骨形成蛋白-2表达载体的构建及其转录活性测定

将骨形成蛋白-2(BMP-2)基因克隆入带HA标签的BMP-2表达载体中,得到带HA标签的重组质粒pGBKT7/HA-BMP-2,将该重组质粒转染酵母AH109、Y187细胞,Western印迹检测表明表达了与预期相对分子质量大小一致的BMP-2.BMP-2表达载体的成功构建为深入研究骨形成蛋白-2在促进骨痂生成过程中的分子机制及其生物学功能打下了基础.     

骨形态发生蛋白-7及抑制性Smads在糖尿病肾病发生发展中的表达变化

本文采用免疫组化、Western blot及荧光实时定量PCR方法,动态观察链脲佐菌素（streptozocin,STZ）诱导的大鼠糖尿病肾病（diabetic nephropathy,DN）发生早期肾脏骨形态发生蛋白-7（bone morphogenetic protein-7,BMP-7）、Smad6、Smad7蛋白及mRNA表达。结果显示,在正常及DN大鼠肾小管均有BMP-7、Smad6、Smad7蛋白表达,以胞浆表达为主。DN大鼠BMP-7、Smad6蛋白表达较正常大鼠明显增多（P〈0．05）,且BMP-7的mRNA表达呈先增加后降低的状态;而Smad7蛋白和mRNA的表达均呈先增加后降低的状态。转化生长因子-β1（transforming growth factor-β1,TGF-β1）及Ⅰ型胶原（collagen typeⅠ,COL-Ⅰ）mRNA在DN大鼠肾脏表达较正常大鼠明显增多（P〈0．05）,且随着糖尿病进展有逐渐增加的趋势。结果提示,作为TGF-β超家族信号分子的一员,BMP-7信号及抑制性Smad通路在DN肾纤维化发生早期可能起重要的反馈性抑制作用。     

软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响

目的：研究软骨寡聚基质蛋白（cartilage oligomeric matrix protein,COMP）过表达对BMP-2诱导骨髓间充质干细胞成骨及成软骨分化的影响。方法：BMP-2诱导骨髓间充质干细胞分化,通过脂质体转染含人COMP基因的质粒使骨髓间充质干细胞过表达COMP,采用实时定量PCR和Western blotting分析COMP基因过表达、成骨相关基因Ⅰ型胶原、RUNX2、骨钙蛋白以及成软骨相关基因Ⅱ型胶原、SOX9、蛋白聚糖、X型胶原的表达变化;通过茜素红染色观察成骨终末阶段矿化结节的生成情况,阿利新蓝染色观察细胞基质蛋白多糖的合成情况。结果：质粒转染后骨髓间充质干细胞COMP基因蛋白和mRNA表达水平显著提高（P<0.05）。COMP基因过表达后,成骨标记基因RUNX2、Ⅰ型胶原（Col1a1）mRNA水平均显著低于对照组（P<0.05）,RUNX2、骨钙蛋白（Osteocalcin）蛋白表达水平明显低于对照组（P<0.05）,而成软骨标记基因SOX9、蛋白聚糖（Aggrecan）mRNA水平均显著高于对照组（P<0.05）,SOX9、Ⅱ型胶原（Col2a1）蛋白表达均明显多于对照组（P<0.05）。细胞成骨茜素红染色弱于对照组,而阿利新蓝染色强于对照组。过表达组细胞X型胶原（Col10a1）基因表达显著低于对照组（P<0.05）,结论：骨髓间充质干细胞COMP基因过表达可抑制BMP-2诱导其成骨分化,促进骨髓间充质干细胞成软骨分化,并抑制软骨细胞的成熟肥大,为软骨组织工程研究提供新的方向。     

骨形态发生蛋白7 在前列腺癌中的表达

目的:探讨骨形态发生蛋白( BMP-7)在前列腺癌组织中的表达及其与临床分期之间的关系.方法:应用免疫印迹法检测30例前列腺癌患者及30例前列腺良性增生患者前列腺组织中BMP-7的表达情况.结果:前列腺癌组织中BMP-7的表达显著高于前列腺良性增生组织,且BMP-7的表达随前列腺癌的临床分期、Gleason分级增高而增加.结论:BMP-7在前列腺癌中的表达明显增高,其表达量与临床分期相关,前列腺癌组织中BMP-7的表达增高提示预后不佳.     

