上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 7: 849 - 857

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.07.002

抗肿瘤药物依托泊苷促进间充质干细胞成骨分化的研究

  • 陆艳青 ,
  • 周兴 ,
  • 李姣 ,
  • 彭建平 ,
  • 王传东 ,
  • 张晓玲
展开
  • 1.广西医科大学，广西生物医药协同创新中心，广西再生医学重点实验室，南宁 530021
    2.上海交通大学医学院附属新华医院骨科，上海 200092
陆艳青（1992—），女，硕士生；电子信箱：835373439@qq.com。

网络出版日期: 2021-08-03

基金资助

国家自然科学基金(81772347);上海市教育委员会高峰高原学科建设计划(20161314);上海交通大学“转化医学交叉研究基金”(YG2019QNB30)

收起

Promotive effect of antitumor drug etoposide on osteogenic differentiation of mesenchymal stem cells

  • Yan-qing LU ,
  • Xing ZHOU ,
  • Jiao LI ,
  • Jian-ping PENG ,
  • Chuan-dong WANG ,
  • Xiao-ling ZHANG
Expand
  • 1.Guangxi Collaborative Innovation Center for Biomedicine, Guangxi Key Laboratory of Regenerative Medicine, Guangxi Medical University, Nanning 530021, China
    2.Department of Orthopedic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Online published: 2021-08-03

Fold

摘要

目的·研究抗肿瘤药物依托泊苷对间充质干细胞（mesenchymal stem cell，MSC）成骨分化的促进作用及可能的机制。方法·体外分离C57BL/6J小鼠骨髓和人骨髓来源MSC。给予小鼠MSC不同浓度（0~12.50 μmol/L）的依托泊苷12、24、36、48和60 h后，采用CCK-8法检测MSC的增殖活性。采用凋亡检测试剂盒检测依托泊苷（0、0.001、0.01、0.1、1 μmol/L）对大鼠成骨性骨肉瘤细胞系UMR-106凋亡的影响。分别以依托泊苷（0、0.001、0.01、0.1、1 μmol/L）处理小鼠和人MSC（同时诱导成骨分化）、UMR-106细胞，通过碱性磷酸酶（alkaline phosphatase，ALP）染色、茜素红染色分析细胞成骨分化情况。通过实时荧光定量PCR技术检测依托泊苷对小鼠MSC成骨分化标志物Alp、骨钙素、Ⅰ型胶原α1链mRNA水平的调控。将0.001 μmol/L依托泊苷与UMR-106细胞共孵育后，通过Illumina Xten高通量转录组测序和生物信息学分析，寻找依托泊苷上调的成骨分化相关靶基因，并通过PCR验证。Western blotting检测依托泊苷（0、0.001、0.01、0.1、1 μmol/L）处理的小鼠MSC中成骨特异性转录因子（osterix，OSX）、矮小相关转录因子2（runt-related transcription factor 2，RUNX2）及DNA结合抑制因子1（inhibitor of DNA binding 1，ID1）的表达变化。结果·依托泊苷对小鼠MSC增殖的影响呈现浓度依赖性；当浓度≥0.20 μmol/L时，依托泊苷开始抑制MSC的增殖，48 h的半数抑制浓度（half-maximal inhibitory concentration，IC50）为2.192 μmol/L，60 h的IC50为1.399 μmol/L。凋亡检测结果显示依托泊苷浓度为0.001~1 μmol/L时UMR-106细胞的凋亡率为16.137%~28.300%。ALP染色、茜素红染色及荧光定量PCR结果显示0.001~0.1 μmol/L依托泊苷对小鼠和人MSC以及UMR-106细胞具有促成骨分化的作用。RNA测序分析表明，依托泊苷可上调UMR-106细胞结缔组织生长因子基因表达。Western blotting分析结果显示，0.001、0.01 μmol/L依托泊苷显著增加小鼠MSC OSX、RUNX2及ID1的表达。结论·抗肿瘤药物依托泊苷可促进MSC成骨分化，该作用可能与其上调ID1、RUNX2、OSX表达有关。

关键词： 依托泊苷; 间充质干细胞; 成骨分化; 骨肿瘤; 骨重建

本文引用格式

陆艳青 , 周兴 , 李姣 , 彭建平 , 王传东 , 张晓玲 . 抗肿瘤药物依托泊苷促进间充质干细胞成骨分化的研究[J]. 上海交通大学学报（医学版）, 2021 , 41(7) : 849 -857 . DOI: 10.3969/j.issn.1674-8115.2021.07.002

Abstract

Objective

·To study the effect and possible mechanism of the anti-tumor drug etoposide in promoting osteogenic differentiation of mesenchymal stem cells (MSCs).

