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北京大学学报（医学版） ›› 2021, Vol. 53 ›› Issue (6): 1171-1177. doi: 10.19723/j.issn.1671-167X.2021.06.027

• 论著 • 上一篇    下一篇

甲磺酸去铁胺促进大鼠颅骨临界骨缺损血管化骨再生的早期连续观察

杜文瑜,杨静文(),姜婷()   

  1. 北京大学口腔医学院·口腔医院,修复科 国家口腔医学中心 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室,北京 100081
  • 收稿日期:2019-12-12 出版日期:2021-12-18 发布日期:2021-12-13
  • 通讯作者: 杨静文,姜婷 E-mail:jingwen.yang@foxmail.com;jt_ketizu@163.com
  • 基金资助:
    国家自然科学基金(81771045);北京大学口腔医院青年科研基金(PKUSS20180106)

Early constant observation of the effect of deferoxamine mesylate on improvement of vascularized bone regeneration in SD rat skull critical size defect model

DU Wen-yu,YANG Jing-wen(),JIANG Ting()   

  1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing 100081, China
  • Received:2019-12-12 Online:2021-12-18 Published:2021-12-13
  • Contact: Jing-wen YANG,Ting JIANG E-mail:jingwen.yang@foxmail.com;jt_ketizu@163.com
  • Supported by:
    National Natural Science Foundation of China(81771045);Research Foundation of Peking University School and Hospital of Stomatology(PKUSS20180106)

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摘要：

目的:连续观察骨缺损愈合早期血管样组织和骨样组织的变化过程,初步了解局部施用甲磺酸去铁胺(deferoxamine mesylate,DFO)对血管化及骨再生的作用,验证DFO维持缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)活性的能力。方法:构建30只6~8周龄雄性SD大鼠颅骨缺损模型,随机分为DFO实验组和生理盐水对照组,于颅骨缺损后第4天分别局部注射200 μmol/L DFO溶液300 μL或生理盐水300 μL,并于缺损后第5、7、10、14和28天,每组每次处死3只大鼠。采用CD31免疫组织化学染色检测血管数量,采用HE染色、Masson染色观察成骨及矿化情况,以HIF-1α免疫组化染色检测HIF-1α蛋白相对表达量。结果:术后第5、7、10、14和28天,实验组血管数量(个)分别为:30.40±12.15、62.00±17.87、73.43±15.63、40.00±7.84和48.71±11.64,对照组血管数量(个)分别为:18.75±6.63、19.13±2.80、51.35±16.21、27.18±7.32和30.88±13.43,各时间点实验组血管数量均显著多于对照组(P<0.05);术后第14和28天,实验组新生骨组织较多,新生骨组织矿化百分比分别为(27.73±5.93)%和(46.53±3.66)%,对照组分别为(11.99±2.02)%和(31.98±4.22)%,这两个时间点实验组矿化百分比显著高于对照组(P<0.001);术后第5、7、10、14和28天,实验组相较对照组的HIF-1α蛋白相对表达量为2.86±0.48、1.32±0.26、1.32±0.32、1.28±0.38、1.05±0.34,仅术后第5天两组间差异有统计学意义(P<0.01)。结论:在骨缺损局部使用DFO可促进血管化及骨再生,可短暂维持HIF-1α蛋白活性。

关键词: 去铁胺, 血管化, 骨生成, 缺氧诱导因子1, α亚基

Abstract:

Objective: To investigate the effect of local administration of deferoxamine mesylate (DFO) on vascularization and osteogenesis and its ability to maintain the activity of hypoxia inducible factor-1α (HIF-1α), by constantly observing early changes of vessel-like structures and bone tissues during bone defects healing. Methods: Skull critical bone defect models were constructed on a total of thirty male SD rats (6-8 weeks old). The rats were randomly divided into experimental group (DFO group) or control group (normal saline group). 300 μL 200 μmol/L DFO solution or normal saline was locally injected on the 4th day after the defect was made. On the 5th, 7th, 10th, 14th, and 28th days after surgery, three rats in each group were sacrificed respectively. HE staining and Masson staining were performed to observe new bone formation and mineralization. HIF-1α immunohistochemistry staining was performed to examine relative expression of protein. Qualitative analysis and comparation were performed by t-tests on relative expression of HIF-1α, numbers of blood vessels and percentages of mineralization tissues of new bone areas. Results: On the 5th, 7th, 10th, 14th and 28th days after surgery, the average numbers of blood vessels were 30.40±12.15, 62.00±17.87, 73.43±15.63, 40.00±7.84, 48.71±11.64 in the DFO group, and 18.75±6.63, 19.13±2.80, 51.35±16.21, 27.18±7.32, 30.88±13.43 in the control group. The number of blood vessels in the DFO group was significantly higher than that of the control group at each time point (P<0.05). The mass of new bone in the DFO group was higher than that in the control group on the 14th and 28th days after surgery. The percentage of mineralization tissues of new bone area on the 14th and 28th days after injection were (27.73±5.93)% and (46.53±3.66)% in the DFO group, and (11.99±2.02)% and (31.98±4.22)% in the control group. The percentage of mineralization tissues in the DFO group was significantly higher than that of the control group at each time point (P<0.001). The relative expression of HIF-1α in the DFO group compared with the control group was 2.86±0.48, 1.32±0.26, 1.32±0.32, 1.28±0.38 and 1.05±0.34 on the 5th, 7th, 10th, 14th and 28th days, with significant expression difference on the 5th day (P<0.01). Conclusion: Use of DFO in bone defects promotes vascularization and osteogenesis in the defect area, and maintains the protein activity of HIF-1α temporarily.

Key words: Deferoxamine, Vascularization, Osteogenesis, Hypoxia inducible factor 1, alpha subunit

中图分类号: 

  • R318.08

图1

SD大鼠颅骨临界骨缺损模型"

图2

CD31标记的血管免疫组织化学染色图像(n=3)"

表1

实验组与对照组的血管计数"

Post-operation days/d Control group ($\overline{x}$±s) DFO group ($\overline{x}$±s) t P
5 18.75±6.63 30.40±12.15 2.439 0.027
7 19.13±2.80 62.00±17.87 6.671 <0.001
10 51.35±16.21 73.43±15.63 4.349 <0.001
14 27.18±7.32 40.00±7.84 3.660 0.002
28 30.88±13.43 48.71±11.64 2.727 0.017

图3

骨缺损区域HE染色(A)及Masson骨胶原染色(B)图像(n=3)"

表2

实验组和对照组矿化程度的测量"

Post-operation days/d Control group ($\overline{x}$±s) DFO group ($\overline{x}$±s) t P
14 (11.99±2.02)% (27.73±5.93)% 5.967 <0.001
28 (31.98±4.22)% (46.53±3.66)% 6.045 <0.001

图4

HIF-1α蛋白表达(DAB ×200,n=3)"

表3

HIF-1α蛋白的相对表达量(倍数)"

Post-operation
days/d		 Relative expression of
HIF-1α (fold change, $\overline{x}$±s)		 t P
5 2.86±0.48 7.660 0.002
7 1.32±0.26 2.131 0.083
10 1.32±0.32 2.007 0.069
14 1.28±0.38 1.454 0.121
28 1.05±0.34 0.196 0.438
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