Exploration of Shh and BMP paracrine signaling in a prostate cancer xenograft

Stromal–epithelial signaling is a critical regulator of normal prostate development and has been speculated to play an equally important role in the development and progression of prostate cancer. Sonic hedgehog (Shh) and bone morphogenetic proteins (BMP-4, BMP-7), expressed by the urogenital sinus epithelium and mesenchyme, exert reciprocal and coordinate effects on outgrowth of nascent prostate ducts. Over-expression of Shh in the LNCaP xenograft was shown previously to accelerate tumor growth by a paracrine mechanism. A survey of BMP regulators expressed in the developing prostate revealed increased Noggin and BMP-7 mRNA in the stromal component of Shh over-expressing xenografts. In vitro studies demonstrated that treatment of LNCaP cells with BMP-4 and BMP-s7 induced Id-1 expression and inhibited tumor cell proliferation. The activity of BMP-4 was abrogated by co-addition of Noggin; the activity of BMP-7 was not. Quantitative analysis of BMP signaling revealed ambivalent results: decreased tumor cell expression of the BMP response gene Id-1 but increased staining for phospho-SMAD 1,5, 8. To directly test whether increased xenograft tumor growth could be explained by Noggin-mediated blockade of BMP-2/4 effects on tumor cell proliferation, we generated LNCaP xenografts containing stromal cells over-expressing Noggin. Tumor cells in these xenografts exhibited decreased Id-1 and reduced SMAD phosphorylation, but tumor growth was not altered. We conclude that tumor cell Shh expression can induce significant changes in expression of BMP ligands and inhibitors in the stromal microenvironment but that acceleration of LNCaP xenograft tumor growth by Shh over-expression cannot be attributed solely to increased Noggin expression in the tumor stroma.     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.     

骨髓间充质干细胞对重症急性胰腺炎胰腺组织修复的促进作用研究

目的观察骨髓间充质干细胞（BMSCs）抑制坏死性凋亡促进修复重症急性胰腺炎（SAP）的可能机理。 方法（1）分离、培养和鉴定大鼠BMSCs;（2）构建牛磺胆酸钠（NaT）诱导的SAP大鼠模型,并分成正常组（NC）、假手术组（Sham）、SAP模型组（SAP）、PBS治疗组（PBS）、BMSCs治疗组和Necrostain-1 （Nec-1）治疗组,并检测胰腺病理评分和血淀粉酶水平;（3）运用Western Blot和qRT-PCR方法检测各组受损胰腺组织内RIPK1、RIPK3、Caspase-8、MLKL蛋白及mRNA表达,各组均数间比较采用单因素方差分析,两两比较采用LSD-t检验。 结果BMSCs可以被诱导分化成骨、软骨、脂肪,并高表达CD44 （99.82﹪）、CD73 （99.87﹪）、CD90 （99.99﹪）、CD105 （99.78﹪）,低表达CD11b （0.65﹪）、CD19 （0.85﹪）、CD34 （0.70﹪）和CD45 （1.20﹪）。SAP组胰腺病理评分（12.90±1.79）及血淀粉酶水平（1052.41±183.12） mU/ ml均高于NC组[评分：0.40±0.52,淀粉酶水平：（236.62±33.21） mU/ ml]和Sham组[评分：0.50±0.53,淀粉酶水平：（242.31±27.94） mU/ ml]（F = 200.275,F = 143.245,P均< 0.001）,且SAP组受损胰腺组织内RIPK1、RIPK3、MLKL表达升高、Caspase-8表达降低（F = 179.905,P < 0.001）;BMSCs组和Nec-1治疗组胰腺病理评分及血淀粉酶水平均低于PBS治疗组[评分：7.20±1.23、7.00±1.05比12.60±1.65,F = 200.275,P < 0.001;淀粉酶水平：（452.21±101.68）mU/ml、（570.18±148.47） mU/ml比（972.77±204.29） mU/ml,F = 143.245,P < 0.001],同时受损胰腺组织内RIPK1、RIPK3、MLKL表达下调、Caspase-8表达升高（F = 179.905,P < 0.001）。 结论BMSCs可能通过抑制坏死性凋亡通路活化来修复SAP。     

N-glycosylation influences the latency and catalytic properties of mammalian purple acid phosphatase

