骨形态发生蛋白-2对人脐带间充质干细胞增殖和分化的影响

目的 观察骨形态发生蛋白-2(BMP-2)对人脐带间充质干细胞(hUCMSCs)增殖和分化的影响.方法 体外培养hUCMSCs,在培养基中加入20 mg/L BMP-2,噻唑蓝(MTT)比色法观察BMP-2对hUCMSCs的增殖效果,流式细胞术检测BMP-2作用后细胞表面STRO-1的表达,逆转录-聚合酶链反应(RT-PCR)测量BMP-2作用后hUCMSCs骨桥蛋白(OPN)、碱性磷酸酶(ALP)、Ⅰ型胶原蛋白(COL1)的mRNA表达变化,碱性磷酸酶染色观察hUCMSCs在BMP-2培养基作用下ALP染色变化,Von kossa染色实验观察BMP-2对hUCMSCs钙结节的形成.结果 细胞在未加BMP-2和加BMP-2培养基中培养1、3、5、7 d,虽然细胞的增殖率上升,但各组间比较差异无统计学意义(P＞0.05),同时发现在2%血清的培养基中培养7 d后,hUCMSCs的增殖促进约10%左右.BMP-2培养7 d后细胞表面STRO-1阳性细胞比例上升明显,由25.1±4.0上升至51.1±6.4,差异有统计学意义(P＜0.01).BMP-2培养条件下COL1 mRNA的表达增强,差异有统计学意义(P＜0.05),OPN mRNA出现表达,ALP mRNA比无BMP-2培养明显增强,差异有统计学意义(P＜0.05).在BMP-2培养条件下ALP染色出现大片细胞阳性染色.在培养28 d,Von kossa染色出现明显的钙结节.结论 BMP-2对hUCMSCs成骨诱导分化作用明显,而增殖作用很弱.     

Synergistic effect of recombinant human bone morphogenic protein-7 and osteogenic differentiation medium on human bone-marrow-derived mesenchymal stem cells in vitro

Purpose  

The purpose of this study was to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) with or without osteogenic differentiation medium (ODM) on osteogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBMSCs) in vitro.     

重组BMP-4对兔骨髓基质干细胞生物学行为的影响

目的应用原核细胞基因工程方法生产出有活性的骨形态发生蛋白-4(bonemorphoge-neticprotein-4,BMP-4),观察其对骨髓基质干细胞生物学行为的影响。方法利用RT-PCR技术,从成熟的人胎盘组织中扩增出长0.34kb编码人BMP-4成熟肽的基因序列,装入表达载体pET-22b( ),转化大肠杆菌BL-21菌株,并诱导目的蛋白表达,SDS-PAGE检测表达蛋白。获得的蛋白制品经小鼠异位成骨测活证实后,诱导培养的兔骨髓基质干细胞,观测细胞形态变化、碱性磷酸酶和骨钙素的含量。结果大肠杆菌中目的蛋白表达量达菌体蛋白的15%,该蛋白可诱导骨髓基质干细胞向成骨细胞分化,形成钙结节,碱性磷酸酶和骨钙素的含量也明显增加。结论大肠杆菌可表达出有活性的BMP-4,该蛋白可诱导骨髓基质干细胞向成骨细胞分化。     

