仙灵骨葆胶囊对骨质疏松症大鼠BMP-2、BMP-4、TGF-β1和PKC蛋白表达影响

目的探讨仙灵骨葆胶囊对骨质疏松症大鼠骨形成蛋白-2(BMP-2)、骨形成蛋白-4(BMP-4)、转化生长因子β1(TGF-β1)和蛋白激酶C(PKC)蛋白表达影响。方法 40只SD大鼠随机分为假手术组、模型组、对照组和实验组,每组大鼠10只。假手术组给予等剂量生理盐水灌胃;模型组:给予等剂量生理盐水灌胃;对照组:给予盖天力牡蛎钙0.3 g/kg灌胃;实验组:给予仙灵骨葆胶囊0.4 g/kg灌胃。各组大鼠灌胃均为每日1次,连续灌胃8周。采用双能X线骨密度仪检测股骨和腰椎骨密度;采用ELISA法检测血清Ca和P含量;采用免疫组化法检测骨组织BMP-2、BMP-4、TGF-β1和PKC蛋白表达。结果除假手术组,其余各组大鼠均造模成功。模型组、对照组、实验组股骨和腰椎骨密度低于假手术组(P0.05);对照组、实验组股骨和腰椎骨密度高于模型组(P0.05);实验组股骨和腰椎骨密度高于对照组(P0.05)。模型组、对照组、实验组血清Ca和P含量低于假手术组(P0.05);对照组、实验组血清Ca和P含量高于模型组(P0.05);实验组血清Ca和P含量高于对照组(P0.05)。模型组、对照组、实验组BMP-2、BMP-4和PKC蛋白表达高于假手术组,而TGF-β1蛋白表达低于假手术组(P0.05);对照组、实验组BMP-2、BMP-4和PKC蛋白表达低于模型组,而TGF-β1蛋白表达高于假手术组(P0.05);实验组BMP-2、BMP-4和PKC蛋白表达低于对照组,而TGF-β1蛋白表达高于对照组(P0.05)。结论仙灵骨葆胶囊可增加骨质疏松症大鼠骨密度,认为可能与下调BMP-2、BMP-4和PKC蛋白表达及上调TGF-β1蛋白表达相关。     

前列腺癌组织中BMP-4的表达及临床意义

目的探讨骨形态发生蛋白-4(bone morphogenetic protein-4,BMP-4)在前列腺癌组织中的表达,分析其表达下调对前列腺癌细胞增殖和侵袭的影响。方法采用免疫组化En Vision法检测58例前列腺癌及40例前列腺良性增生(benign prostatic hyperplasia,BPH)组织中BMP-4蛋白的表达;利用Western blot法检测前列腺癌细胞(LNcap、PC-3、Du145)和BPH细胞中BMP-4蛋白的表达;将BMP-4 siRNA和对照siRNA分别转染LNcap,分为未处理组、对照siRNA组和BMP-4 siRNA组,采用Western blot法检测三组LNcap细胞中BMP-4、MMP-2和MMP-9的表达量;利用CCK8分别检测三组细胞的增殖能力;采用Tanswell小室实验检测三组细胞的侵袭能力。结果前列腺癌组织中BMP-4蛋白阳性率高于BPH组织(P 0. 05),BMP-4蛋白在前列腺癌细胞中的表达量高于BPH细胞。BMP-4 siRNA组细胞中BMP-4蛋白表达低于未处理组和对照siRNA组; BMP-4 siRNA组细胞增殖能力及细胞侵袭能力均低于未处理组和对照siRNA组。BMP-4 siRNA组细胞中MMP-2和MMP-9蛋白表达量低于未处理组和对照siRNA组。结论 BMP-4高表达于前列腺癌组织中,与前列腺癌细胞增殖和侵袭相关,有望成为前列腺癌潜在的治疗靶点。     

