TGF⁃β1 p38MAPK及BMP⁃7蛋白在肝多房棘球蚴病患者肝组织中的表达及意义

目的　检测肝多房棘球蚴病患者肝组织中转化生长因子⁃β1（transforming growth factor⁃β1, TGF⁃β1）、p38MAPK及骨形态发生蛋白⁃7（bone morphogenetic protein⁃7, BMP⁃7）表达水平,探讨其在肝多房棘球蚴病肝纤维化中的潜在作用。方法　以20例肝多房棘球蚴病患者为研究对象,分别采集距肝脏病灶0.5 cm内（A组）、距肝脏病灶0.5～1.5 cm（B组）肝组织及距肝脏病灶2 cm及以上的正常肝组织（C组）。肝组织标本分别行HE和Masson染色观察纤维化病理变化,采用Western blotting检测肝组织中TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平,分析TGF⁃β1、p38MAPK及BMP⁃7蛋白表达与肝纤维化的相关性。结果　HE染色结果显示,A组和B组肝组织中肝细胞结构排列紊乱、肝小叶结构有不同程度破坏,可见不同程度肝细胞变性、萎缩、坏死及纤维组织增生,并有嗜酸性粒细胞浸润;C组肝组织无异常病理改变,肝细胞结构形态正常、大小均匀,未见明显排列紊乱,肝小叶结构清晰,无或轻度细胞变性、坏死及炎性细胞浸润。Masson染色结果显示,A组和B组肝组织可见汇管区较多纤维结缔组织增生,出现不同程度小叶内纤维化;C组肝组织无明显异常病理改变。A、B、C组肝组织中TGF⁃β1（P < 0.001）、p38MAPK（P < 0.01）及BMP⁃7蛋白（P < 0.05）表达水平差异均有统计学意义,A组和B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均显著高于C组（P均< 0.05）,B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平亦显著高于C组（P均< 0.05）。TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均与肝纤维化程度呈正相关（r = 0.866、0.702、0.801,P均< 0.05）,不同纤维化程度肝组织中TGF⁃β1（F = 72.580,P < 0.01）、p38MAPK（[χ2] = 31.705,P < 0.01）及BMP⁃7蛋白（[χ2] = 48.388,P < 0.01）表达水平差异均有统计学意义。TGF⁃β1蛋白与p38MAPK、BMP⁃7蛋白表达水平均呈显著正相关（r = 0.607、0.702,P均 < 0.001）,BMP⁃7与p38MAPK蛋白表达水平亦呈显著正相关（r = 0.456,P < 0.001）。结论　TGF⁃β1、p38MAPK和BMP⁃7蛋白通过相互作用、共同介导了肝多房棘球蚴病肝纤维化发生。     

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL–TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL–TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.     

Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFβ3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells

Chondrogenic promotion by rhGDF5 with or without rhTGFβ3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFβ3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFβ1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFβtype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFβRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFβ1 or rhTGFβ3 treatment. ALK6 protein expression also showed increase by rhTGFβ3. rhTGFβ1/rhTGFβ3 induced ALK6 up-regulation was inhibited by SD-208, a TGFβ type I receptor inhibitor. Chondrogenesis by rhTGFβ1/rhTGFβ3 or the combination between rhTGFβ1/rhTGFβ3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFβ1/rhTGFβ3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFβ3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFβs in the synergy between rhTGFβs and rhBMPs.     

