摘要
目的本实验旨在探讨异补骨脂素对成骨细胞增殖和分化的影响及其可能的作用机制。方法常规复苏培养前成骨细胞株OCT1细胞,分别给予异补骨脂素低剂量(10μg·mL^(-1))、中剂量(30μg·mL^(-1))和高剂量(60μg·mL^(-1))进行干预48 h,同步采用骨形态发生蛋白2(Bone morphogenetic protein 2,BMP2)纯化蛋白(50 ng·mL^(-1))为阳性对照。MTT法观察细胞增殖情况;实时荧光定量RT-PCR反应检测BMP2、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)和成骨细胞特异性转录因子(Osterix,Osx)mRNA的表达;Western blot检测Runx2和Osx蛋白的表达;从BMP2^(loxp/loxp)小鼠中分离培养原代颅盖骨成骨细胞,利用体外条件性基因敲除技术敲除BMP2基因后,给予最佳浓度的异补骨脂素干预48 h后,采用实时荧光定量RTPCR反应检测Runx2和Osx基因表达的情况,并利用碱性磷酸酶(Alkaline phosphatase,ALP)染色观察成骨细胞中ALP活性变化的情况。结果MTT法检测结果显示,异补骨脂素能促进成骨细胞的增殖,但以低剂量(10μg·mL^(-1))组最明显(P<0.05);实时荧光定量RT-PCR反应结果表明,异补骨脂素低剂量(10μg·mL^(-1))和中剂量(30μg·mL^(-1))能促进成骨细胞BMP2 mRNA的表达,以前者作用最佳(P<0.05)。异补骨脂素低剂量(10μg·mL^(-1))可明显促进成骨细胞Runx2 mRNA的表达(P<0.05)。异补骨脂素低剂量(10μg·mL^(-1))和中剂量(30μg·mL^(-1))能促进成骨细胞Osx mRNA的表达。Western blot检测结果显示,异补骨脂素低剂量(10μg·mL^(-1))和中剂量(30μg·mL^(-1))能促进成骨细胞中Runx2蛋白的表达。此外,异补骨脂素低剂量(10μg·mL^(-1))、中剂量(30μg·mL^(-1))和高剂量(60μg·mL^(-1))均可提高Osx蛋白的表达量。体外条件性基因敲除实验结果显示,异补骨脂素诱导的成骨细胞Runx2和Osx基因以及ALP染色高表达呈BMP2的依赖性。结论异补骨脂素可以促进成骨细胞增殖和分化,并可能通过BMP2/Runx2/Osx信号通路而发挥作用。
Objective The purpose of this study was to examine effects of Isopsoralen on the osteoblast proliferation and differentiation and find its possible molecular mechanisms for anti-osteoporosis.Methods OCT-1 cells were cultured with common methods.While growing well,cells were cultured with 3 doses(10μg·mL^(-1),30uμg·mL^(-1)and 60μg·mL^(-1))of Isopsoralen for 48 h,or with purified bone morphogenetic protein 2(BMP2)protein(50 ng·mL^(-1)).We first determined the effect of Isopsoralen on cell proliferation by MTT assay.The real time RT-PCR was also used to quantify changes in the mRNA levels of several genes,such as BMP2,Runt-related transcription factor 2(Runx2),and Osterix(Osx).We also used the Western blot analysis to evaluate the expression of Runx2 and Osx proteins.At last we used the BMP2^(loxp/loxp)mice to isolate the primary calvaria osteoblasts,cultured with Isopsoralen of the best dose for 48 h after the in vitro conditional gene knockout technology,and tested the gene expressions of Runx2 and Osx.And the alkaline Phosphatase(ALP)staining was also performed.Result Isopsoralen(10μg·mL^(-1))can promote osteoblast proliferation obviously.From the real time RT-PCR analysis,Isopsoralen can enhance the BMP2 mRNA levels,the effect of 10μg·mL^(-1)was the best,and 30μg·mL^(-1)followed.In addition,we found that Isopsoralen(10μg·mL^(-1))can enhance the Runx2 mRNA levels significantly.We also found that lower doses of Isopsoralen can enhance the Osx mRNA levels,the effect of 30μg·mL^(-1)was the best,and 10μg·mL^(-1)followed.From the Western blot analysis,low doses of Isopsoralen(10μg·mL^(-1)and 30μg·mL^(-1))can stimulate the expression of Runx2 protein.Besides,three doses of Isopsoralen can stimulate the expression of Osx protein,and the effect of 10μg·mL^(-1)and 30μg·mL^(-1)are better.Finally,the results of in vitro conditional gene knockout experiment showed that the overexpression of Runx2 and Osx genes in osteoblasts,as well as ALP staining,induced by Isopsoralen are BMP2 dependent.Conclusions In this study,we firstly demonstrate that Isopsoralen can stimulate osteoblast proliferation and differentiation by mediating BMP2/Runx2/Osx signaling pathway.
作者
张有为
黄晨
黄廷锐
唐德志
Zhang Youwei;Huang Chen;Huang Tingrui;Tang Dezhi(Centre Hospital of Baoji,Baoji 721008,China;Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2023年第8期2677-2683,共7页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
上海市科学技术委员会中药创新与传承领域项目(20S21902000):“滋肾阴颗粒”治疗原发性骨质疏松症的临床前研究,负责人:唐德志。
