过表达SHOX2对BMP9诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响

基础医学与临床 ›› 2021, Vol. 41 ›› Issue (1): 1-7.

• 研究论文 • 下一篇

过表达SHOX2对BMP9诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响

张平¹, 袁晓慧¹, 黄华坤¹, 杨春梅¹, 张露露¹, 罗小辑², 罗进勇^1*

1.重庆医科大学检验医学院临床诊断教育部重点实验室,重庆 400016;
2.重庆医科大学附属第一医院骨科, 重庆 400016

收稿日期:2019-04-17 修回日期:2020-03-28 出版日期:2021-01-05 发布日期:2020-12-30
通讯作者: ^*luojingyong@sina.com
基金资助:
国家自然科学基金(81874001);重庆市基础科学与前沿技术研究项目 (cstc2017jcyjAX0196)

Effects of SHOX2 over-expression on BMP9-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2

ZHANG Ping¹, YUAN Xiao-hui¹, HUANG Hua-kun¹, YANG Chun-mei¹, ZHANG Lu-lu¹, LUO Xiao-ji², LUO Jin-yong^1*

1. Key Laboratory of Clinical Laboratory Medical Diagnostics, Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016;
2. Department of Orthopedics, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

Received:2019-04-17 Revised:2020-03-28 Online:2021-01-05 Published:2020-12-30
Contact: ^*luojingyong@sina.com

摘要/Abstract

摘要： 目的研究矮小身材同源框2(SHOX2)对骨形态发生蛋白9(BMP9)诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响。方法构建SHOX2重组腺病毒(Ad-SHOX2),实验分为对照(NC)组、Ad-SHOX2组、BMP9组和BMP9+Ad-SHOX2组。碱性磷酸酶(ALP)评估早期成骨;钙盐沉积检测晚期成骨;显微镜观察细胞增殖;Western blot检测成骨相关转录因子(RUNX2)、增殖细胞核抗原(PCNA)、ERK1/2磷酸化及总蛋白表达。结果成功构建Ad-SHOX2。Ad-SHOX2组与BMP9组较NC组ALP活性、钙盐结节及RUNX2蛋白表达明显上升(P＜0.05)。相较于以上两组,BMP9+Ad-SHOX2组ALP活性增高(P＜0.05),低感染率BMP9+Ad-SHOX2组钙盐结节减少。高感染率组则较BMP9组钙盐沉积增多,与Ad-SHOX2组无明显差异。同时,BMP9+Ad-SHOX2组RUNX2蛋白表达高于BMP9组,低于Ad-SHOX2组(P＜0.05)。显微镜观察Ad-SHOX2组与BMP9组细胞密度均高于NC组,PCNA蛋白表达上调(P＜0.05)。且BMP9+Ad-SHOX2组PCNA蛋白表达高于BMP9组(P＜0.05)。此外,Ad-SHOX2组及BMP9+Ad-SHOX2组较NC组T-ERK1/2和p-ERK1/2蛋白表达增高(P＜0.05)。结论无外源性成骨刺激因子诱导条件下,SHOX2可能通过调控MAPK/ERK信号通路促进C3H10T1/2细胞增殖及成骨分化,但其与BMP9对成骨分化的调控作用是否可能互为拮抗,有待进一步研究。