胃癌患者血清BMP-4、HGF的表达及其与淋巴结转移的关系研究

目的:探讨血清骨形成蛋白-4(BMP-4)、肝细胞生长因子(HGF)水平与胃癌患者淋巴结转移的相关性.方法:选择本院收治的80例胃癌患者为研究对象.患者入院时均接受血清BMP-4、HGF水平检测,并择期进行手术治疗,以术后病理检查为依据判断患者淋巴结转移情况并分组,询问并记录患者基线资料,分析血清BMP-4、HGF水平与胃癌患者淋巴结转移的关系.结果:病理检查结果显示,51例发生淋巴结转移(63.75％)、29例未发生转移(36.25％).转移组患者入院时血清BMP-4、HGF水平分别为(12.68±1.35)ng·mL-1、(1184.68±90.06)pg·mL-1高于未转移组(10.14±1.01)ng·mL-1、(986.40±75.38)pg·mL-1.低、中分化患者占比90.20％(46/51)高于未转移组51.72％(15/29)(P<0.05);经Logistic回归显示,血清BMP-4、HGF过表达与胃癌患者淋巴结转移密切相关.结论:血清BMP-4、HGF过表达与胃癌患者淋巴结转移密切相关.     

特发性矮小症患儿rhGH治疗前后血清BMP-2、TAZ水平变化及其预后价值

目的 探讨特发性矮小症(ISS)患儿rhGH治疗前后骨形态发生蛋白-2(BMP-2)、转录联合激活因子(TAZ)水平变化及其预后价值。方法 选取2019年3月至2020年3月本院儿科收治的儿童120例，收集整理其基本临床资料。以治疗前血清BMP-2、TAZ水平均值设置为基础值，接受rhGH治疗1个月(30d)后血清BMP-2、TAZ水平升高至基础值30%以上为敏感组(n=81),其他设置为非敏感组(n=39)。ELISA法测试患儿血清BMP-2、TAZ水平，并检测计算患儿生长发育指标、骨代谢指标、甲状腺指标、空腹血糖及糖化血红蛋白等指标。结果 敏感组rhGH治疗1个月、12个月、24个月后BMP-2、TAZ、GV、PAH、BAP、PINP水平较治疗前显著提高，且随时间变化显著增加(P<0.05);β-CTX水平较治疗前显著降低，且随时间变化显著降低(P<0.05)。敏感组与不敏感组治疗前后TSH、FT₃、FT₄、空腹血糖、糖化血红蛋白均无显著变化(P<0.05)。治疗1个月、12个月、24个月后敏感组血清BMP-2、TAZ水...     

雌激素对骨质疏松性骨折愈合过程BMP-4基因表达的影响

目的：研究雌激素对骨质疏松性骨折愈合骨形态发生蛋白-4（bone morphogenetic protein-4,BMP-4）基因定位分布的影响。 方法： 选用健康SD大鼠，随机分为卵巢切除（OVX）组、假手术对照（sham）组和雌激素替代治疗组（OVXE）。制成胫骨骨折愈合模型并分别于骨折后不同时点处死大鼠取材。通过光镜、电镜观察形态学和超微结构变化；采用原位杂交方法观察雌激素对BMP-4基因表达影响。 结果： (1)OVX组在骨痂形态及成骨细胞和破骨细胞活性与OVXE组比较有明显差异，较sham组差异更大。（2）骨折后1-3 d BMP-4 mRNA表达强度:sham组相似文献   