Methods

·Primary cultured MSCs were isolated from the bone marrow of C57BL/6J mice and humans. After the mouse MSCs were given different concentrations (0?12.50 μmol/L) of etoposide for 12, 24, 36, 48 and 60 h, the cell proliferation ability was detected by CCK-8 kit assay. The apoptosis detection kit was performed to detect apoptosis of osteoblast/osteosarcoma cell line UMR-106 treated by etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L). The mouse and human MSCs (simultaneously inducing osteogenic differentiation), and UMR-106 cells were treated with etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L), whose osteogenic differentiation was analyzed by alkaline phosphatase (ALP) staining and alizarin red staining. The mRNA levels of osteogenic differentiation markers i.e., Alp, osteocalcin, and collagen type Ⅰ α 1 chain were analyzed by real-time quantitative PCR. After the UMR-106 cells were treated with etoposide (0.001 μmol/L), Illumina Xten′s high-throughput transcriptome sequencing and bioinformatics analysis were used to find the target gene related with osteogenic differentiation, which was then verified by PCR. Western blotting was used to detect the expression of osterix (OSX), runt-related transcription factor 2 (RUNX2) and DNA binding inhibitor 1 (ID1) in the etoposide (0, 0.001, 0.01, 0.1, 1 μmol/L) treated mouse MSCs.

Results

·The effect of etoposide on the proliferation of mouse MSCs was concentration-dependent. When the concentration was ≥0.20 μmol/L, etoposide inhibited the proliferation of MSCs. The half-maximal inhibitory concentrations (IC50) for 48 h and 60 h were 2.192 μmol/L and 1.399 μmol/L, respectively. Apoptosis detection results showed that the apoptotic rates of UMR-106 cells treated with 0.001?1 μmol/L etoposide were 16.137%?28.300%. The results of ALP staining, alizarin red staining and PCR showed that etoposide at 0.001?0.1 μmol/L significantly promoted the osteogenic differentiation of mouse and human MSCs and UMR-106 cells. RNA sequencing analysis showed that etoposide up-regulated connective tissue growth factor expression in the UMR-106 cells. Western blotting results showed that 0.001 and 0.01 μmol/L etoposide significantly increased the protein expression of OSX, RUNX2 and ID1 in the mouse MSCs.

Conclusion

·The anti-tumor drug etoposide can promote the osteogenic differentiation of MSCs, which may be related to the up-regulation of ID1, RUNX2 and OSX expressions.

Key words： etoposide (VP-16); mesenchymal stem cell (MSC); osteogenic differentiation; bone tumor; bone remodeling