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase or uteroferrin, contains two potential consensus N-glycosylation sites at Asn(97) and Asn(128). In this study, endogenous rat bone PAP was found to possess similar N-glycan structures as rat recombinant PAP heterologously expressed in baculovirus-infected Sf9 insect cells. PAP from Sf9 cells was shown to contain two N-linked oligosaccharides, whereas PAP expressed by mammalian CHO-K1 cells was less extensively glycosylated. The extent of N-glycosylation affected the catalytic properties of the enzyme, as N97Q and N128Q mutants, containing a single oligosaccharide chain, exhibited a lower substrate affinity and catalytic activity compared to those of the fully glycosylated PAP in the native, monomeric state. The differences in substrate affinity and catalytic activity were abolished and partially restored, respectively, by proteolytic cleavage in the loop domain, indicating that the extent of N-glycosylation influences the interaction of the repressive loop domain with catalytically important residues.     

Shear strength and toughness of trabecular bone are more sensitive to density than damage

Microdamage occurs in trabecular bone under normal loading, which impairs the mechanical properties. Architectural degradation associated with osteoporosis increases damage susceptibility, resulting in a cumulative negative effect on the mechanical properties. Treatments for osteoporosis could be targeted toward increased bone mineral density, improved architecture, or repair and prevention of microdamage. Delineating the relative roles of damage and architectural degradation on trabecular bone strength will provide insight into the most beneficial targets. In this study, damage was induced in bovine trabecular bone samples by axial compression, and the effects on the mechanical properties in shear were assessed. The damaged shear modulus, shear yield stress, ultimate shear stress, and energy to failure all depended on induced damage and decreased as the architecture became more rod-like. The changes in ultimate shear strength and toughness were proportional to the decrease in shear modulus, consistent with an effective decrease in the cross-section of trabeculae based on cellular solid analysis. For typical ranges of bone volume fraction in human bone, the strength and toughness were much more sensitive to decreased volume fraction than to induced mechanical damage. While ultimately repairing or avoiding damage to the bone structure and increasing bone density both improve mechanical properties, increasing bone density is the more important contributor to bone strength.     

带蒂皮瓣移植在小腿开放性骨折中的临床研究

目的:探讨不同带蒂皮瓣移植术式在小腿开放性骨折中的临床应用效果。方法:35例小腿开放性骨折患者,完善术前准备后,根据小腿皮肤软组织的缺损部位、面积、深度分别选择腓肠神经营养皮(肌)瓣、隐神经营养逆行皮瓣、局部旋转皮(肌)瓣修复创面。结果:所有移植皮瓣中,1处移植失败(坏死面积>1/2),2处皮瓣远端1-2.5cm坏死,经清创换药后愈合,余患者皮瓣均成活。从皮瓣成活的优良率和出血量上对比,三组间无明显差别,从手术时间上对比,局部旋转皮(肌)瓣组优于另两组(P<0.05)。结论:在小腿开放性骨折伴皮肤软组织缺损的治疗中,完善的术前准备及根据皮损形态选择适当的皮瓣是获得满意疗效的基础。     

Bone formation in cartilage produced by transplanted epiphyseal chondrocytes

Summary Chondrocytes were isolated from rat epiphyseal cartilage, cultured in vitro, and exposed to exogenous tracers which accumulated in their lysosomes. The cells were then injected into the posterior tibial muscle of animals from the same outbred strain, where they reconstructed calcifying hyaline cartilage. The mineralization of the tissue was followed by ingrowth of blood capillaries from the host bed. Macrophage-like cells surrounding the vessels phagocytized degenerated chondrocytes and unmineralized matrix, whereas multinucleated chondroclasts removed some of the mineralized cartilage matrix. Mesenchyme-like cells accompanying the invading vessels attached to the remaining septa of calcified cartilage matrix and developed into osteoblasts depositing bone matrix on the surface of these septa. The apparent lack of inherent tracer labeling of the lysosomes in the different bone cells indicate that they were derived from the host. No signs of transformation of chondrocytes into bone cells were observed.When isolated rat epiphyseal chondrocytes were injected into the wall of the hamster cheek pouch, calcifying cartilage was reconstructed without signs of subsequent ossification. Transplantation of cartilage reconstructed in the hamster into the dorsal muscles of rats was, however, followed by formation of bone by a sequence analogous to that described above. Such an osteogenetic response was also obtained when the cartilage had been devitalized before transplantation.These experiments show that calcified cartilage, developing in or grafted into an intramuscular site, is able to induce and serve as a substrate for endochondral bone formation, similar to that occurring during normal development. They further indicate that bone induction by calcified cartilage does not require the presence of living chondrocytes.Financial support was obtained from the Swedish Medical Research Council (proj. no. 03355), the King Gustaf V 80th Birthday Fund, and from the funds of Karolinska Institutet. The authors thank Karin Blomgren for technical assistance and Inger Lohmander-Åhrén and Eva Pettersson for secretarial helpOn leave from the Department of Histology and Embryology, Medical Academy, Warsaw, Poland     