重组骨形态发生蛋白-7在骨损伤疾病治疗中的研究进展

骨形态发生蛋白-7(BMP-7)是具有强诱骨活性的蛋白质因子,已通过基因工程技术在体外得到表达,较长时间以来不断被应用于骨损伤疾病的研究,得到了确切的治疗效果。通过载体将BMP-7基因转入真核细胞,与生物聚合载体复合后植入体内,能表达并分泌活性的BMP-7,诱导骨细胞的生成,促进骨组织的修复,成为一种新的有效的治疗手段。     

重组人BMP-2在烟草不同组织中的表达

骨形态发生蛋白（BMPs）是一类调节骨组织发育的生长因子。BMP-2是BMP家族中诱骨活性最强的。在骨组织工程研究和临床应用中需要大量的BMP-2。因此,研究出一种能够有效地大量生产BMP-2的方法是十分必要的。随着植物分子生物学的进展,转基因植物被用作一种生物反应器来生产目的蛋白。以gus作为报告基因,研究了重组人bmp-2基因在烟草中的表达。通过GUS活性检测、半定量PCR和Western blotting分析了根、茎、叶组织中基因表达的水平,结果显示融合蛋白在根和茎组织中表达量显著高于叶组织。由于根和茎组织中蛋白组成与叶组织相比相对简单,提示其更易于进行目的蛋白的纯化。     

可调控小鼠尿激酶原激活剂真核表达载体的构建及其在Huh7细胞中的表达

目的：建立基于Tet-on系统的可调控真核细胞表达小鼠尿激酶原激活剂（uPA）的诱导表达系统。方法：提取C57小鼠肾组织总RNA,RT-PCR扩增uPAcDNA序列;提取基因组DNA,扩增uPA编码区后最后一个外显子序列,构建pTRE2-uPAcDNA-700载体,将其与pTet-on瞬时共转染Huh7细胞系,24h后用强力霉素诱导表达,诱导后36h分别收集细胞和培养上清（诱导组）,提取细胞总RNA并进行细胞uPA转录水平的检测;采用溶圈法对细胞分泌至培养上清中uPA的生物活性进行检测,同时以转染但未诱导Hun7（未诱导组）和正常培养Huh7（正常对照组）细胞及培养上清作为对照。结果：与未诱导组和正常对照组相比较,仅诱导组在转录水平上扩增出目的条带;溶圈法证实转染的细胞不仅表达uPA,而且表达的蛋白具有一定的生物活性。结论：构建了可调控的uPA真核诱导表达系统,为进一步制备可调控肝细胞表达uPA转基因小鼠及进一步揭示uPA肝损伤机理奠定了基础。     

重组人BMP-2生物活性的鉴定

目的鉴定在大肠杆菌中表达的重组人骨形态发生蛋白2（recombinant human bone morphogeneticprotein-2,rhBMP-2）的生物学活性。方法构建重组人BMP-2的原核表达质粒,在大肠杆菌中诱导表达目的蛋白,纯化复性后,采用细胞及动物实验对其生物活性进行鉴定。结果细胞实验表明,重组人BMP-2具有诱导MC3T3-E1前成骨细胞向成骨细胞分化的特性。动物实验显示,重组人BMP-2在大鼠肌肉内具有诱导异位成骨功能。结论制备的重组人BMP-2具有较好的生物学活性。     

骨形成蛋白(BMP)-2/4，-5与IA型BMP受体在口腔正常上皮、良性病变及癌变中的表达分析

认识BMP及其受体与口腔正常上皮及其癌变的关系。有助于深入了解口腔上皮癌变的机理。本文用免疫组织化学方法对BMP-2/4,-5与BMPR-IA在口腔颊部粘膜正常上皮,良性病变和癌变中的表达进行观察和半定量分析。标本包括：9例正常上皮（normal buccal muosa,NB)。8例慢性炎症（nonspecific chronic inflammation,NCI),7例过度角化（hyperkeratosis,HK)。5例乳头状瘤（squamous cell papilloma,SCP)。29例鳞癌（squamous cell carcinoma,SCC)。10例癌旁上皮（epithelium immediately adjacent to carcinoma,EAC)以及6例硬腭粘膜上皮（normal mucosa of hard palate,NHP）。结果显示：BMP-2/4,-5与BMPR-IA在口腔粘膜的正常与良性病变上皮中有弱的和不均一的阳性表达,NB与NHP无明显差别,而除3例SCC外,其它SCC几乎均有程度不一的阳性表达,在EAC中的表达接近于SCC,二者明显高于正常与良性组,此外,转移在淋巴结中的癌细胞的BMP-2/4与BMP-5阳性程度略高于原发灶的癌细胞,本文认为;BMP-2/4,5与BMPR-IA可能参与调控口腔上皮的癌变。     

