微丝解聚在低强度脉冲超声促进骨髓间充质干细胞成骨分化中的作用

杨丽; 姚欢; 朱慧; 何依蔓; 杨珂; 王冬

doi:10.16016/j.2097-0927.202110052

您当前的位置：

首页 >

文章列表页 >

微丝解聚在低强度脉冲超声促进骨髓间充质干细胞成骨分化中的作用

生物医学工程 | 更新时间：2026-01-09

- 微丝解聚在低强度脉冲超声促进骨髓间充质干细胞成骨分化中的作用
- Role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells promoted by low intensity pulsed ultrasound
- 陆军军医大学学报 2022年44卷第9期页码：943-949
- 作者机构：
  
  1.400016 重庆，重庆医科大学附属第一医院超声科
  2.400014 重庆，重庆医科大学附属儿童医院儿童发育疾病教育部重点实验室
- 作者简介：
  
  [王冬，E-mail：wang57554@163.comWANG Dong, E-mail: wang57554@163.com
- 基金信息：
  
  国家自然科学基金面上项目(81771845)
- DOI：10.16016/j.2097-0927.202110052
  中图分类号： R322.71;R329.21;R363.274
- 收稿：2021-10-12，
  
  修回：2021-11-28，
  
  纸质出版：2022-05-15
- 稿件说明：
移动端阅览
杨丽, 姚欢, 朱慧, 等. 微丝解聚在低强度脉冲超声促进骨髓间充质干细胞成骨分化中的作用[J]. 陆军军医大学学报, 2022,44(9):943-949.

Li YANG, Huan YAO, Hui ZHU, et al. Role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells promoted by low intensity pulsed ultrasound[J]. Journal of Army Medical University, 2022, 44(9): 943-949.
杨丽, 姚欢, 朱慧, 等. 微丝解聚在低强度脉冲超声促进骨髓间充质干细胞成骨分化中的作用[J]. 陆军军医大学学报, 2022,44(9):943-949. DOI： 10.16016/j.2097-0927.202110052.

Li YANG, Huan YAO, Hui ZHU, et al. Role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells promoted by low intensity pulsed ultrasound[J]. Journal of Army Medical University, 2022, 44(9): 943-949. DOI： 10.16016/j.2097-0927.202110052.

摘要

目的

研究微丝解聚在低强度脉冲超声(low intensity pulsed ultrasound，LIPUS)促进骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)成骨分化时的作用。

方法

培养小鼠BMSCs，随机分为4组：空白对照组；LIPUS组；LIPUS+DMSO组；LIPUS+CytoD(细胞松弛素D，Cytochalasin D)组。碱性磷酸酶(alkaline phosphatase，ALP)染色检测ALP活性。免疫荧光染色观察RUNX2的表达和细胞定位。Western blot检测成骨分化相关基因Ⅰ型胶原(Collagen1)、骨桥蛋白(osteopontin，OPN)、Runt相关转录因子2(runt-related transcription factor 2，RUNX2)的蛋白表达变化以及细胞中丝状肌动蛋白(filamentous actin，F-actin)与球状/单体肌动蛋白(globular/monomeric actin，G-actin)的含量。通过激光共聚焦显微镜观察细胞形态变化及微丝骨架结构变化。

结果

与空白对照组相比，LIPUS辐照能显著增加ALP活性，促成骨相关基因Collagen1 (

0.01)、

OPN

(

0.05)、RUNX2 (

0.01)的蛋白表达量升高，其中RUNX2表达量升高约1.5倍(

0.01)。荧光染色显示LIPUS处理后RUNX2在细胞核内表达明显增加，细胞略缩小，同时可见细胞质内点状G-actin分布，Western blot实验分析F-actin/G-actin的比值发现LIPUS组明显小于对照组(

0.05)。此外，与LIPUS+DMSO组相比，LIPUS+CytoD组能促进胞内微丝的解聚，F-actin/G-actin的比值明显下降(

0.05)，ALP活性明显增加(

0.05)，RUNX2的细胞核内表达明显增加(

0.05)，成骨相关基因的蛋白表达水平明显增高(

0.05)。

结论

LIPUS可以促进BMSCs成骨分化，其机制可能与LIPUS促进微丝的解聚有关。

Abstract

Objective

To investigate the role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) promoted by low intensity pulsed ultrasound (LIPUS).

Methods

Mouse BMSCs were randomly divided into 4 groups: blank control group

LIPUS group

LIPUS+DMSO group and LIPUS+cytochalasin D (CytoD) group. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining. The expression and cellular localization of runt-related transcription factor 2 (RUNX2) were observed by immunofluorescence staining. Western blotting was performed to determine the protein changes of osteogenic differentiation-related genes

type I collagen (collagen 1)

osteopontin (OPN) and runt-related transcription factor 2 (RUNX2)

as well as the contents of filamentous actin (F-actin) and globular actin (G-actin) in the cells. The changes of cell morphology and microfilament cytoskeleton structure were observed with confocal laser scanning microscopy.

