人骨形态发生蛋白-2完整成熟肽基因的克隆

目的：克隆人骨形态发生蛋白－２（ｈＢＭＰ－２）完整肽基因。方法：依据Ｇｅｎｂａｎｋ中ｈＢＭＰ－２的序 化学合成两条引物,从人胎儿骨组织中提取得到的总ＲＮＡ中,通过反转录聚合酶链式反应（ＲＴ－ＰＣＲ）得到ｈＢＭＰ－２完整成熟肽基因。将所得的基因片段插入克隆载体ｐＵＣ－１９并转化大肠杆菌ＪＭ１０９,提取重组质粒,酶切并测序。结果：ＤＮＡ琼脂糖凝胶电泳显示：ＰＣＲ产物为一长约４００ｂｐ的带,阳性克隆质粒     

JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells

Bone morphogenetic protein-2 (BMP-2) is a multi-functional growth factor belonging to the transforming growth factor β superfamily that has a broad range of activities that affect many different cell types. BMP-2 induces odontoblastic differentiation of human dental pulp cells (DPCs), but the underlying mechanism remains unclear. In this study, we investigated the potential role of the JNK mitogen-activated protein kinases (MAPK) pathway in BMP-2-induced odontoblastic differentiation of DPCs. The levels of phosphorylated and unphosphorylated JNK MAPK were quantified by Western blot analysis following treatment with BMP-2 and the JNK inhibitor SP600125. The role of JNK MAPK in the BMP-2-induced odontoblastic differentiation of DPCs was determined by measuring alkaline phosphatase (ALP) activity and by examining the expression of odontoblastic markers using quantitative real-time polymerase chain reaction analysis. The effect of JNK MAPK silencing on odontoblastic differentiation was also investigated. BMP-2 upregulated the phosphorylation of JNK in DPCs in a dose- and time-dependent manner. Early markers of odontoblastic differentiation, including ALP activity, osteopontin and dentin matrix protein-1, were not inhibited by the JNK inhibitor. However, the JNK inhibitor, SP600125, significantly inhibited late-stage differentiation of odontoblasts, including the gene expression of osteocalcin, dentin sialophosphoprotein and bone sialoprotein, and also reduced the formation of mineralized nodules in BMP-2-treated DPCs. Consistent with this observation, silencing of JNK MAPK also decreased late-stage odontoblastic differentiation. Taken together, these findings suggest that JNK activity is required for late-stage odontoblastic differentiation induced by BMP-2.     

激活素A和骨形成蛋白-4诱导人羊膜上皮细胞转分化为心肌样细胞的实验研究

近年来羊膜上皮细胞(AECs)被认为是心肌细胞移植较好的种子细胞之一。研究表明,激活素A(ActivinA)和骨形成蛋白-4(BMP-4)能够诱导人胚胎干细胞向心肌细胞分化。本研究通过体外分离、培养并鉴定人AEC后,采用Activin A和BMP-4作为诱导剂,观察它们能否诱导AEC向心肌样细胞转分化。应用定量RT-PCR、Western blot、免疫细胞化学等方法来检测基因及蛋白表达变化。结果显示,人AEC能够表达上皮细胞特异性蛋白角蛋白19(CK19)。经Activin A和BMP-4共同诱导培养14d后,AEC心肌特异性转录因子Nkx2.5和特异性结构蛋白α-actinin的mRNA表达水平明显升高,同时α-actinin的蛋白表达水平亦明显增加。本实验提示,ActivinA和BMP-4在体外能够诱导人AEC向心肌样细胞转分化,同时该诱导方法可能成为诱导AEC向心肌样细胞转分化的一个新方法。     

Exogenously added BMP-6, BMP-7 and VEGF may not enhance the osteogenic differentiation of human adipose stem cells

Abstract

In the present study bone morphogenetic protein (BMP)-6 alone or in synergy with BMP-7 and vascular endothelial growth factor (VEGF) were tested with human adipose stem cells (hASCs) seeded on cell culture plastic or 3D bioactive glass. Osteogenic medium (OM) was used as a positive control for osteogenic differentiation. The same growth factor groups were also tested combined with OM. None of the growth factor treatments could enhance the osteogenic differentiation of hASCs in 3D- or 2D-culture compared to control or OM. In 3D-culture OM promoted significantly total collagen production, whereas in 2D-culture OM induced high total ALP activity and mineralization compared to control and growth factors groups, but also high cell proliferation. In this study, hASCs did not respond to exogenously added growth although various parameters of the study set-up may have affected these findings contradictory to the previous literature.     