实时荧光定量聚合酶链反应筛选重组人骨形态发生蛋白-2诱导比格犬骨髓基质干细胞成骨分化差异表达的miRNAs

目的 利用实时荧光定量聚合酶链反应(Q-PCR)方法筛选骨形态发生蛋白-2(rhBMP-2)诱导比格犬骨髓基质干细胞(BMSCs)成骨分化的miRNAs,探讨过表达miRNAs对成骨分化的影响.方法 选取4只比格犬分离BMSCs原代,取培养至第3代BMSCs分为2组:对照组(用基础培养基培养)和实验组(在基础培养基中加入rhBMP-2培养7d,诱导细胞成骨分化).采用碱性磷酸酶染色法验证细胞成骨分化情况,采用Q-PCR法筛选成骨分化细胞与对照组细胞差异表达的miRNAs;随机选择其中一种低表达miRNA(miR-125b),采用Lipofectamine2000进行转染,其中实验组1转染miR-125bmimics以过表达miR-125b,其阴性对照实验组2转染mimics-NC,验证过表达miR-125b 7 d后对rhBMP-2诱导的比格犬BMSCs成骨分化的影响. 结果 成功建立比格犬BMSCs培养方法;rhBMP-2诱导比格犬BMSCs成骨分化7d后,进行Q-PCR筛选,获得表达差异相对于对照组达到2倍以上的miRNAs 41个,其中表达上调的有13个,表达下调的有28个;实验组1相对于实验组2,细胞中miR-125b表达上调4.13倍(P＜0.05).转染7d后,ALP染色结果显示:实验组1细胞质中深蓝色粗大颗粒明显少于实验组2. 结论 采用Q-PCR方法筛选比格犬BMSCs成骨分化的miRNAs系统高效、准确.筛选获得的miRNAs中,miR-125b过表达能够显著抑制比格犬BMSCs的成骨分化.     

Bone morphogenetic protein-2 regulates proliferation of human mesenchymal stem cells

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein-2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum-free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein-2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence-activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein-2 treatment in serum-free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein-2-treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well-developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein-2 treatment in serum-free medium.     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

重组人骨形态发生蛋白-2诱导脂肪成体干细胞成异位软骨的研究

目的探索重组人骨形态发生蛋白-2（rhBMP-2）诱导脂肪成体干细胞异位软骨的生成能力。方法将获取的兔脂肪组织机械分割，通过Ⅰ型胶原酶消化后得到脂肪成体干细胞。经rhBMP-2诱导培养14d的脂肪成体干细胞微球种植在BALB／C裸鼠肌袋内。术后4、8周各处死2只动物，取材固定、脱钙、包埋后切片染色。镜下观察。结果经过rhBMP-2连续诱导培养14d的脂肪成体干细胞已具有软骨细胞的表型（细胞基质中富含蛋白多糖和Ⅱ型胶原），并不向成骨方向分化（Von kossa染色阴性）。术后4、8周有软骨细胞形成。结论rhBMP-2具有诱导脂肪成体干细胞向软骨细胞分化的潜能。     

聚乙二醇化骨形态发生蛋白-2基因转染兔骨髓间充质干细胞及其表达测定

目的：聚乙二醇化骨形态发生蛋白-2基因（PEG/BMP-2）纳米颗粒转染兔骨髓间充质干细胞（rBMSCs）,检测骨形态发生蛋白-2（BMP-2）在靶细胞中的表达。方法：原代分离培养rBMSCs,分别采用PEG/BMP-2、脂质体/BMP-2转染细胞,采用流式细胞仪检测转染效率,采用Western Blot和real time RT-PCR方法检测BMP-2表达。结果：成功制备出PEG/BMP-2纳米颗粒并将PEG/BMP-2转染至rBMSCs,转染细胞中BMP-2呈高表达,且与脂质体转染法相比,具有更高的转染效率。结论：PEG/BMP-2纳米颗粒转染rBMSCs可高表达BMP-2,为骨缺损治疗修复提供新的治疗方法。     