Results

As compared with the blank control group

LIPUS irradiation sig

nificantly enhanced the activity of ALP and upregulated the protein expression of osteogenic related genes Collagen1 (

0.01)

OPN (

0.05) and RUNX2 (

0.01)

with about a 1.5-fold increase in RUNX2 level (

0.01). Immunofluorescence staining showed that RUNX2 expression was obviously increased in the nucleus after LIPUS irradiation

and the cells shrank slightly with punctate distribution of G-actin in the cytoplasm. Western blotting revealed that the ratio of F-actin/G-actin was remarkably lower in the LIPUS group than the control group (

0.05). In addition

in comparison with the LIPUS+DMSO group

the LIPUS+CytoD group had greatly promoted the depolymerization of intracellular microfilaments

reduced ratio of F-actin/G-actin (

0.05)

enhanced activity of ALP (

0.05)

upregulated expression of RUNX2 in the nucleus (

0.05)

and improved expression levels of the osteogenic related genes (

0.05).

Conclusion

LIPUS promotes the osteogenic differentiation of BMSCs

which may be related to LIPUS facilitating the depolymerization of microfilaments.

关键词

Keywords

references

ANGLE S R, SENA K, SUMNER D R, et al . Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound[J ] . Ultrasonics, 2011, 51(3): 281-288. DOI: 10.1016/j.ultras.2010.09.004.

LI X, WANG M, JING X, et al . Bone marrow-and adipose tissue-derived mesenchymal stem cells: characterization, differentiation, and applications in cartilage tissue engineering[J ] . Crit Rev Eukaryot Gene Expr, 2018, 28(4): 285-310. DOI: 10.1615/critreveukaryotgeneexpr.2018023572.

MARSELL R, EINHORN T A. The biology of fracture healing[J]. Injury, 2011, 42(6): 551-555. DOI: 10.1016/j.injury.2011.03.031.

XIAO W, XU Q, ZHU Z, et al . Different performances of CXCR4, integrin-1β and CCR-2 in bone marrow stromal cells (BMSCs) migration by low-intensity pulsed ultrasound stimulation[J ] . Biomed Tech (Berl), 2017, 62(1): 89-95. DOI: 10.1515/bmt-2015-0166.

LIU X, HOU W Q, HE L, et al . AMOT130/YAP pathway in topography-induced BMSC osteoblastic differentiation[J ] . Colloids Surf B Biointerfaces, 2019, 182: 110332. DOI: 10.1016/j .colsurfb.2019.06.061.

HE Y B, LIU S Y, DENG S Y, et al . Mechanical stretch promotes the osteogenic differentiation of bone mesenchymal stem cells induced by erythropoietin[J ] . Stem Cells Int, 2019, 2019: 1839627. DOI: 10.1155/2019/1839627.

JIANG X X, SAVCHENKO O, LI Y F, et al . A review of low-intensity pulsed ultrasound for therapeutic applications[J ] . IEEE Trans Biomed Eng, 2019, 66(10): 2704-2718. DOI: 10.1109/TBME.2018.2889669.

TABUCHI Y, HASEGAWA H, SUZUKI N, et al . Low-intensity pulsed ultrasound promotes the expression of immediate-early genes in mouse ST2 bone marrow stromal cells[J ] . J Med Ultrason (2001), 2020, 47(2): 193-201. DOI: 10.1007/s10396-020-01007-9.

SENA K, ANGLE S R, KANAJI A, et al . Low-intensity pulsed ultrasound (LIPUS) and cell-to-cell communication in bone marrow stromal cells[J ] . Ultrasonics, 2011, 51(5): 639-644. DOI: 10.1016/j.ultras.2011.01.007.

KABSCH W, MANNHERZ H G, SUCK D, et al . Atomic structure of the actin: DNase I complex[J ] . Nature, 1990, 347(6288): 37-44. DOI: 10.1038/347037a0.

SINGHVI R, KUMAR A, LOPEZ G P, et al . Engineering cell shape and function[J ] . Science, 1994, 264(5159): 696-698. DOI: 10.1126/science.8171320.

PRASAD A, ALIZADEH E. Cell form and function: interpreting and controlling the shape of adherent cells[J]. Trends Biotechnol, 2019, 37(4): 347-357. DOI: 10.1016/j.tibtech.2018.09.007.

VERSAEVEL M, GREVESSE T, GABRIELE S. Spatial coordination between cell and nuclear shape within micropatterned endothelial cells[J]. Nat Commun, 2012, 3: 671. DOI: 10.1038/ncomms1668.