过表达BMP9对人肺鳞状细胞癌细胞NCI-H520迁移和侵袭的影响

目的:探讨骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)对人肺鳞状细胞癌细胞NCIH520迁移和侵袭的影响及其机制。方法:采用RT-PCR和Western blot法分别检测NCI-H520细胞和正常人支气管上皮(HBE)细胞中BMP9的mRNA和蛋白表达水平;用Ad BMP9感染NCI-H520细胞,RT-PCR和Western blot法验证BMP9的变化;应用划痕愈合实验检测各组细胞的迁移能力,Transwell小室检测迁移和侵袭能力,并用RTPCR和Western blot法检测迁移相关因子基质金属蛋白酶2(MMP2)的mRNA和蛋白水平。Western blot法检测BMP-Smad经典通路中Smad1/5的磷酸化水平(p-Smad1/5)。用BMP特异性拮抗剂Ad Noggin与Ad BMP9共处理NCI-H520细胞,检测p-Smad1/5及细胞迁移能力的变化。结果:BMP9在NCI-H520细胞中的mRNA和蛋白水平均比在HBE细胞中低;Ad BMP9感染的NCI-H520细胞中BMP9的mRNA和蛋白水平均明显升高,细胞迁移和侵袭能力均明显下降,MMP2的mRNA和蛋白水平下降,且p-Smad1/5的表达水平明显上调。在NCI-H520细胞中,Noggin能有效逆转BMP9导致的p-Smad1/5升高和细胞迁移抑制能力。结论:外源性BMP9转入肺鳞癌细胞NCI-H520可明显抑制其迁移侵袭的能力,该抑制作用可能与BMP-Smad信号通路的激活有关。     

Biomimetic modification of the TiO(2)/glass composite Ecopore with heparinized collagen and the osteoinductive factor BMP-2

The porous TiO(2)/glass composite Ecopore has potential applications in hard tissue replacement. We describe the modification of Ecopore with the growth factor bone morphogenetic protein-2 (BMP-2) to add osteoinductive properties. Ecopore covalently coated with BMP-2 caused a weak induction of alkaline phosphatase in murine embryonal fibroblasts. In a rabbit bone defect model, BMP-2-coated Ecopore had moderately higher bone apposition rates and ingrown bone quantities at 6 weeks after implantation. To overcome loss of function due to chemical surface coupling, we filled the pore system of Ecopore with heparinized collagen sponge and loaded this secondary matrix with BMP-2. Heparinization of collagen filling increased the BMP-2 loading capacity of the matrix approximately 1.28-fold. Within 96 h, 17.0+/-0.1 and 10.1+/-0.2% of the used BMP-2 was released from non-modified and heparinized Ecopore/collagen, respectively, indicating that the heparin modification retarded BMP-2 release. Revealed by energy-dispersive X-ray spectroscopy analysis of implant cross-sectional areas, BMP-2-loaded Ecopore/collagen had significantly higher bony ingrowth quantities in rabbits, with the heparinized modification yielding the highest value (16.09+/-3.51%, p<0.005) compared with the non-heparinized matrix (10.72+/-4.07%, p<0.05) and the BMP-2-free controls (5.60+/-1.47%). This suggested a beneficial effect of the biomimetic modification of Ecopore with heparinized collagen for bone healing and integration.     