关键词: 矮小身材同源框2(SHOX2), 骨形态发生蛋白9(BMP9), 成骨分化, C3H10T1/2细胞

Abstract: Objective To investigate the effects of short stature homeobox 2(SHOX2) on bone morphogenetic protein 9(BMP9)-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2. Methods After construction of SHOX2 recombinant adenoviruse (Ad-SHOX2), normal control(NC) group, Ad-SHOX2 group, BMP9 group, as well as BMP9+Ad-SHOX2 group were set up. Early osteogenic ability was evaluated with alkaline phosphatase(ALP); Late osteogenic ability was detected by Ca²⁺ deposition; Cell proliferation was observed using microscope; Meanwhile, Western blot was employed to confirm protein expression of proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2(RUNX2), T-ERK1/2 and p-ERK1/2. Results Ad-SHOX2 was constructed successfully. Ad-SHOX2 and BMP9 groups produced higher ALP activity, more Ca²⁺ accumula-tion, and higher RUNX2 expression in comparison with NC group (P＜0.05). Compared with Ad-SHOX2 and BMP9 groups, ALP activity in BMP9+Ad-SHOX2 group was enhanced (P＜0.05). Ad-SHOX2(low infection)+BMP9 group dampened Ca²⁺ accumulation remarkably. And that with high infection stimulated Ca²⁺ accumulation compared with BMP9 group, but changeless in contrast with Ad-SHOX2 group. Meanwhile, RUNX2 expression in BMP9+Ad-SHOX2 group was higher compared with BMP9 group's, but no change was found in Ad-SHOX2 group (P＜0.05). Cell growth density in Ad-SHOX2 and BMP9 groups was greater than NC group's, consistent with protein level of PCNA(P＜0.05). And protein expression of PCNA in BMP9+Ad-SHOX2 group was increased compared with BMP9 group's(P＜0.05). Additionally, protein expressions of T-ERK1/2 and p-ERK1/2 in both Ad-SHOX2 group and BMP9+Ad-SHOX2 group were up-regulated compared with NC group (P＜0.05). Conclusions SHOX2 may promote proliferation and osteogenic differentiation of C3H10T1/2 cells via regulating MAPK/ERK signaling pathway in the absence of exogenous osteoinductive factors. However, its effects on osteogenic differentiation with BMP9 potentially function with antagonistic effect and this is target for further research.

Key words: short stature homeobox2(SHOX2), bone morphogenetic protein 9(BMP9), osteogenic differentiation, C3H10T1/2 cell

中图分类号:

Q254

张平, 袁晓慧, 黄华坤, 杨春梅, 张露露, 罗小辑, 罗进勇. 过表达SHOX2对BMP9诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响[J]. 基础医学与临床, 2021, 41(1): 1-7.

ZHANG Ping, YUAN Xiao-hui, HUANG Hua-kun, YANG Chun-mei, ZHANG Lu-lu, LUO Xiao-ji, LUO Jin-yong. Effects of SHOX2 over-expression on BMP9-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2[J]. Basic & Clinical Medicine, 2021, 41(1): 1-7.

参考文献

[1] Dimitriou R, Jones E, McGonagle D,et al. Bone regeneration: current concepts and future directions[J]. BMC Med, 2011,9:66-66.
[2] Wang X, Wang Y, Gou W, et al. Role of mesenchymal stem cells in bone regeneration and fracture repair: a review[J]. Int Orthop, 2013,37:2491-2498.
[3] Salazar VS, Gamer LW, Rosen V. BMP signalling in skeletal development, disease and repair[J]. Nat Rev Endocrinol, 2016,12:203-221.
[4] Luu HH, Song WX, Luo X, et al. Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells[J]. J Orthop Res, 2007,25:665-677.
[5] Bobick BE, Cobb J. Shox2 regulates progression through chondrogenesis in the mouse proximal limb[J]. Cell Sci, 2012,125:6071-6083.
[6] Clement-Jones M, Schiller S, Rao E, et al. The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome[J]. Hum Mol Genet, 2000,9:695-702.
[7] Luo J, Deng ZL, Luo X, et al. A protocol for rapid generation of recombinant adenoviruses using the AdEasy system[J]. Nat Protoc, 2007,2:1236-1247.
[8] Cobb J, Dierich A, Huss-Garcia Y, et al. A mouse model for human short-stature syndromes identifies Shox2 as an upstream regulator of Runx2 during long-bone developmen[J]. Proc Natl Acad Sci U S A, 2006,103:4511-4515.
[9] Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation[J]. Cell, 1997,89:747-754.
[10] Matsushita T, Chan YY, Kawanami A, et al. Extra-cellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in osteoblast differentiation and in support-ing osteoclastogenesis[J]. Mol Cell Biol, 2009,29:5843-5857.
[11] Xiao G, Jiang D, Thomas P, et al.MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1[J]. J Biol Chem, 2000,275:4453-4459.
[12] Ge C, Xiao G, Jiang D, et al. Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor[J]. J Biol Chem, 2009, 284:32533-32543.
[13] Elefteriou F, Benson MD, Sowa H, et al.ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae[J]. Cell Metab, 2006,4:441-451.

过表达SHOX2对BMP9诱导的小鼠间充质干细胞系C3H10T1/2成骨分化的影响

Effects of SHOX2 over-expression on BMP9-induced osteogenic differentiation of mouse mesenchymal stem cell line C3H10T1/2

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