BMP-2对不同血清浓度下OPG-/-小鼠骨髓间充质干细胞增殖和分化的作用

研究骨形成蛋白-2(Bone Morphogenetic Protein 2,BMP-2)对在不同血清浓度条件下OPG-/-小鼠骨髓间充质干细胞(Bone Marrow Derived Stroma Cells,BMSCs)的增殖和分化作用。取OPG-/-小鼠股骨,分离骨髓基质细胞进行体外培养。在各自培养条件下分为实验组(BMP-2组)和空白对照组(CON组),倒置相差显微镜下观察细胞形态变化,MTT法测定细胞增殖率,PNPP法测定碱性磷酸酶(AlkalinePhosphatase,ALP)的表达。BMP-2组和CON组在含有10%、15%和20%FBS的培养条件下,第3、5和7d的增殖较无FBS和5%FBS条件下明显(P<0.05),而15%和20%FBS条件下增殖又较10%FBS显著(P<0.05);BMP-2组对ALP表达的影响与CON存在较大差异,BMP-2组在所有不同血清浓度条件下的第3、5和7 dALP活性均高于CON组(P<0.05)。BMP-2组对OPG-/-小鼠BMSCs无增殖作用,较高血清浓度是其增殖的主要条件,但在ALP的分化活性方面作用显著。     

腺病毒载体介导的BMP-2转基因诱导成骨研究进展

骨形态发生蛋白2可诱导具有成骨分化潜能的间充质细胞向成骨细胞分化.腺病毒载体介导的BMP-2转基因方法被认为是最有效的BMP-2转基因诱导成骨手段.本文参考了大量的有关英文文献,从BMP-2腺病毒载体及其介导的BMP-2转基因基本途径、成骨诱导研究所取得的成果和存在的问题等几个方面,重点回顾了近五年来的研究进展,并对这一领域的前景进行了一些展望.     

TSP-1与BMP-7在慢性鼻-鼻窦炎鼻黏膜组织中的表达及意义

目的观察血小板反应蛋白-1(TSP-1)、骨形态发生蛋白-7(BMP-7)在慢性鼻-鼻窦炎(CRS)患者鼻黏膜组织中的表达变化,探讨其在CRS发病机制中的意义。方法采用酶联免疫吸附方法检测TSP-1、BMP-7及转化生长因子β1在鼻黏膜组织中的含量变化。结果随着CRS不伴鼻息肉型患者分期的增高,TSP-1和TGF-β1水平逐渐升高(P0.05),BMP-7水平逐渐下降(P0.05)。随着CRS伴鼻息肉型患者分期的增高,TSP-1和TGF-β1水平均明显下降(P0.05),BMP-7水平均明显升高(P0.05)。结论 TSP-1与BMP-7在CRS中表达异常,可能在CRS发病机制中发挥作用。     

DM2患者血清CRP、APN及血浆BMP-2水平变化的临床意义

糖尿病是由多种病因引起的以慢性高血糖为特征的代谢紊乱症候群,其主要的病理基础是体内胰岛素绝对或相对缺乏以及靶组织对胰岛素不敏感[1].而本病的主要风险在于长期的慢性高血糖可导致的心、脑血管疾病、慢性难愈性感染、甚至引起神经系统的慢性病变.近来的研究表明,其病情的发生及进展与患者的自然免疫、低度炎症等因素密切相关[2].为进一步研究糖尿病及其并发症与相关血清指标变化的关系,本文测定了60例糖尿病患者的血清CRP、脂联素(APN)及骨形态发生蛋白-2(BMP-2)水平,现报道如下.     

BMP-7对高糖环境下肾小管上皮细胞Id2和E2A蛋白表达的影响

 目的: 通过观察高糖环境下给予外源性骨形态发生蛋白-7(BMP-7)对肾小管上皮细胞(NRK-52E)表达分化抑制因子2(Id2)和转录因子E2A的影响,探讨BMP-7减轻高糖诱导的肾小管纤维化病变的可能机制。方法: 将体外培养的肾小管上皮细胞NRK-52E分为3组:对照(control)组、高糖(HG)组和不同浓度BMP-7(10 μg/L和20 μg/L)干预组,HG组和干预组分别设12 h、24 h和48 h 3个时点。Western blot方法检测Id2、E2A、上皮细胞钙粘蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(Col-Ⅰ)蛋白的表达,real-time PCR方法检测Id2 mRNA的表达。结果: 与control组相比,HG组Id2的mRNA和蛋白水平及E-cadherin的蛋白水平明显下调,E2A、α-SMA和Col-Ⅰ的蛋白水平明显上调(P < 0.05);与HG组相比,20 μg/L BMP-7干预组Id2的mRNA和蛋白水平及E-cadherin的蛋白水平均较同时点显著上调,E2A、α-SMA和Col-Ⅰ的蛋白水平显著下调(P < 0.05)。相关性分析结果显示,Id2蛋白与E2A蛋白表达呈显著负相关(P < 0.05)。结论: BMP-7可阻断高糖诱导的肾小管上皮细胞的纤维化,其机制可能与促进Id2蛋白并抑制E2A蛋白的表达有关。     