骨形态形成蛋白-7及其受体在单侧输尿管梗阻大鼠模型中表达的研究

目的：探讨单侧输尿管梗阻（UUO）大鼠模 型不同时点肾脏中骨形态形成蛋白-7（BMP-7）及其受体BMPR-Ⅱ、ALK-2、ALK-3、ALK-6 mR NA表达水平的动态变化及意义。方法:60只Wistar大鼠随机分为正常大鼠 组、假手术组和单侧输尿管梗阻组，分别于术后1 d、3 d、7 d、14 d处死，采用RT-PCR方 法检测肾组织BMP-7、BMPR-Ⅱ、ALK-2、ALK-3、ALK-6的mRNA表达水平，免疫组织化学方法 检测肾小管间质中BMP-7的蛋白定位和表达。结果:与对照组相比，输尿 管梗阻组肾组织BMP-7及其受体BMPR-Ⅱ、ALK-2、ALK-3的mRNA表达于术后第1 d即出现下降 并随梗阻时间延长递减，而ALK-6的mRNA表达水平变化不明显。免疫组化结果显示BMP－7蛋 白在对照组中高度表达，主要分布在肾小管及肾间质，肾小球内基本无表达。在梗阻组随梗阻时间延长、肾间质损害加重，BMP－7蛋白表达呈进行性下降。结论:在肾间质损害过程中BMP-7及其受体BMPR-Ⅱ、ALK-2、ALK-3于病变早期即出现表达下调，早于肾间质纤维化病理改变的出现，BMP-7及其受体表达下调可能参与介导肾小管间质纤维化的发生发展。     

Heterodimeric BMP-2/7 exhibits different osteoinductive effects in human and murine cells

As robust osteoinductive cytokines, bone morphogenetic proteins (BMPs) play a significant role in bone tissue engineering. Constituted of two different polypeptides, heterodimeric BMPs are more effective than the homodimers in bone formation. While most studies focused on the murine cell lines, such as murine preosteoblasts MC3T3-E1, the role of heterodimeric BMPs in the osteogenic differentiation of human cells remains uncertain, which hinders their application to practical treatment. In this study, we compared the osteoinductive effects of BMP-2/7 heterodimer in human adipose-derived stem cells (hASCs) with their homodimers BMP-2 and BMP-7, in which MC3T3-E1 cells were utilized as a positive control. The results indicated that BMP-2/7 was not a stronger inducer during the osteogenic differentiation of hASCs as that for MC3T3-E1, and extracellular-signal-regulated kinase signaling played a role in the different effects of BMP-2/7 between hASCs and MC3T3-E1. Our study demonstrates the osteoinductive effects of heterodimeric BMP-2/7 present in a cell-specific pattern and cautions should be taken when applying heterodimeric BMP-2/7 to clinical practice.     

MicroRNA在粒细胞增殖和分化中的调控作用

微小RNA（microRNA,miRNA）是高度保守的非蛋白编码的RNA,可调节基因的表达。研究表明骨髓原始及幼稚造血细胞的增殖与分化异常是很多血液系统疾病的骨髓学特点;而miRNA参与骨髓造血调控,并且与血液系统疾病的发生存在密切的关系。本文着重综述miRNA对粒细胞增殖和分化的调控。     

Spatial patterning of BMP-2 and BMP-7 on biopolymeric films and the guidance of muscle cell fate

In the cellular microenvironment, growth factor gradients are crucial in dictating cell fate. Towards developing materials that capture the native microenvironment we engineered biomimetic films that present gradients of matrix-bound bone morphogenetic proteins (BMP-2 and BMP-7). To this end layer-by-layer films composed of poly(l-lysine) and hyaluronan were combined in a simple microfluidic device enabling spatially controlled growth factor diffusion along the film. Linear long-range gradients of both BMPs induced the trans-differentiation of C2C12 myoblasts towards the osteogenic lineage in a dose dependent manner with a different signature for each BMP. The osteogenic marker alkaline phosphatase (ALP) increased in a linear manner for BMP-7 and non-linearly for BMP-2. Moreover, an increased expression of the myogenic marker troponin T was observed with decreasing matrix-bound BMP concentration, providing a substrate that it is both osteo- and myo-inductive. Lastly, dual parallel matrix-bound gradients of BMP-2 and -7 revealed a complete saturation of the ALP signal. This suggested an additive or synergistic effect of the two BMPs. This simple technology allows for determining quickly and efficiently the optimal concentration of matrix-bound growth factors, as well as for investigating the presentation of multiple growth factors in their solid-phase and in a spatially controlled manner.     

