【作者】 周怡君； 姜鸣欣； 史册； 孙宏晨；

【Author】 Zhou Yijun;Jiang Mingxin;Shi Shi;Sun Hongchen;Hospital of Stomatology, Jilin University;Norman Bethune Stomatology College of Jilin University;

【机构】 吉林大学口腔医院； 吉林大学白求恩口腔医学院；

【摘要】 目的:进行性骨化性纤维发育不良(fibrodysplasia ossificans progressiva, FOP)主要表现为全身软组织广泛性、进行性、不可逆的异位骨化(heterotopic ossification, HO),目前药物治疗效果有限,手术治疗又可能造成新的HO。95%的FOP是由活化素I型受体(activin receptor type I, ACVR1)R206H突变引起的,但与ACVR1和ACVR1 R206H相互作用的具体分子及其相互作用后是如何传递信号通路的,目前尚不清楚。传统的探究蛋白相互作用的方法具有一定局限性,且商业ACVR1抗体及抑制剂的特异性不佳。近期研究发现工程化的抗坏血酸过氧化物酶(APEX)可在完成膜结构中、在有限的时间空间内对酶附近的蛋白质进行生物素标记,应用该方法可有利于深入了解ACVR1和ACVR1 R206H互作分子及其下游相关的信号通路,探讨FOP发生的分子机制,为后续药物研发提供新的思路。方法:构建Acvr1基因敲除小鼠胚胎成纤维细胞永生化细胞系(iMEF A2KO);Acvr1-Apex2慢病毒和Acvr1 R206H-Apex2慢病毒感染iMEF A2KO,PCR、qRT-PCR、Western blot、成骨向诱导及染色检测Acvr1-Apex2和Acvr1 R206H-Apex2基因在细胞系中的整合、表达及蛋白功能。Western blot检测APEX2的激活情况。结果:成功构建Acvr1基因敲除小鼠胚胎成纤维细胞永生化细胞系(iMEF A2KO),细胞表达MEF相关标志物,具有多向分化能力,可连续传代50代以上,Acvr1 m RNA的表达维持在较低水平。Acvr1-Apex2慢病毒和Acvr1 R206H-Apex2慢病毒感染细胞后,PCR、qRT-PCR及Western blot结果显示Acvr1-Apex2和Acvr1 R206H-Apex2基因成功整合至各自细胞基因组内并表达。相较于"A-A"组(细胞感染Acvr1-Apex2慢病毒),"R-A"组(细胞感染Acvr1 R206H-Apex2慢病毒)细胞ALP活性增强、矿化结节量增多。激活APEX2后,相较于iMEF A2KO组或未激活组中只有两条内源性生物素蛋白条带的情况,"A-A"组和"R-A"组中可见大量标记蛋白条带,且两组间条带略有差异。结论:基于Acvr1基因敲除小鼠胚胎成纤维细胞永生化细胞系,成功构建ACVR1-APEX2及ACVR1 R206H-APEX2表达体系,在激活APEX2后可捕捉周围互作蛋白,可用于后续分析差异蛋白、探究发病机制及研发治疗药物。更多还原

【Abstract】 Purpose: Fibrodysplasia ossificans progressiva(FOP) is characterized by extensive,progressive and irreversible heterotopic ossification(HO) of systemic soft tissue. At present, the effect of drug therapy is limited, and surgical treatment may cause new HO.95% of FOP is caused by mutations in the activin receptor type I(ACVR1) R206H, but the specific molecules that interact with ACVR1 and ACVR1 R206H and how the signaling pathways are transmitted after interaction are still unclear. Traditional approaches to exploring protein interactions have some limitations, and the specificity of commercial ACVR1 antibodies and inhibitors is poor. Recent studies have found that engineered ascorbate peroxidase(APEX) can biotin label proteins near the enzyme in the completion of membrane structure within a limited time and space. The application of this method can help to further understand ACVR1 and ACVR1 R206H interaction molecules and their downstream related signaling pathways, explore the molecular mechanism of FOP, and provide new ideas for subsequent drug development.Methods: Immortalized Acvr1 knockout mouse embryonic fibroblast cell line(iMEF A2KO) was constructed. Acvr1-apex2 lentivirus and Acvr1 R206H-Apex2 lentivirus were used to infect iMEF A2KO. The integration, expression and protein function of Acvr1-apex2 and Acvr1 R206H-Apex2 genes in cell lines were detected by PCR,qRT-PCR, Western blot, osteogenic induction and staining. Western blot analysis of APEX2 activation.Results: The immortalized cell line of Acvr1 knockout mouse embryonic fibroblasts(iMEF A2KO) was successfully constructed. The cells expressed MEF-related markers and had multidirectional differentiation ability, which could be passed continuously for more than50 generations, and the expression of Acvr1 m RNA was maintained at a low level. After Acvr1-apex2 lentivirus and Acvr1 R206H-Apex2 lentivirus infected cells, PCR, qRT-PCR and Western blot results showed that Acvr1-apex2 and Acvr1 R206H-Apex2 genes were successfully integrated into their respective cell genomes and expressed. Compared with the "A-A" group(cells infected with Acvr1-apex2 lentivirus), the "R-A" group(cells infected with Acvr1 R206H-Apex2 lentivirus) increased the activity of ALP and the number of mineralized nodules. After activating APEX2, a large number of labeled protein bands were observed in the "A-A" and "R-A" groups, and the bands were slightly different between the two groups, compared with only two bands in the iMEF A2KO group or the non-activated group.Conclusion: Based on the immortalized cell line of Acvr1 knockout mouse embryonic fibroblasts, the expression system of ACVR1-APEX2 and ACVR1 R206H-APEX2 is successfully constructed, which can capture surrounding interacting proteins after activation of APEX2, and can be used for subsequent analysis of differential proteins,exploration of pathogenesis and development of therapeutic drugs.更多还原