前列腺癌骨转移灶中BMP-2、BMP-4、BMP-7的表达及临床意义

目的检测BMP-2、BMP-4、BMP-7在前列腺癌骨转移灶中的表达,探讨其在前列腺癌成骨性转移中的作用。方法采用免疫组化EnVision法检测28例前列腺癌骨转移病例及17例良性前列腺增生(benign prostate hyperplasia,BPH)病例中BMP-2、BMP-4、BMP-7的表达并对其进行对比分析。结果 BMP-2在所有前列腺癌骨转移灶及BPH病例中均表达,二者中其阳性率及表达强度无明显差异(P>0.05)。BMP-4在前列腺癌骨转移灶及BPH中的阳性率无明显差异(P>0.05),但在前者中BMP-4的表达强度明显高于后者(P<0.05)。BMP-7在前列腺癌骨转移灶中的阳性率及表达强度均明显高于BPH(P<0.05)。在BPH的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核同时阳性的表达率分别为13.3%、7.1%和11.1%,在前列腺癌骨转移灶的阳性表达病例中,BMP-2、BMP-4、BMP-7细胞质与细胞核的同时阳性的表达率均为100%,且细胞核的表达强度明显高于细胞质。结论 BMP-4、BMP-7在前列腺癌骨转移灶中高表达,提示其在前列腺癌的成骨性转移中可能起重要作用。     

骨形态发生蛋白-7及其受体的信号转导

骨形态发生蛋白-7(BMP-7)是BMP家族中的一员,具有高效的骨诱导活性,属于转化生长因子-β(TGF-β)超家族成员。BMP-7受体属于转化生长因子β(TGF-β)受体超家族的成员,是一种膜蛋白受体。BMP-7首先与Ⅱ型跨膜丝/苏氨酸激酶受体(ActRⅡ、ⅡB)结合,受体的蛋白激酶活性被激活,再与Ⅰ型受体(主要是ALK2)结合,形成异源二聚体,催化Ⅰ型受体GS区的丝氨酸和苏氨酸残基磷酸化。Ⅰ型受体被激活后,作用于Smad1、Smad5或Smad8的C端SSXS模体,使其磷酸化,再与Smad4形成复合物,进入核内与多种转录因子相互作用发挥基因调控作用。     

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.     

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4–PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.     

Heat treatment of BMP-2 depots on implant materials generates an immobilized layer of BMP-2 with pronounced bioactivity

Bone cells seeded directly on depots of bone morphogenetic protein-2 (BMP-2) increase alkaline phosphatase (ALP) expression. Heating of such BMP-2 depots to 100 degrees C augmented the intensity of this local ALP induction. To understand this unexpected finding, we investigated the effect of heat treatment on BMP-2 depots more closely. Using a novel bioassay based on ALP-induction of remote cells, we found that the amount of released bioactive BMP-2 from heat-treated depots decays within days and could be described by an exponential function. From this function, we expected that pre-incubation of BMP-2 depots in culture medium for 4 weeks renders them insufficient to induce ALP. However, preincubated, heat-treated depots still induced maximal ALP, unless treated with the selective BMP-2 inhibitor noggin. Furthermore, heat treatment of BMP-2 depots generated a layer of immunoreactive BMP-2 at the surface of the carrier. In contrast, BMP-2 was washed off completely if heat treatment of adsorbed protein was omitted. Results show that heat treatment generates both a soluble pool of BMP-2 and a material-bound layer of BMP-2 in which the protein is protected against degradation. Therefore, heat treatment appears useful to locally immobilize BMP-2 on various implant surfaces.     