人骨形态发生蛋白2和9对人胃癌MNK-45细胞增殖、凋亡和迁移的影响及其机制

 目的：研究人骨形态发生蛋白2和9（BMP2和BMP9）对人胃癌MNK-45细胞增殖、凋亡和迁移的影响,并初步探讨其作用机制。方法：用重组腺病毒AdBMP2和AdBMP9感染MNK-45细胞,采用MTT法、Hoechst 33258染色、流式细胞术(FCM)、 划痕愈合实验和Transwell小室检测BMP2和BMP9 对MNK-45细胞增殖、凋亡和迁移能力的影响, Western blotting检测总GSK-3β、磷酸化GSK-3β（p-GSK-3β）及β-catenin的表达水平。结果：(1）MTT显示AdBMP2和AdBMP9转染后,MNK-45细胞的增殖自第3 d开始被抑制并呈时间依赖性;(2）Hoechst 33258染色与FCM一致显示上调BMP2和BMP9表达可以促进MNK-45细胞的凋亡;(3） 划痕愈合与Transwell迁移实验结果一致提示上调BMP2和BMP9表达均可抑制MNK-45细胞的迁移;(4）Western blotting显示BMP2组和BMP9组的p-GSK-3β均较GFP组升高,但对β-catenin的表达量无明显影响（P>0.05）;上述结果与对照组相比差异均有统计学意义（P<0.05或P<0.01）。结论：上调BMP2和BMP9表达对MNK-45细胞的增殖具有抑制作用,其机制与β-catenin无明显关系。     

急性心肌缺氧对乳鼠心肌细胞脑钠尿肽表达的影响及其作用机制

目的: 观察急性缺氧损伤对乳鼠心肌细胞脑钠尿肽(BNP)表达水平的影响并探讨其可能作用机制。方法: 原代乳鼠心肌细胞缺氧、无糖、无血清培养以模拟心肌缺血损伤,利用CCK-8法测细胞存活率、ELISA法测白细胞介素-6(IL-6)和BNP的表达;以IL-6直接干预体外培养的心肌细胞, 采用RT-PCR、Western blotting和ELISA方法观察BNP、转化生长因子β₁(TGF-β₁)、Smad2 mRNA的转录和蛋白的表达。结果: 缺氧显著上调IL-6和BNP的表达,且两者呈线性正相关;IL-6直接干预可剂量和时间依赖性地上调心肌细胞BNP的mRNA和蛋白表达水平,同时TGF-β₁和Smad2的表达水平亦增加;而针对TGF-β₁的中和抗体能够部分地抑制由IL-6引起的BNP表达增加。结论: 急性心肌缺氧可直接上调BNP的表达水平,这种调节效应与TGF-β₁/Smad2信号通路上调有关。     

骨髓间充质干细胞成脂成骨平衡调控研究进展

骨质疏松症（OP）是一种全身性的骨代谢疾病,主要表现为骨量减少、骨质脆性增加、骨骼微结构退化。随着对骨质疏松症发病机制的深入研究,骨髓间充质干细胞（BMSCs）分化失衡（成骨分化减少,成脂分化增多）是引发骨质疏松症的关键。我们总结了BMSCs成脂成骨分化相关的信号通路,充分理解这些信号通路,有利于更好的阐明骨质疏松症的发病机制,为临床医生有效地治疗骨质疏松症提供帮助。     

Mutation of a cleavage site adjacent to the mature domain leads to increase in secreted mature BMP-2 with reduced activity

Proteolytic cleavage of precursor bone morphogenetic protein (proBMP) is an important step in generating the active mature BMP. ProBMP-2 contains two proprotein convertase (PC) recognition sites (S1 and S2) and is postulated to be cleaved by PCs at those sites. Cell lines expressing proBMP-2, with a silenced S1 site (mS1) that inhibited PC cleavage, secreted the 20-kDa form BMP-2, while cells expressing wild type (wt) BMP-2 secreted 18- and 20-kDa mature BMP-2 N-terminal isoforms. The mS1 cells secreted 15-fold more mature BMP-2 than the wt, despite their similar mRNA levels. Mutant-secreted BMP-2 demonstrated biological activity in vitro; however, its activity was reduced compared with wt. These data demonstrate that proBMP-2 can be cleaved at an alternative cleavage site without prior S1 site cleavage in cell lines overexpressing BMP-2 and more importantly suggest that the presence of the 2-kDa linker peptide can affect activity and secretion of the mature protein.     

