factors required for bone remodeling, which are considered to be excellent candidate cells for bone regeneration. Bone morphogenetic protein 2 (BMP2) and ADSCs have a synergistic effect on bone regeneration and can significantly enhance the osteogenic differentiation of ADSCs.

OBJECTIVE: To explore the efficacy of conditioned medium of ADSCs combined with BMP2 on postmenopausal osteoporosis in a rat model.

METHODS: Healthy and female Sprague-Dawley rats aged 8-10 months (n=75) were obtained from the Animal Center of Shenyang Medical College. Ovariectomy was performed to induce postmenopausal osteoporosis in 60 rats, and the remaining 15 rats underwent surgeries without removal of the ovaries (sham group). Ovariectomized rats were randomized into four groups: osteoporosis group, conditioned medium group, BMP2 group, and conditioned medium+BMP2 group, followed by injection of DMEM medium, ADSCs conditioned medium, BMP2, and ADSCs conditioned medium+BMP2 via the tail vein, respectively. After 12 weeks of treatment, femurs and serum samples of each group were taken. The number and structure of trabecular bone and trabecular spacing were detected histologically. Serum P1NP, ALP, TRAP, OPG, RANKL levels were detected by ELISA. Western blot and real-time PCR were used to detect RANKL and OPG at protein and mRNA levels. Cytokine chip analysis of cytokines in ADSCs conditioned medium was performed.

RESULTS AND CONCLUSION: Compared with the sham group, increased trabecular spacing, fewer trabeculae and marked trabecular disconnection were observed in osteoporosis rats. The trabeculae in conditioned medium+BMP2 group appeared to be more complete and continuous with less widened spacing compared with the osteoporosis, conditioned medium and BMP2 groups. Serum levels of P1NP and ALP were dramatically higher, while TRAP level group was decreased significantly in the conditioned medium+BMP2 group compared with the other groups (both P < 0.05). In the conditioned medium+BMP2 group, RANKL/OPG ratio was reduced significantly compared with the other groups (P < 0.01), further promoting bone formation. ADSCs conditioned medium contained a variety of cytokines that were essential for bone formation and remodeling, including bone morphogenetic proteins 4 and 7, leukemia inhibitory factor, brain-derived neurotrophic factor, osteoprotegerin, and insulin-like growth factor 1. These results demonstrate that ADSCs conditioned medium combined with BMP2 can mitigate osteoporosis induced by ovariectomy and it may be an attractive strategy to treat postmenopausal osteoporosis.

Key words: postmenopausal osteoporosis, adipose-derived mesenchymal stem cells, conditioned medium, bone morphogenetic protein 2, osteogenic differentiation, bone regeneration

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