骨形态发生蛋白2对后纵韧带骨化的影响及其可能机制

中华创伤杂志 Pub Date : 2019-07-15 DOI:10.3760/CMA.J.ISSN.1001-8050.2019.07.005

Yuan He, Liang Yan, Liang Li, Songchuan Zhao, D. Hao, B. He

{"title":"骨形态发生蛋白2对后纵韧带骨化的影响及其可能机制","authors":"Yuan He, Liang Yan, Liang Li, Songchuan Zhao, D. Hao, B. He","doi":"10.3760/CMA.J.ISSN.1001-8050.2019.07.005","DOIUrl":null,"url":null,"abstract":"Objective \nTo investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. \n \n \nMethods \nThe expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. \n \n \nResults \nTwo fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. \n \n \nConclusion \nThe Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. \n \n \nKey words: \nOssification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms","PeriodicalId":10161,"journal":{"name":"中华创伤杂志","volume":"35 1","pages":"605-611"},"PeriodicalIF":0.0000,"publicationDate":"2019-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Effects of bone morphogenetic protein 2 on ossification of posterior longitudinal ligament and possible mechanism\",\"authors\":\"Yuan He, Liang Yan, Liang Li, Songchuan Zhao, D. Hao, B. He\",\"doi\":\"10.3760/CMA.J.ISSN.1001-8050.2019.07.005\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Objective \\nTo investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. \\n \\n \\nMethods \\nThe expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. \\n \\n \\nResults \\nTwo fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. \\n \\n \\nConclusion \\nThe Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. \\n \\n \\nKey words: \\nOssification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms\",\"PeriodicalId\":10161,\"journal\":{\"name\":\"中华创伤杂志\",\"volume\":\"35 1\",\"pages\":\"605-611\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-07-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"中华创伤杂志\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3760/CMA.J.ISSN.1001-8050.2019.07.005\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"中华创伤杂志","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3760/CMA.J.ISSN.1001-8050.2019.07.005","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}

引用次数: 0

摘要

目的探讨骨形态发生蛋白2（BMP2）在后纵韧带骨化中的作用及其与转化生长因子-β（TGF-β）/Smad信号通路的关系。方法构建野生型pcDNA3.1-BMP2（WT）、突变型pcDNA3.1%BMP2（37G）、突变株pcDNA3.1-BMP（190T）和突变株pcDNA 3.1-BMP（37G/190T）的表达载体，并用琼脂糖凝胶电泳进行鉴定。将构建的载体转染到脂质体介导的小鼠胚胎成纤维细胞C3H10T1/2中，检测BMP2的表达。根据转染情况分为六组：（1）非转染组；（2）空载体pcDNA3.1转染组；（3） pcDNA3.1-bmp2（WT）转染组；（4） pcDNA3.1-bmp2（37G）转染组；（5） pcDNA3.1-bmp2（190T）转染组；（6） pcDNA3.1-bmp2（37G/190T）转染组。根据是否包括BMP2多态性来定义实验组和对照组。因此，非转染组和空载体pcDNA3.1转染组为对照组，其他组为实验组。通过蛋白质印迹检测阳性细胞克隆中磷酸化Smad1/5/8和Smad4的表达，并通过定量检测试剂盒检测碱性磷酸酶（ALP）。比较实验组之间的蛋白质表达。结果从pcDNA3.1-BMP2中切出的两个片段经琼脂糖电泳分别为1.2kb和5.4kb。直接测序结果与靶基因序列一致。成功转染BMP2基因并在C3H10T1/2细胞中稳定表达。Western印迹显示转染后实验组磷酸化Smad1/5/8蛋白的表达显著增加，经野生型或突变型pcDNA3.1-BMP2转染的Smad4蛋白表达明显高于对照组（P＜0.05），BMP2基因外显子2 Ser37Ala（T/G）多态性与稳定转染的C3H10T1/2细胞中ALP活性呈正相关。结论BMP2基因外显子2 Ser37Ala（T/G）多态性通过TGF-β/Smad信号通路促进OPLL骨化，其可能机制是上调Smad4和ALP的蛋白表达。关键词：骨化，后纵韧带；骨形态发生蛋白2；信号转导；单核苷酸多态性

