摘要
目的探讨小分子药物嘌吗啡胺(PM)靶向激活Hedgehog(Hh)信号通路后对骨髓来源间充质干细胞(BMSCs)成骨-成脂分化平衡的影响及其机制。方法通过细胞计数试剂盒-8法评估PM对BMSCs增殖的细胞毒性,并通过检测不同浓度PM处理下BMSCs细胞内Gli1转录水平,选择合适的PM处理浓度。随后通过诱导BMSCs成骨、成脂分化,检测ALP表达(ALP染色)、钙结节(Von Kossa矿化结节染色法)评估PM对BMSCs成骨诱导的影响,采用油红O染色检测PM对BMSCs成脂诱导的影响。最后通过qRT-PCR法验证PM干预下BMSCs成骨-成脂分化过程中相关核心转录因子、特征基因的变化。结果10μmol/L及以下浓度的PM对BMSCs增殖无明显毒性反应,并且2μmol/L PM即可显著激活BMSCs细胞内Hh信号通路(P<0.01)。在2μmol/L PM干预下,BMSCs成骨分化诱导时ALP表达、钙结节形成较对照组显著增强,而成脂分化诱导时脂滴的形成受到了明显的抑制。在此基础之上,qRT-PCR结果提示2μmol/L PM可明显促进BMSCs成骨分化过程中核心转录因子Sp7以及特征性基因ALP、整合素结合唾液酸蛋白(Ibsp)的转录,并抑制成脂分化过程中核心转录因子过氧化物酶体增殖物激活受体γ(Pparg)、CCAAT增强子结合蛋白α(Cebpa)以及特征基因围脂滴蛋白1(Plin1)、脂联素(Adipoq)、脂肪酸转位酶(Cd36)的转录。结论PM可通过激活Hh信号通路,调控成骨-成脂分化相关核心转录因子,促进BMSCs成骨分化并抑制其成脂分化。
Objective To investigate the effect of purmorphamine(PM)on Hedgehog(Hh)signaling pathway and osteo-blastogenesis and adipogenesis balance of bone marrow-derived mesenchymal stem cells(BMSCs).Methods The cyto-toxicity of PM on BMSCs proliferation was evaluated by CCK-8,and the appropriate concentration of PM was selected by detecting Gli1 transcription levels in BMSCs cells under different concentrations of PM intervention.Then,the effect of PM on BMSCs osteogenic induction was evaluated by ALP staining and Von Kossa staining,and the effect of PM on BMSCs adipocyte induction was detected by Oil red O staining.Finally,qRT-PCR was used to verify the expression changes of related core transcription factors and characteristic genes in BMSCs during osteoblast and adipocyte differentiation under PM intervention.Results PM showed no significant toxicity to BMSCs proliferation under 10μmol/L concentration,and 2μmol/L PM significantly activated Hh signaling pathway in BMSCs(P<0.01).After 2μmol/L PM treatment,ALP expression and calcium nodule formation of BMSCs were significantly increased during osteogenic differentiation compared with the control group,while lipid droplet formation was significantly inhibited during adipogenesis.Furthermore,qRT-PCR results showed that 2μmol/L PM significantly promoted the transcriptions of core transcription factor Sp7 and characteristic genes ALP and bone sialoprotein(Ibsp)during BMSCs osteogenic differentiation,and inhibited the transcriptions of core transcription factors peroxisome proliferator-activated receptorsγ(Pparg),CCAAT enhancer-binding proteinα(Cebpa)and characteristic genes perilipin 1(Plin1),adiponectin(Adipoq)and fatty acid translocase(Cd36)during adipocyte differentiation.Conclusion PM promotes osteoblast differentiation and inhibits adipocyte differentiation of BMSCs by activating Hh signaling pathway and regulating relative core transcription factors.
作者
雷新环
朱忠
杨海兰
袁赤亭
郭宇华
章礼炜
LEI Xinhuan;ZHU Zhong;YANG Hailan;YUAN Chiting;GUO Yuhua;ZHANG Liwei(Department of Orthopedics,Taizhou Hospital of Zhejiang Province,Taizhou 317000,China;不详)
出处
《浙江医学》
CAS
2023年第17期1812-1817,1841,I0004,共8页
Zhejiang Medical Journal
基金
浙江省基础公益研究计划项目(LGF20H250004)。