人骨形成蛋白7基因重组2型腺相关病毒载体的构建及其在骨髓间充质干细胞中的表达

目的构建人骨形成蛋白7基因（hBMP7）重组腺相关病毒载体,并观察其在兔骨髓间充质干细胞中的表达。方法将骨形成蛋白7基因片段克隆入穿梭质粒pUC18获得重组质粒pUC18-hBMP7。KpnⅠ和鼠Ⅱ双酶切质粒pUC18-hBMP7／与pSNAV,用T4DNA连接酶连接分别回收的两片段后转化大肠杆菌DH5α感受态细胞,获得重组质粒PSNAV—hBMP7／,转染BHK-21细胞,筛选培养,用能表达Rap和Cap的重组Ⅰ型单纯疱疹病毒HSVl-rc／△UL2感染此细胞,裂解细胞收获病毒液。采用氯仿处理-PEG／NaCl沉淀-氯仿抽提法分离浓缩、纯化与测定病毒滴度。用rAAV2-hBMP7／和rAAV2-EGFP在体外分别转染兔骨髓间充质干细胞。流式细胞仪、RT-PCR和Western—blot方法检测兔骨髓间充质干细胞中hBMP-7基因的转录和表达。结果成功构建具有感染活性的重组腺相关病毒载体rAAV2-hBMP7／,病毒载体对兔骨髓间充质干细胞早期转染效率可达99．8％,hBMP7／在兔骨髓间充质干细胞中可得到转录和表达。结论成功构建了人骨形成蛋白7基因重组腺相关病毒载体,rAAV2-hBMP7／载体在体外可转染兔骨髓间充质干细胞,并获得较高的转染效率。     

The effect of recombinant human osteogenic protein-1 (bone morphogenetic protein-7) impregnation on allografts in a canine intercalary bone defect.

The utility of cortical allografts in repairing large bone defects is limited by their slow and incomplete incorporation into host bone. In order to determine the effects of recombinant human osteogenic protein-1 (rhOP-1) impregnation on allograft incorporation, we used a canine intercalary bone defect model. Bilateral resection of a 4 cm segment of the femoral diaphysis and reconstruction with structural bone allografts were performed. In one limb, the allograft was soaked in solution with rhOP-1 for 1 h before implantation. In the other limb, the allograft was soaked in the same solution without rhOP-1. Dynamic load-bearing, radiographic analysis, biomechanical testing, and histomorphometric analysis were conducted. Radiographic analysis showed significantly larger periosteal callus area in the rhOP-1 treated group at week 2. The rhOP-1 significantly increased allograft bone porosity and significantly increased the number of active osteons in the allografts. There were no significant differences between the rhOP-1 treated and non-treated allografts in load bearing and biomechanical analyses. These findings indicate that rhOP- I increases intercalary allograft remodeling without deleterious effects in mechanical and functional strength.     

Clinical evaluation of recombinant human bone morphogenetic protein-2

Recombinant human bone morphogenetic protein-2 is an osteoinductive protein that plays a pivotal role in bone growth and regeneration. Several hundred studies were conducted in the past 7 years in numerous animal models to establish unequivocally the efficacy, safety, mechanism of action, pharmacokinetics, and surgical handling properties of recombinant human bone morphogenetic protein-2, building a solid foundation for clinical development programs. Pilot clinical trials have shown the feasibility and safety of recombinant human bone morphogenetic protein-2 treatment, and defined the effective dose for its use in open long bone fractures and for augmentation or preservation of the alveolar bone in the dental ridge. Prospective observational clinical studies helped define clinical efficacy end points, identify significant variables, and estimate appropriate population sample size for pivotal clinical trials. Pivotal clinical trials of recombinant human bone morphogenetic protein-2 are underway in patients with open tibial shaft fractures and in patients with a deficiency of the alveolar ridge.     