Effect of bone sialoprotein on proliferation and osteodifferentiation of human bone marrow-derived mesenchymal stem cells in vitro

We performed this study to investigate the effects of recombinant human bone sialoprotein (BSP) on the proliferation and osteodifferentiation of human BMSCs(hBMSCs). The hBMSC cultures were divided into 4 groups: control group, 10⁻¹⁰ M BSP group (BSP group), osteogenic medium group (10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 mg/L ascorbic acid, OM group) and BSP + OM group (OM plus10⁻¹⁰ M BSP). Compared with the control group, cell growth of the other three groups slowed down, while fluorescence at the G₀/G₁ phase increased. After 28 days, in the OM group and the BSP + OM group, the proportion of STRO-1-positive cells decreased by 22.7% and 38.4% and ALP activity increased by 50% and 71.43%, respectively. CD271 mRNA expression decreased while Cbfa1, osteocalcin and osterix mRNA levels increased in the OM and BSP + OM groups, and the mRNA level change was greater in the BSP + OM group. After 28 days, the number of nodules in the BSP + OM group was 112.5% more than that in the OM group, but nodules did not formed in the control or BSP group. We conclude that BSP is capable of inhibiting hBMSCs proliferation and enhancing their osteogenic differentiation and mineralization in the presence of OM.     

微创踝关节融合术治疗老年创伤性踝关节炎中的临床效果及对患者氧化损伤与骨代谢的影响

目的:研究微创踝关节融合术治疗老年创伤性踝关节炎中的临床效果及对患者氧化损伤与骨代谢的影响。方法:收集2014年3月至2015年3月我院收治的94例老年创伤性踝关节炎患者,按随机数表法分为实验组和对照组,每组各45例。两组患者在手术前均进行常规检查,对照组采用常规开放式踝关节融合术,实验组采用微创踝关节融合术。对比两组治疗后血清氧化损伤指标肌红蛋白(MYO)、缺血修饰白蛋白(IMA)、总抗氧化能力(TAC)、丙二醛(MDA)水平,骨代谢指标碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、甲状旁腺素(PTH)、骨钙素(BGP)、降钙素(CT)水平,视觉疼痛模拟评分(VAS)、美国矫形外科足踝协会(AOFAS)评分及不良反应的发生情况。结果:治疗后,实验组血清MYO、IMA、MDA水平显著低于对照组[(20.48±2.59)ng/mL vs.(27.07±2.97)ng/m L,(65.68±8.20)U/L vs.(74.27±9.01)U/L,(5.01±1.03)nmol/L vs.(9.64±2.17)nmol/L](P0.05),血清TAC水平显著高于对照组[(11.40±2.50)kU/L vs.(7.36±1.03)kU/L](P0.05);血清ALP、BGP、CT水平均显著高于对照组[(103.28±12.47)U/L vs.(90.53±10.02)U/L,(11.08±1.42)ng/L vs.(8.01±1.23)ng/L,(61.39±5.87)ng/L vs.(50.28±4.92)ng/L](P0.05),ACP、PTH水平均显著低于对照组[(5.21±0.60)U/L vs.(8.03±0.92)U/L,(42.95±5.38)ng/L vs.(60.49±6.92)ng/L](P0.05);VAS评分显著低于对照组[(1.06±0.23)分vs.(3.79±0.67)分](P0.05),AOFAS评分显著高于对照组[(73.02±6.28)分vs.(65.58±5.13)分](P0.05);不良反应总发生率显著低于对照组[6.66%(3/45) vs. 20.41%(10/49)](P0.05)。结论:微创踝关节融合术可调节老年创伤性踝关节炎患者的骨代谢,增强骨密度,减少术后不良反应,有利于改善患者预后。