敲低细丝蛋白B对小鼠颅顶成骨前体细胞增殖、迁移及凋亡的影响

背景:细丝蛋白B(FLNB)能将肌动蛋白细胞骨架交联成动态结构,对细胞的定向移动至关重要,可调控软骨细胞的增殖、分化及凋亡的发生。然而,细丝蛋白B对成骨细胞增殖、迁移及凋亡的影响尚未报道。目的:探究细丝蛋白B对MC3T3-E1细胞的增殖、迁移及凋亡的影响。方法:构建敲低细丝蛋白B表达的腺病毒载体(sh-FLNB1、sh-FLNB2、sh-FLNB3),感染小鼠颅顶成骨前体细胞(MC3T3-E1),嘌呤霉素药筛,通过Western Blot以及RT-PCR技术检测敲低效率,选取敲低细丝蛋白B效率最佳的MC3T3-E1细胞株作为稳转细胞株。将细胞分为Blank组、mc3t3组、sh-NC组(空载体)、sh-FLNB组(sh-FLNB慢病毒),Blank组为α-MEM完全培养基无细胞;mc3t3组为单纯α-MEM完全培养基培养;sh-NC组、sh-FLNB组为含有嘌呤霉素2.5μg/mL的α-MEM完全培养基培养。培养3 d采用CCK-8法和划痕实验检测MC3T3-E1细胞增殖和迁移能力;流式细胞仪检测细胞凋亡情况;RT-PCR进一步验证细胞凋亡相关基因的表达。结果与结论:①Western Blot及RT-PCR结果显示,sh-FLNB3组细丝蛋白B敲低效率最佳,差异有显著性意义(P<0.0001),以此作为后续实验稳转细胞株;②CCK-8数据显示,敲低细丝蛋白B后MC3T3增殖能力显著减弱(P<0.05);③划痕实验显示,敲低细丝蛋白B后MC3T3的迁移能力明显下降;④流式细胞仪及RT-PCR显示,敲低细丝蛋白B后,MC3T3-E1细胞凋亡率增加,促凋亡因子Bax表达显著上升,抑凋亡因子Bcl-2表达下降(P<0.05);⑤结果说明,细丝蛋白B敲低可降低MC3T3-E1细胞的增殖能力,细胞迁移能力下降,并增加细胞凋亡的发生。

Filamin B:The next hotspot in skeletal research?

Filamin B （FLNB） is a large dimeric actin-binding protein which crosslinks actin cytoskeleton filaments into a dynamic structure. Lip to present, pathogenic mutations in FLNB are solely found to cause skeletal deformities, indicating the important role of FLNB in skeletal development. FLNB-related disorders are classified as spondylocarpotarsal synostosis （SCT）, Larsen syndrome （LS）, atelosteogenesis （AO）, boomerang dysplasia （BD）, and isolated congenital talipes equinovarus, presenting with scoliosis, short- limbed dwarfism, clubfoot, joint dislocation and other unique skeletal abnormalities. Several mecha- nisms of FLNB mutations causing skeletal malformations have been proposed, including delay of ossi- fication in long bone growth plate, reduction of bone mineral density （BMD）, dysregulation of muscle differentiation, ossification of intervertebral disc （IVD）, disturbance of proliferation, differentiation and apoptosis in chondrocytes, impairment of angiogenesis, and hypomotility of osteoblast, chondrocyte and fibroblast. Interventions on FLNB-related diseases require prenatal surveillance by sonography, gene testing in high-risk carriers, and proper orthosis or orthopedic surgeries to correct malformations including scoliosis, cervical spine instability, large joint dislocation, and clubfoot. Gene and cell therapies for FLNB-related diseases are also promising but require further studies.

Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B

Hepatitis B virus(HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein,and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA(pg RNA), and improved the secretion level of hepatitis B surface antigen(HBsAg) and hepatitis B e antigen(HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.