Strict Regulation of Gene Expression Utilizing a Dual-control Gene Expression Vector System

可严格调控性是体现原核表达载体优越性的重要指标。构建了一种双控双调节原核表达载体系统,用双载体控制调节目的基因的表达,即SP6启动子（promoter）＋乳糖（lac）调节基因表达系统和araB启动子（promoter）＋ara C调节基因表达系统,分别由乳糖类似物IPTG和阿拉伯糖（L-arabinose）诱导目的基因的表达。该系统由2个表达载体共同完成目的基因的表达。pE SP-1为主表达载体,即目的基因克隆到此表达载体上,由SP6启动子（promoter）＋乳糖（lac）调节基因调控;pA RA-SP6为辅助表达载体,该载体通过SP6 RNA聚合酶的表达来控制调节主表达载体的启动子（SP6）,由araB启动子（promoter）＋ara C调节基因调控。实验结果显示该双控双调节表达载体系统控制严格,并且表达蛋白的量具有可调控性。     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-7 IN Escherichia coli

Bone morphogenetic protein-7 (BMP-7) is a multifunctional cytokine of the transforming growth factor β superfamily, which induces bone formation and plays an important role during bone tissue repair and embryonic development. In this study, human BMP-7 (hBMP-7) cDNA was cloned and expressed in Escherichia coli, and its yield was approximately 30% of the total bacterial protein. After the bacteria were lysed by ultrasonication and repeated washing, inclusion bodies were extracted and dissolved using a high-strength denaturant. The monomer of rhBMP-7 was purified by ion-exchange chromatography, and the purity coefficient was approximately 96%. The protein was renatured with refolding buffers at different pH values. The renatured rhBMP-7 dimer protein in this study increased the alkaline phosphatase activity of NIH3T3 cells. This study may be helpful for the in vitro production and biomedical application of rhBMP-7 protein expressed in an E. coli expression system.     

Mesenchymal stem cells from osteoporotic patients reveal reduced migration and invasion upon stimulation with BMP-2 or BMP-7

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression.     

Osteogenic protein-1 (OP-1, BMP-7) induces osteoblastic cell differentiation of the pluripotent mesenchymal cell line C2C12

The effects of Osteogenic Protein-1 (OP-1, BMP-7) on the differentiation of the pluripotent mesenchymal cell line, C2C12, were examined. OP-1 at 50 ng/ml partially inhibited myotube formation in C2C12 cells, while OP-1 at 200 ng/ml completely inhibited myotube formation and induced the formation of cells displaying osteoblastic morphology. High concentrations of OP-1 elevated the alkaline phosphatase (AP) activity dramatically, both as a function of time and OP-1 concentration. Osteocalcin (OC) mRNA expression was detected as early as 8 days in OP-1-treated cultures and subsequently increased considerably. Expression of bone sialoprotein (BSP) mRNA was low in control cultures and stimulated by OP-1. Collagen type I mRNA expression was enhanced by OP-1 during the early days in culture, but gradually decreased thereafter. MyoD mRNA expression, high in control cultures, was suppressed by OP-1 in a dose- and time-dependent manner. OP-1 enhanced ActR-I mRNA expression and significantly elevated the mRNA expressions of BMP-1, BMP-4, BMP-5, GDF-6, and GDF-8. The present results indicate that OP-1 is a potent inducer of C2C12 differentiation into osteoblastic cells.     

The bone morphogenetic protein family and osteogenesis.

The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP-2 through BMP-7. These molecules are part of the TGF-beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF-betas and the BMPs. The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud.(ABSTRACT TRUNCATED AT 250 WORDS)     

Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.     

Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.