参考文献

1 Gianferante DM, Mirabello L, Savage SA. Germline and somatic genetics of osteosarcoma-connecting aetiology, biology and therapy[J]. Nat Rev Endocrinol, 2017, 13(8): 480-491.
2 Reed DR, Hayashi M, Wagner L, et al. Treatment pathway of bone sarcoma in children, adolescents, and young adults[J]. Cancer, 2017, 123(12): 2206-2218.
3 Sarkar N, Bose S. Controlled delivery of curcumin and vitamin K2 from hydroxyapatite-coated titanium implant for enhanced in vitro chemoprevention, osteogenesis, and in vivo osseointegration[J]. ACS Appl Mater Interfaces, 2020, 12(12): 13644-13656.
4 Pan SS, Yin JH, Yu LD, et al. 2D MXene-integrated 3D-printing scaffolds for augmented osteosarcoma phototherapy and accelerated tissue reconstruction[J]. Adv Sci (Weinh), 2020, 7(2): 1901511.
5 Takeuchi A, Yamamoto N, Hayashi K, et al. Joint-preservation surgery for pediatric osteosarcoma of the knee joint[J]. Cancer Metastasis Rev, 2019, 38(4): 709-722.
6 Whelan JS, Davis LE. Osteosarcoma, chondrosarcoma, and chordoma[J]. J Clin Oncol, 2018, 36(2): 188-193.
7 Dang WT, Li T, Li B, et al. A bifunctional scaffold with CuFeSe2 nanocrystals for tumor therapy and bone reconstruction[J]. Biomaterials, 2018, 160: 92-106.
8 Marina NM, Smeland S, Bielack SS, et al. Comparison of MAPIE versus MAP in patients with a poor response to preoperative chemotherapy for newly diagnosed high-grade osteosarcoma (EURAMOS-1): an open-label, international, randomised controlled trial[J]. Lancet Oncol, 2016, 17(10): 1396-1408.
9 Goorin AM, Schwartzentruber DJ, Devidas M, et al. Presurgical chemotherapy compared with immediate surgery and adjuvant chemotherapy for nonmetastatic osteosarcoma: Pediatric Oncology Group Study POG-8651[J]. J Clin Oncol, 2003, 21(8): 1574-1580.
10 Gorthi A, Romero JC, Loranc E, et al. EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma[J]. Nature, 2018, 555(7696): 387-391.
11 Xu R, Yallowitz A, Qin A, et al. Targeting skeletal endothelium to ameliorate bone loss[J]. Nat Med, 2018, 24(6): 823-833.
12 Debnath S, Yallowitz AR, McCormick J, et al. Discovery of a periosteal stem cell mediating intramembranous bone formation[J]. Nature, 2018, 562(7725): 133-139.
13 Liu Q, Yu YX, Reisdorf RL, et al. Engineered tendon-fibrocartilage-bone composite and bone marrow-derived mesenchymal stem cell sheet augmentation promotes rotator cuff healing in a non-weight-bearing canine model[J]. Biomaterials, 2019, 192: 189-198.
14 Sui BD, Hu CH, Liu AQ, et al. Stem cell-based bone regeneration in diseased microenvironments: challenges and solutions[J]. Biomaterials, 2019, 196: 18-30.
15 Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics[J]. Nat Rev Genet, 2009, 10(1): 57-63.
16 Ivkovic S, Yoon BS, Popoff SN, et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development[J]. Development, 2003, 130(12): 2779-2791.
17 Lambi AG, Pankratz TL, Mundy C, et al. The skeletal site-specific role of connective tissue growth factor in prenatal osteogenesis[J]. Dev Dyn, 2012, 241(12): 1944-1959.
18 Arnott JA, Lambi AG, Mundy C, et al. The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis[J]. Crit Rev Eukaryot Gene Expr, 2011, 21(1): 43-69.
19 Liao X, Bu Y, Jiang SS, et al. CCN2-MAPK-Id-1 loop feedback amplification is involved in maintaining stemness in oxaliplatin-resistant hepatocellular carcinoma[J]. Hepatol Int, 2019, 13(4): 440-453.
20 Bhattacharya A, Baker NE. A network of broadly expressed HLH genes regulates tissue-specific cell fates[J]. Cell, 2011, 147(4): 881-892.
21 Anido J, Sáez-Borderías A, Gonzàlez-Juncà A, et al. TGF?β receptor inhibitors target the CD44high/Id1high glioma-initiating cell population in human glioblastoma[J]. Cancer Cell, 2010, 18(6): 655-668.
22 Jeong JW, Kim M, Lee J, et al. ID1-mediated BMP signaling pathway potentiates glucagon-like peptide-1 secretion in response to nutrient replenishment[J]. Int J Mol Sci, 2020, 21(11): E3824.
23 Chen WC, Chung CH, Lu YC, et al. BMP-2 induces angiogenesis by provoking integrin α6 expression in human endothelial progenitor cells[J]. Biochem Pharmacol, 2018, 150: 256-266.
24 梁承伟, 朱炯, 沈海敏, 等. 依托泊苷诱导骨肉瘤细胞凋亡的实验研究[J]. 中国新药与临床杂志, 2006, 25(9): 718-721.
25 熊齐胜, 韩迎祥, 汪学松, 等. 骨肉瘤的分化疗法与研究进展[J]. 中国现代医学杂志, 2020, 30(11): 48-51.
Options
文章导航

/

〈 〉