UHLER C, SHIVASHANKAR G V. Geometric control and modeling of genome reprogramming[J]. Bioarchitecture, 2016, 6(4): 76-84. DOI: 10.1080/19490992.2016.1201620.

PEGORARO A F, JANMEY P, WEITZ D A. Mechanical properties of the cytoskeleton and cells[J]. Cold Spring Harb Perspect Biol, 2017, 9(11): a022038. DOI: 10.1101/cshperspect.a022038.

LI L, YANG Z, ZHANG H, et al . Low-intensity pulsed ultrasound regulates proliferat ion and differentiation of osteoblasts through osteocytes[J ] . Biochem Biophys Res Commun, 2012, 418(2): 296-300. DOI: 10.1016/j.bbrc.2012.01.014.

RUTTEN S, NOLTE P A, KORSTJENS C M, et al . Low-intensity pulsed ultrasound affects RUNX2 immunopositive osteogenic cells in delayed clinical fracture healing[J ] . Bone, 2009, 45(5): 862-869. DOI: 10.1016/j.bone.2009.07.012.

MAEDA S, TSUDA E, MIZUKAMI H, et al . Histological evaluation of low-intensity pulsed ultrasound on osteochondritis dissecans of the humeral Capitellum[J ] . Asia Pac J Sports Med Arthrosc Rehabilit Technol, 2015, 2(2): 56-62. DOI: 10.1016/j.asmart.2014.12.004.

LESSEY E C, GUILLUY C, BURRIDGE K. From mechanical force to RhoA activation[J]. Biochemistry, 2012, 51(38): 7420-7432. DOI: 10.1021/bi300758e.

HEIDEMANN S R, KAECH S, BUXBAUM R E, et al . Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts[J ] . J Cell Biol, 1999, 145(1): 109-122. DOI: 10.1083/jcb.145.1.109.

LECUIT T, LENNE P F. Cell surface mechanics and the control of cell shape, tissue patterns and morphogenesis[J]. Nat Rev Mol Cell Biol, 2007, 8(8): 633-644. DOI: 10.1038/nrm2222.

WICKSTEAD B, GULL K. The evolution of the cytoskeleton[J]. J Cell Biol, 2011, 194(4): 513-525. DOI: 10.1083/jcb.201102065.

YU H Y, TAY C Y, LEONG W S, et al . Mechanical behavior of human mesenchymal stem cells during adipogenic and osteogenic differentiation[J ] . Biochem Biophys Res Commun, 2010, 393(1): 150-155. DOI: 10.1016/j.bbrc.2010.01.107.

GONZÁLEZ-CRUZ R D, FONSECA V C, DARLING E M. Cellular mechanical properties reflect the differentiation potential of adipose-derived mesenchymal stem cells[J]. Proc Natl Acad Sci USA, 2012, 109(24): E1523-E1529. DOI: 10.1073/pnas.1120349109.

CHEN L, SHI K K, FRARY C E, et al . Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells[J ] . Stem Cell Res, 2015, 15(2): 281-289. DOI: 10.1016/j.scr.2015.06.009.

MISHRA P, MARTIN D C, ANDROULAKIS I P, et al . Fluorescence imaging of actin turnover parses early stem cell lineage divergence and senescence[J ] . Sci Rep, 2019, 9: 10377. DOI: 10.1038/s41598-019 -46682-y.

MATSUOKA F, TAKEUCHI I, AGATA H, et al . Morphology-based prediction of osteogenic differentiation potential of human mesenchymal stem cells[J ] . PLoS One, 2013, 8(2): e55082. DOI: 10.1371/journal.pone.0055082.

MARKLEIN R A, LO SURDO J L, BELLAYR I H, et al . High content imaging of early morphological signatures predicts long term mineralization capacity of human mesenchymal stem cells upon osteogenic induction[J ] . Stem Cells, 2016, 34(4): 935-947. DOI: 10.1002/stem.2322.

SAKAI D, KⅡ I, NAKAGAWA K, et al . Remodeling of actin cytoskeleton in mouse periosteal cells under mechanical loading induces periosteal cell proliferation during bone formation[J ] . PLoS One, 2011, 6(9): e24847. DOI: 10.1371/journal.pone.0024847.

SEN B E, XIE Z H, UZER G, et al . Intranuclear actin regulates osteogenesis[J ] . Stem Cells, 2015, 33(10): 3065-3076. DOI:10.1002/stem.2090.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

新型二甲双胍碳点促进炎症微环境下人牙周膜干细胞成骨分化

LPS诱导主动脉瓣膜间质细胞成骨分化的作用及机制研究

体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响

过表达Notch配体Delta-like基因的MLO-Y4骨细胞株的建立及其对骨髓基质细胞成骨分化的作用

TGF-βⅡ型受体小分子抑制剂ITD-1对骨髓基质细胞成骨分化和血管形成的作用