油酸诱导SW872前脂肪细胞分化及分化过程中促酰化蛋白的分泌

目的探讨油酸对SW872前脂肪细胞的诱导分化作用及SW872细胞分化过程中促酰化蛋白的分泌。方法体外培养SW872细胞,0.6mmol/L油酸作为诱导分化剂刺激SW872前脂肪细胞,油红O染色观察细胞形态的变化及脂滴的聚集,化学比色法测定甘油三酯的总量,ELISA方法测定培养上清中促酰化蛋白的含量。结果SW872细胞分化之前呈成纤维细胞样,胞内无脂滴,甘油三酯的含量较低;加入0.6mmol/L油酸诱导分化刺激72h时,SW872细胞已经完全分化,胞浆中脂滴明显增多,油红O染色可见胞浆中丰富的脂肪染色,甘油三酯的含量是诱导分化前的14倍(P<0.01)。诱导分化之前,SW872细胞分泌ASP的量非常低,几乎不能检测出;诱导分化72h,ASP的含量增加到1.12ng/mg细胞总蛋白质。结论油酸能诱导SW872前脂肪细胞分化为成熟的脂肪细胞;ASP的分泌与脂肪细胞的分化程度密切相关。     

Effect of nano-structured bioceramic surface on osteogenic differentiation of adipose derived stem cells

Tissue engineering strategies to construct vascularized bone grafts potentially revolutionize the treatment of massive bone loss. The surface topography of the grafts plays critical roles on bone regeneration, while adipose derived stem cells (ASCs) are known for their capability to promote osteogenesis and angiogenesis when applied to bone defects. In the present study, the effects of hydroxyapatite (HAp) bioceramic scaffolds with nanosheet, nanorod, and micro-nano-hybrid (the hybrid of nanorod and microrod) surface topographies on attachment, proliferation and osteogenic differentiation, as well as the expression of angiogenic factors of rat ASCs were systematically investigated. The results showed that the HAp bioceramic scaffolds with the micro-/nano-topography surfaces significantly enhanced cell attachment and viability, alkaline phosphatase (ALP) activity, and mRNA expression levels of osteogenic markers and angiogenic factors of ASCs. More importantly, the biomimetic feature of the hierarchical micro-nano-hybrid surface topography showed the highest stimulatory effect. The activation in Akt signaling pathway was observed in ASCs cultured on HAp bioceramics with nanorod, and micro-nano-hybrid surface topographies. Moreover, these induction effects could be repressed by Akt signaling pathway inhibitor LY294002. Finally, the in vivo bone regeneration results of rat critical-sized calvarial defect models confirmed that the combination of the micro-nano-hybrid surface and ASCs could significantly enhance both osteogenesis and angiogenesis as compared with the control HAp bioceramic scaffold with traditional smooth surface. Our results suggest that HAp bioceramic scaffolds with micro-nano-hybrid surface can act as cell carrier for ASCs, and consequently combine with ASCs to construct vascularized tissue-engineered bone.     