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice

onemorphogenetic proteins (BMPs)haveapowerfulcapacitytoelicitnewboneformation .ThereareseveraldeliverymethodsofBMPsintreatingbonedefects ,oneofwhichisgenetherapy .Retrovirus,adenovirusandadeno associatedvirushavebeenutilizedtodeliverBMPgene .1,2 Sincethedirectuseofthesevectorshasseveraldisadvantages ,wehavedevelopedexvivo genetherapytechniquewhichinvolvestheisolationandcultivationofautologousbonemarrow derivedmesenchymalstemcells (MSCs) ,transfectionofthecellsinvitroandimplantationofthesec…     

Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响

目的 观察经典Wnt/β-catenin以及骨形态发生蛋白(BMP)信号通路对大鼠骨髓来源的间充质干细胞(BMSCs)体外增殖以及向成骨方向分化的影响.方法 应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分4组:对照组、Wnt组、BMP组、Wnt3a和BMP-2联合组,在不同时间点用细胞计数试剂盒-8(CCK-8)法检测细胞增殖活性,碱性磷酸酶(ALP)活性定量测定、Yon Kossa染色观察细胞向成骨分化和基质矿化程度.逆转录-聚合酶链反应(RT-PCR)检测成骨特异性标志物表达.结果 培养第7天,CCK-8测吸光度值联合组为0.845,Wnt组为0.738,明显高于对照组0.409(P＜0.05).培养第6天和第12天,ALP活性吸光度值联合组为63.8和144.3,BMP-2组为40.8和104.1,均明显高于对照组7.3和18.9(P ＜0.05).培养14 d后,联合组骨钙素mRNA表达量高于对照组,而Runx2及Osterix表达量与其他组比较增高不显著.培养3周后,Yon Kossa染色显示联合组钙结节数量及大小均高于对照组.结论 Wnt3a因子和骨形态发生蛋白-2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化.     

Safety of recombinant human bone morphogenetic protein-2 after spinal laminectomy in the dog

STUDY DESIGN: This was a randomized, blinded trial of the safety of the application of recombinant human bone morphogenetic protein (rhBMP)-2 or autologous bone graft onto a laminectomy defect of the dog in the presence or absence of a dural membrane puncture. OBJECTIVE: To test the safety of rhBMP-2 in an application in which direct contact of the material with neural tissue occurs. SUMMARY OF BACKGROUND DATA: Application of rhBMP-2 in laboratory animals stimulates local bone formation to effect spinal fusion and healing of segmental bone defects. The use of rhBMP-2 as a bone graft substitute in spinal fusion would eliminate donor site morbidity and may augment the rate of successful fusion. Because rhBMP-2 may unintentionally come in contact with neural tissue, the consequences of such a safety issue must be addressed in an animal model before human trials. METHODS: Twenty skeletally mature beagles underwent spinal exposure followed by bilateral laminectomy at L5. In half of the dogs, a puncture wound was made to the dura with the expression of cerebrospinal fluid at the site of the puncture. In randomly selected animals, the exposed dural elements received either autologous bone graft with the bone removed from the laminectomy site or an implant of the rhBMP-2 device. The animals was observed for 12 weeks with periodic clinical examinations and monthly computed tomographic scans. RESULTS: There was no clinical, radiographic, or histologic evidence of neurologic abnormalities in these animals. The rhBMP-2 stimulated bone growth in the laminectomy defect and came into direct contact with the dural membrane. There was no evidence of abnormal mineralization within the thecal sac or in the spinal cord itself. CONCLUSIONS: The rhBMP-2 implant stimulated bone formation in the laminectomy site. Neither autologous bone, rhBMP-2, nor the dural puncture had deleterious consequences for the animals.     