不同来源成体干细胞BMP-2基因转染并微囊化后细胞活性及分泌量观察

目的比较不同来源成体干细胞BMP-2基因转染并微囊化后的存活情况,并检测分泌至微囊外的BMP-2蛋白数量。方法从大鼠的骨髓、脂肪和滑膜组织内分离培养不同类型的成体干细胞;骨髓间充质干细胞（BMSCs）、脂肪干细胞（ADSCs）和滑膜干细胞（SMSCs）。利用高压静电装置制备海藻酸钠-聚赖氨酸-海藻酸钠（APA）微囊,在荧光显微镜下观察绿色荧光蛋白（GFP）标记细胞在微囊内的存活情况。利用含有BMP-2基因的重组腺病毒感染不同来源的成体干细胞后微囊化包裹,用酶联免疫吸附法（ELISA）检测分泌到微囊外的BMP-2蛋白量。结果包裹4周后荧光显微镜观察发现,BMSCs、ADSCs和SMSCs可在微囊内长期存活。ELISA检测发现,3种转基因细胞在微囊化后都可以持续分泌BMP-2蛋白,其中BMSCs分泌最多,在第2、3、4周和其他2种细胞相比有明显差异（P〈0．01）。结论3种来源的成体干细胞都可以作为BMP-2基因给药促进骨再生研究的候选细胞,其中以骨髓来源的间充质干细胞为首选。     

Inhibition of endogenous antagonists with an engineered BMP-2 variant increases BMP-2 efficacy in rat femoral defect healing

Bone morphogenetic proteins (BMP) have been used successfully by orthopedic clinicians to augment bone healing. However, these osteoinductive proteins must be applied at high concentrations to induce bone formation. The limited therapeutic efficacy may be due to the local expression of BMP antagonists such as Noggin that neutralize exogenous and endogenous BMPs. If so, inhibiting BMP antagonists may provide an attractive option to augment BMP induced bone formation. The engineered BMP-2 variant L51P is deficient in BMP receptor type I binding, but maintains its affinity for BMP receptor type II and BMP antagonists including Noggin, Chordin and Gremlin. This modification makes L51P a BMP receptor-inactive inhibitor of BMP antagonists. We implanted β-tricalcium phosphate ceramics loaded with BMP-2 and/or L51P into a critical size defect model in the rat femur to investigate whether the inhibition of BMP antagonist with L51P enhances the therapeutic efficacy of exogenous BMP-2. Our study reveals that L51P reduces the demand of exogenous BMP-2 to induce bone healing markedly, without promoting bone formation directly when applied alone.     

Directional BMP-2 for functionalization of titanium surfaces

Efficient immobilization of biomacromolecules on material surfaces is a key to development in areas of regenerative medicine and tissue engineering. However, strong and irreversible immobilization of cytokines on surfaces often diminishes their biological functionality. A destructive hydrophobic interaction between the material surface and the biomolecule may underlie this inactivation. Alternatively, dissociation of the cytokine from the material may be necessary for signal transduction. Here we propose a new method for immobilizing cytokines on material surfaces: a material-binding artificial peptide is used to mediate reversible interaction between the cytokine and the material surface. We created artificial proteins that contained three copies of a Ti-binding motif, and fused them to the N-terminal of BMP-2. The engineered BMP-2 showed reversible binding to Ti surfaces and induced BMP signaling activity. When a hydrophobic protein devoid of the Ti-binding motif was fused to BMP-2, the protein tightly bound to Ti surfaces but showed little BMP activity, confirming the importance of the mode of immobilization.