细丝蛋白B在钙化性主动脉瓣疾病中的表达及其临床意义

目的 研究细丝蛋白B(FLNB)在钙化性心脏瓣膜中的表达及其临床意义。方法 2021年8月~2022年1月在我院手术切除的钙化性主动脉瓣标本9例,同期因主动脉夹层行手术切除的正常主动脉瓣膜标本8例作为对照。组织切片茜素红染色观察瓣膜钙化结节。采用免疫组化分析方法对瓣膜中细丝蛋白B的表达进行分析。利用冰冻组织标本进行蛋白质免疫印迹检测,验证相应分子表达水平间差异。结果 钙化瓣膜切片经茜素红染色可以看见明显钙化结节。免疫组化检查显示细丝蛋白B表达于细胞质,钙化瓣膜高于正常瓣膜,钙化组平均光密度为(6.57±1.65)%;对照组为(0.91±0.52)%,两组比较差异有统计学意义(P<0.05),蛋白质免疫印迹结果与之一致。结论 细丝蛋白B在钙化瓣膜中表达增高,可能参与了瓣膜钙化的病理进程。

细丝蛋白A与蛋白激酶B在小肠腺癌中的表达与意义

目的探讨细丝蛋白A(filamin A,FLNA)和蛋白激酶B(protein kinase B,PKB)在小肠腺癌组织中的表达及其临床意义。方法应用免疫组织化学法、逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)和蛋白质印迹法(Western印迹法),检测56例小肠腺癌组织及正常小肠组织(距腺癌组织边缘5 cm以上)中FLNA、PKB的表达情况。结果小肠腺癌组织中FLNA mRNA表达低于正常小肠组织,而PKB mRNA的表达水平高于正常小肠组织(P<0.05)。小肠腺癌组织中FLNA表达低于正常小肠组织,而PKB的表达水平高于正常小肠组织(P<0.05)。FLNA、PKB的表达与患者性别、年龄、肿瘤组织学分级无关(P>0.05),而与肿瘤淋巴结转移、临床肿瘤分期和肿瘤浸润深度相关(P<0.05);Spearman秩相关系数显示FLNA与PKB表达呈负相关(r=-0.289,P=0.008)。结论 FLNA、PKB参与小肠腺癌的发生、发展过程,磷脂酰肌醇3-激酶/PKB信号通路的活化可作为临床潜在治疗靶点。

Rare loss-of-function variants in FLNB cause non-syndromic orofacial clefts

Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb^(−/−) embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.

成人拉森综合征1例报道及文献复习

拉森综合征(Larsen syndrome,LRS)是一种罕见的骨骼系统遗传性疾病,最早报道于1950年[1],主要临床表现为大关节脱位,包括髋关节、膝关节、肘关节脱位,足部畸形,身材矮小,以及典型的面部特征,常出现于儿童期。脊柱侧凸、后凸畸形常见于LRS,其进展会引起患儿心肺呼吸功能发育受限或神经系统症状。北京协和医院于2021年10月收治1例成年LRS患者。

GnRH缺陷疾病致病基因RNF216互作蛋白的筛选

目的通过酵母双杂交实验筛选与环指蛋白216(ring finger protein 216,RNF216)相互作用的蛋白,进一步阐明RNF216在GnRH缺陷疾病中的作用。方法构建pGBKT7-RNF216重组表达载体,将其转化到Y2HGold酵母中,与人cDNA文库进行杂交,筛选与RNF216相互作用的蛋白,然后在Y2HGold酵母中进行验证。结果成功构建了pGBKT7-RNF216重组表达载体,并在Y2HGold酵母中成功表达;通过酵母双杂交实验,筛选到了一个与RNF216相互作用的蛋白——丝状蛋白B(filamin B,FLNB),并在Y2HGold酵母中验证了它们之间的相互作用。结论成功筛选到一个与RNF216相互作用的FLNB蛋白,RNF216可能通过调节FLNB或FLNB/FLNA异源二聚体影响GnRH神经元的增殖和迁移。