慢病毒介导的降钙素基因相关肽转染对大鼠骨髓间充质干细胞内皮分化的影响

 目的：研究慢病毒介导的降钙素基因相关肽（calcitonin gene-related peptide,CGRP）基因转染对大鼠骨髓间充质干细胞（mesenchymal stem cells,MSCs）内皮分化功能的影响。方法：用密度梯度离心结合贴壁法培养大鼠骨髓MSCs,携带CGRP的慢病毒载体转染MSCs,未转染MSCs作为对照,应用ELISA法检测CGRP蛋白表达;实验分为转染Lenti-CGRP的MSCs（CGRP）组、转染Lenti-CGRP的MSCs+CGRP受体拮抗剂（CGRP8-37）组（CGRP +CGRP8-37）及未经转染的对照组。应用免疫细胞化学检测细胞CD31和Ⅷ因子相关抗原的表达以鉴定其分化能力;细胞计数法观察转染CGRP后MSCs增殖的影响;Matrigel实验观察转染CGRP对MSCs形成管腔样结构能力的影响。结果：MSCs转染CGRP基因经诱导分化后的CD31和Ⅷ因子相关抗原阳性细胞数量增多,与对照组相比有显著差异(P<0.05);转染CGRP组细胞数量增长较快,与对照组相比差异有统计学意义(P<0.05);Matrigel实验显示转染CGRP组细胞微血管新生明显,与对照组相比差异有统计学意义(P<0.05)。而CGRP+CGRP8-37组上述作用明显被抑制。结论: 转染CGRP基因能促进MSCs向内皮分化,并能促进内皮细胞增殖,在一定的条件下,可促进微血管新生。     

Expression of growth differentiation factor 9 (GDF9) and its receptor in adult cat testis

Oocyte-secreted growth differentiation factor (GDF) 9 plays an essential role during follicle maturation through actions on granulosa cells. Despite its critical role in female reproduction, GDF9 expression, signalling and function are less well characterized during spermatogenesis. The purpose of this study was to investigate temporal and spatial expression and potential cellular targets of GDF9 in the adult cat testis. Our result confirmed that GDF9 is stage-specifically localized in the cytoplasm of round spermatids and pachytene spermatocytes of the cat seminiferous epithelium. In particular, activin receptor-like kinase (ALK) 5, the type I receptor of GDF9, is principally localized in the cytoplasm of round spermatids. Smad2/3, signal transducers for GDF9 signalling pathway, is mainly immunolocalized in the cytoplasm of germ cells, Sertoli cells and Leydig cells, but the expression in germ cells are weaker than in Sertoli cells. The expression pattern of ALK5 and Smad2/3 show that GDF9-ALK5-Smad2/3 may not be the only signalling pathway for testicular cell to respond to GDF9. Overall, our results demonstrate that GDF9 is a germ cell-specific factor in the adult cat testis, and that GDF9 regulates the tight junctions of Sertoli cells by paracrine secretion, and regulates the germ cells by autocrine secretion.     

持续张应力对骨髓基质干细胞增殖及骨向分化的影响

目的通过体外加力装置研究持续牵张应力对大鼠骨髓基质干细胞(bone marrow stromal cells,BMSCs)增殖及骨向分化能力的影响。方法选用3月龄健康SD雌性大鼠,采用全血贴壁培养法分离及培养BMSCs。取生长良好的第3～5代细胞接种于Flexercell应力加载系统(10%、1 Hz),根据应力作用时间不同分为1、6、12、24 h组和48 h组。观察并分析持续牵张力对于大鼠BMSCs形态、增殖活性以及成骨能力变化的影响。结果 (1)随着加力时间的延长,与对照组相比,实验组细胞形态呈现一定规律性,细胞长轴多垂直于受力径向。(2)10%持续张应力作用可抑制BMSCs增殖活性。(3)持续张应力可增高碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(collagenI,COLⅠ)、核心结合因子Cbfa1(core binding factor a1,又名Runx2)mRNA的表达量,且呈现时间依赖性。其中实验组ALP表达量在24 h明显高于相应对照组,COLⅠ表达量在24 h及48 h均明显高于对照组,Runx2表达量在6 h与对照组相比显著增高(P<0.05)。骨钙素(osteocalcin,OC)含量在加力起始阶段显著高于对照组,随时间推移逐渐下降,48 h时明显低于对照组(P<0.05)。(4)持续张力可以促使Runx2蛋白水平增高,且在6 h实验组明显高于对照组(P<0.05)。之后缓慢下降,在24 h时显著低于对照组水平(P<0.05)。结论持续牵张力作用下BM-SCs细胞形态呈现一定规律性排列,其增殖活性受到抑制,但早期成骨向分化能力却显著提高。