侧壁开窗法上颌窦底提升术植入Bio-Oss后骨生成的mRNA表达谱

 目的：探讨侧壁开窗法上颌窦底提升术这种特殊的引导性骨再生（guided bone regeneration, GBR）过程的转录组特点。方法：3只Beagle犬,随机选择一侧经侧壁开窗行上颌窦底提升术,同时植入Bio-Oss骨粉,术后2周,对另一侧用相同的方法行上颌窦底提升术。第2次术后2周,收集植入的Bio-Oss骨粉组织块,提取总RNA,全基因组表达谱芯片检测分析上颌窦底提升后2周和4周时的差异表达基因。利用基因本体论（gene ontology, GO）对差异表达的基因进行功能分类,以及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）进行信号通路分析。结果：在2周时表达升高的基因与刺激反应、免疫-炎症反应和细胞增殖有关,同时,与神经元分化、破骨细胞分化和早期血管化过程相关的基因也高表达。在4周时,与细胞代谢和发育相关的基因表达升高,重要的是,与血管生成和骨骼发育相关的基因都高表达,与骨骼发育相关的基因包括骨生成相关的生长和分化因子［转化生成因子β1（transforming growth factor beta 1, TGFB1）和骨形态发生蛋白4（bone morphogenetic protein 4, BMP4）］、软骨和骨胞外基质［软骨黏附素（chondroadherin, CHAD）、Ⅰ型胶原α1链（collagen type I, alpha 1 chain, COL1A1）、Ⅰ型胶原α2链（collagen type I, alpha 2 chain, COL1A2）、Ⅴ型胶原α2链（collagen type Ⅴ, alpha 2 chain, COL5A2）、核心蛋白聚糖（decorin, DCN）、骨连接蛋白（osteonectin, SPARC）和骨钙素（osteocalcin, BGLAP）］等相关的基因。信号通路分析显示4周时表达升高的基因与转化生长因子β/骨形态发生蛋白（transforming growth factor beta/bone morphogenetic protein, TGF-β/BMP)信号通路有关。无翅型MMTV整合位点家族（wingless-type MMTV integration site family, WNT）信号通路中的关键基因WNT5A和卷曲蛋白受体9（frizzled receptor 9, FZD9）在2周时表达升高,而连环蛋白β1（catenin beta 1, CTNNB1）在4周时表达升高。结论：侧壁开窗法上颌窦底提升术2周后转录表达谱变化以免疫-炎症反应和破骨反应下调、骨生成相关基因表达升高为特点,骨再生的过程还与神经生成和血管生成过程相关。在骨再生的过程中,TGF-β/BMP和WNT信号通路起重要作用,WNT信号通路的作用复杂。     

腺病毒介导的BMP9过表达抑制人骨肉瘤细胞系143B的增殖与迁移

目的 探讨骨形态发生蛋白9(BMP9)过表达对人骨肉瘤细胞系143B的生物学行为的影响.方法 用RTPCR和Western blot检测骨肉瘤细胞系中BMP9的内源性表达,用表达BMP9的腺病毒重组体(adBMP9)感染内源性表达BMP9相对较低的骨肉瘤细胞系,Transwell法检测细胞侵袭,划痕愈合实验检测细胞迁移,MTT法和结晶紫染色法检测细胞的增殖,Hoechst 33258染色法检测凋亡,平板集落形成实验检测集落形成能力.结果 adBMP9可以明显减弱骨肉瘤细胞系143B的增殖、迁移、侵袭及集落形成能力(P＜0.05),并促进细胞凋亡.结论 adBMP9能抑制人骨肉瘤细胞系143B的增殖和迁移,促进细胞凋亡.     

Potential Roles of Bone Morphogenetic Protein 9 in the Odontogenic Differentiation of Dental Pulp Cells

IntroductionThe differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing.MethodsFluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation.ResultsBMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment.ConclusionsBMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.     

非综合征型先天缺牙致病基因和分子机制的研究进展

先天缺牙是牙齿发育过程中常见的牙数目发育异常,对患者的颌面部发育及美观和咀嚼功能产生严重的影响。根据有无伴发全身症状,先天缺牙可分为综合征型先天缺牙与非综合征型先天缺牙。近几年发现新的相关基因和新的突变位点及分子机制已成为目前非综合征型先天缺牙基因研究的主要方向。本文通过对近年来文献的回顾,对与非综合征型先天缺牙主要相关的Wnt/β-catenin信号通路、TGF-β/BMP信号通路、PAX9基因和MSX1基因、EDA/EDAR/NF-κb信号通路的分子机制以及相互调节的紧密联系进行综述,为未来先天缺牙的防治提供了新的理论基础。非综合征型先天缺牙致病基因的分子机制的研究目前甚少,对于其机制的精准探索将成为先天缺牙未来主要的研究方向之一。     

BMP2 Regulation of CXCL12 Cellular,Temporal, and Spatial Expression Is Essential During Fracture Repair

The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)‐dependent fracture healing occurs through a tight control of chemokine C‐X‐C motif‐ligand‐12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site‐specific response of CXCL12⁺‐BMP2⁺ endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2^cKO/+) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2^cKO/+ mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2^cKO/+ mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2^cKO/cKO endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2‐expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12⁺‐BMP2⁺ perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12⁺‐BMP2⁺ to osteogenesis while departing their supportive role to angiogenesis. Our findings have far‐reaching implications for understanding mechanisms regulating the selective recruitment of distinct cells into the repairing niches and the development of novel pharmacological (by targeting BMP2/CXCL12) and cellular (MSCs, endosteal cells) interventions to promote fracture healing. © 2015 American Society for Bone and Mineral Research.     

TGF‐β & BMP Receptors Endoglin and ALK1: Overview of their Functional Role and Status as Antiangiogenic Targets

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF‐β superfamily, TGF‐β1, TGF‐β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell–cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1‐mediated cells signaling.