rhBMP-2在兔桡骨远端骨缺损模型中成骨效应研究

目的 评价大肠杆菌DH5α高度表达的重组人骨形态发生蛋白-2（rhBMP-2）促进兔桡骨远端骨缺损骨愈合的有效性和剂量-成骨关系.方法 72只成年雄性新西兰白兔按照所设rhBMP-2剂量随机等分为空白对照组、0.5mg组和1.0mg组,右桡骨远端截骨构建骨缺损模型,分别于骨缺损处植入吸附rhBMP-2的纤维胶原蛋白载体复合物.术后2周、4周和8周每组分别处死动物8只,根据钼钯X线摄片、微CT检测、三点弯曲最大承载力测定和组织细胞学检查评估rhBMP-2成骨效应及剂量-成骨关系.结果 3个rhBMP-2剂量组术后2周、4周、8周Lane-Merchant评分所示骨愈合率有统计学差异（P＜0.05）.根据微CT检测,所有标本正常骨密度无个体差异（P＞0.078）,3个rhBMP-2剂量组术后2周、4周、8周缺损区骨密度均有统计学差异（P＜0.05）;3个rhBMP-2剂量组术后2周、4周、8周三点弯曲最大承载力均有统计学差异（P＜0.05）.组织细胞学检查结果也符合以上结果.结论 rhBMP-2（0.5 mg、1.0 mg）能有效促进兔桡骨远端骨缺损愈合,其成骨能力在一定剂量范围内存在剂量依赖性.纤维胶原蛋白载体有着良好的生物相容性,但降解速度相对较快,对rhBMP-2缓释、提高局部有效浓度维持时间的作用有限.     

重组人骨形态发生蛋白-2对鼠雪旺细胞增殖及生长相关蛋白GAP-43表达的影响

目的 观察重组入骨形态发生蛋白-2( rhBMP-2)对体外培养的乳鼠雪旺细胞增殖及生长相关蛋白( GAP-43)表达的影响。方法 将纯化的雪旺细胞分两组,一组设为对照,另一种加含终质量浓度为5 μg/L rhBMP-2的DMEM/F12培养液培养,在培养后0、12、24、36、48、72 h分别用噻唑蓝(MTT)比色法检测不同时间点的A值并绘制生长曲线;用BrdU法测定雪旺细胞增殖率;用Western blot法检测GAP-43蛋白的表达水平。结果 经含5μg/L rhBMP-2培养液培养的雪旺细胞,在24、36、48 h细胞增殖率明显高于对照组,差异有统计学意义(P＜0.05);实验组中GAP-43在24、36、48 h的表达也显著高于对照组(P＜0.05)。结论 rhBMP-2有促进雪旺细胞分裂增殖和GAP-43蛋白表达的作用,可能是其促进周围神经再生的重要机制之一。     

Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow.

Mesenchymal stem cells (MSCs) residing in bone marrow (BM) are the progenitors for osteoblasts and for several other cell types. In humans, the age-related decrease in bone mass could reflect decreased osteoblasts secondary to an age-related loss of osteoprogenitors. To test this hypothesis, BM cells were isolated from vertebral bodies of thoracic and lumbar spine (T1-L5) from 41 donors (16 women and 25 men) of various ages (3-70 years old) after death from traumatic injury. Primary cultures were grown in alpha modified essential medium with fetal bovine serum for 13 days until adherent cells formed colonies (CFU-Fs). Colonies that stained positive for alkaline phosphatase activity (CFU-F/ALP+) were considered to have osteogenic potential. BM nucleated cells were plated (0.5, 1, 2.5, 5, or 10 x 106 cells/10-cm dish) and grown in dexamethasone (Dex), which promotes osteoblastic differentiation. The optimal plating efficiency using BM-derived cells from donors of various ages was 5 x 106 cells/10-cm dish. BM-derived cells were also grown in the absence of Dex at this plating density. At the optimal plating density, in the presence of Dex, the number of CFU-F/ALP+ present in the BM of the younger donors (3-36 years old) was 66.2 +/- 9.6 per 106 cells (mean +/- SEM), but only 14.7 +/- 2.6 per 106 cells in the older donors (41-70 years old). With longer-term culture (4-5 weeks) of these BM cells in medium containing 10 mM beta-glycerophosphate and 100 microg/ml ascorbic acid, the extracellular matrix mineralized, a result consistent with mature osteoblastic function. These results demonstrate that the number of MSCs with osteogenic potential (CFU-F/ALP+) decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number. Our results are particularly important in that the vertebrae are a site of high turnover osteoporosis and, possibly, the earliest site of bone loss in age-related osteoporosis.