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Effects of bone morphogenetic protein 2 on ossification of posterior longitudinal ligament and possible mechanism

Objective To investigate the effect of bone morphogenetic protein 2(BMP2)on ossification of the posterior longitudinal ligament (OPLL) and its relationship with transforming growth factor-β (TGF-β)/Smad signaling pathway. Methods The expression vectors of wild type pcDNA3.1-BMP2 (WT), mutant pcDNA3.1-BMP2 (37G), mutant pcDNA3.1-BMP2 (190T) and mutant pcDNA3.1-BMP2 (37G/190T) were constructed and identified by agarose gel electrophoresis. The constructed vector was transfected into mouse embryonic fibroblasts C3H10T1/2 mediated by liposome to detect the expression of BMP2. Six groups were divided according to the transfection situation: (1) the non-transfection group; (2) empty vector pcDNA3.1 transfection group; (3) pcDNA3.1-bmp2 (WT) transfection group; (4) pcDNA3.1-bmp2 (37G) transfection group; (5) pcDNA3.1-bmp2 (190T) transfection group; (6) pcDNA3.1-bmp2 (37G/190T) transfection group. The experimental and control group were defined according to whether BMP2 polymorphism was included. Therefore, the non-transfection group and empty vector pcDNA3.1 transfection group were control groups, and the other groups were experimental groups. The expression of phosphorylated Smad1/5/8 and Smad4 in positive cell clones were detected by western blotting, and the alkaline phosphatase (ALP) was detected by quantitative detection kits. The protein expressions were compared among the experimental groups. Results Two fragments digested from pcDNA3.1-BMP2 represented 1.2 kb and 5.4 kb by agarose electrophoresis. The direct sequencing results were in accordance with target gene sequence. BMP2 gene was successfully transfected and stably expressed in C3H10T1/2 cells. Western blotting showed that the expression of phosphorylated Smad1/5/8 protein in the experimental groups was increased significantly after transfection, with significant difference between the experimental groups and the control groups (P 0.05). The expressions of Smad4 protein transfected by wild or mutation type pcDNA3.1-BMP2 were significantly higher than those in the control groups (P 0.05). However, there were significant differences between the two groups and other experimental groups (P<0.05). The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene was positively correlated with ALP activity in stably transfected C3H10T1/2 cells. Conclusion The Ser37Ala (T/G) polymorphism in exon 2 of BMP2 gene promotes OPLL ossification through TGF-β/Smad signaling pathway, the possible mechanism for which is to up-regulate the protein expressions of Smad4 and ALP. Key words: Ossification, posterior longitudinal ligament; Bone morphogenetic protein 2; Signal transduction; Single nucleotide polymorphisms

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

中华创伤杂志

自引率

0.00%

发文量

11327

期刊介绍： Chinese Journal of Trauma (International Standard Serial Publication Number: ISSN 1001-8050, Domestic Uniform Serial Publication Number: CN 50-1098/R) was founded in September 1985, which is the only high-level medical professional academic journal that can comprehensively and systematically reflect the achievements and development trends of China's traumatology medicine, and has a wide academic influence in China's traumatology medicine community. It has a wide range of academic influence in China's trauma medicine. Chinese Journal of Trauma is a source journal of China Science and Technology Paper Statistics, a source journal of China Science Citation Database (CSCD), a core journal of China Comprehensive Medicine and Health Care, a source journal of China Academic Journals Comprehensive Evaluation Database (CAJCED), a full-text journal of China Journal Full-text Database (CJFD), a core academic journal of China Center for Scientific Evaluation (RCCSE), a core academic journal of China Traumatology and Traumatology Center (CTC), a core academic journal of China Traumatology Center (RCCSE). RCCSE) core academic journals; Chinese Biomedical Journal Database (CMCC), Chinese Biomedical Journal Citation Database (CBJCED), China Journal Network (CJN), China Academic Journals (CD-ROM), Chinese Academic Journals Abstracts (Chinese Edition), Chemical Abstracts of the United States (CA), Index Copernicus of Poland (IC), and Japan Institute of Science and Technology Database (JICST), World Health Organization Western Pacific Region Medical Search (WPRIM) and Russian Journal of Abstracts (ΡЖ) included journals.