SATB2促进BMP9诱导C2C12细胞的成骨分化

目的:研究特异AT序列结合蛋白2(Special AT-rich sequence-binding protein 2,SATB2)在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导的间充质干细胞(mesenchymal stem cells,MSCs)C2C12成骨分化中的作用。方法:首先用Ad-BMP9感染C2C12细胞,RT-PCR检测SATB2在基因水平上的表达变化,Western blot检测SATB2在蛋白水平上的变化;构建过表达SATB2重组腺病毒Ad-SATB2,感染C2C12细胞后,Western blot验证Ad-SATB2表达情况;用AdSATB2处理BMP9诱导的C2C12细胞,碱性磷酸酶(ALP)活性和染色检测成骨早期ALP的变化,茜素红S染色检测成骨晚期指标钙盐沉积的变化。结果:Ad-BMP9处理C2C12细胞后,比对照组SATB2在基因水平和蛋白水平上表达量上调;Ad-SATB2可以在细胞中成功表达SATB2蛋白;用Ad-SATB2处理BMP9诱导的C2C12细胞后,ALP以及钙盐的沉积与对照比较均上调。结论:SATB2可以促进BMP9诱导的间充质干细胞C2C12的成骨分化。     

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.     

重组人骨形态发生蛋白－2成熟肽在大肠杆菌中的表达及其诱导成骨活性

报告了人骨形态发生蛋白－２（ＢＭＰ－２）成熟肽的基因克隆,及其在大肠杆菌中的表达。用ＡｆｌⅡ酶剪除ＢＭＰ－２ｃＤＮＡ中的信号肽及前肽部分的序列,将编码成熟肽的ｃＤＮＡ３′端０．３６ｋｂ基因片段克隆于大肠杆菌表达载体ｐＭＸ－１质粒的ＡＴＧ下游,受控于ＰＬＰＲ启动子。重组子以大肠杆菌ＤＨ５α为宿主细胞进行温度诱导表达。工程菌经４２℃６ｈ诱导后,在ＳＤＳ－ＰＡＧＥ上出现一条新蛋白带,分子量约为１２ｋＤ,约占菌体总蛋白的２０％。主要以包涵体形式存在的表达产物经初步纯化后,可获得纯度较高的重组人骨形态发生蛋白－２成熟肽（ｒｈＢＭＰ－２ｍ）。小鼠肌肉植入试验发现。ｒｈＢＭＰ－２ｍ植入后的不同时间,在局部出现间质细胞的聚集和增生、软骨细胞及软骨基质的生成和硬质骨的形成,证明ｒｈＢＭＰ－２ｍ具有明显的诱导新骨形成的作用。     

尿酸影响人骨髓间充质干细胞成骨分化过程中BMP-2 表达

目的:探讨在人骨髓间充质干细胞(h BMSCs)成骨分化过程中,不同浓度尿酸(UA)对骨形态形成蛋白-2(BMP-2)表达的影响。方法:以全骨髓贴壁培养法分离h BMSCs,将生长状态良好的第3代h BMSCs分为5组,分别为空白对照组(加入完全培养基)和成骨诱导组(加入成骨诱导液及含0 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8 mmol/L尿酸的完全培养基)。连续干预诱导14d后,用倒置显微镜观察细胞形态的变化,通过观察茜素红染色情况及检测碱性磷酸酶(ALP)活性进行成骨情况的检测。RT-PCR技术检测各组细胞BMP-2 mR NA的表达情况。结果:第3代h BMSCs大多为形态单一的长梭形,呈旋涡状生长;干预诱导后的细胞逐渐变成不规则的立方形,局部形成团块状结节,以含尿酸浓度为0.8 mmol/L的成骨诱导培养基最为显著。连续干预14d后,空白对照组茜素红染色为阴性,而各成骨诱导组细胞茜素红染色结果为阳性,提示干预诱导后的细胞为成骨细胞。碱性磷酸酶活性随尿酸浓度的增加和干预时间的延长而增强(P<0.05)。RT-PCR检测结果显示,空白对照组无BMP-2 mR NA的表达。成骨诱导组随培养基中尿酸浓度的增加,BMP-2 mR NA表达逐渐增强,呈浓度依赖性(P<0.05)。结论:尿酸上调h BMSCs向成骨细胞分化过程中BMP-2 mR NA的表达。     

Immunolocalization of vascular endothelial growth factor on intramuscular ectopic osteoinduction by bone morphogenetic protein-2

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.     

人骨形态发生蛋白12对人骨肉瘤细胞的生物学作用

为研究人骨形态发生蛋白(human bone morphogenetic proteins,hBMP)12对人骨肉瘤细胞株MG63和U2OS的作用,分别用hBMP12重组腺病毒(AdBMP12)以及含重组hBMP12(recombinant hBMP12,rhBMP12)的条件培养液干预人骨肉瘤细胞MG63和U2OS,利用台盼蓝拒染法、TUNEL法、吖啶橙/溴乙啶(AO/EB)荧光双染法、Transwell小室和碱性磷酸酶活性测定法分别检测细胞增殖、凋亡、迁移以及成骨分化能力的变化.与相应对照组相比,AdBMP12和含rhBMP12的条件培养液的干预致两种骨肉瘤细胞株细胞存活率降低,并呈一定的时间依赖性;凋亡率均随时间延长而增加,并且两种检测方法的结果一致;不同时间点的细胞穿膜数均明显减低;碱性磷酸酶活性在干预3d后开始逐渐增加,至第9d仍可观测到.以上差异均有统计学意义(P<0.01).提示无论是以腺病毒介导基因转入还是重组蛋白直接作用方式,hBMP12都可以抑制人骨肉瘤细胞株MG63和U2OS的增殖和迁移,并诱导其凋亡和向成骨细胞分化.     

百令胶囊对多囊卵巢综合征患者卵泡液骨形态蛋白、生长分化因子-9及胰岛素样生长因子的影响

目的:探讨百令胶囊对多囊卵巢综合征患者卵泡液骨形态蛋白(BMP)、生长分化因子(GDF)-9及胰岛素样生长因子(IGF)的影响。方法:选择2013年7月至2016年7月我院接诊的90例多囊卵巢综合征患者,通过随机数表法分为观察组(n=45)和对照组(n=45),对照组使用达英-35治疗,观察组在此基础上使用百令胶囊。比较两组糖代谢指标、内分泌指标、卵泡液BMP-15、GDF-9、IGF-1水平的变化以及临床疗效。结果:治疗后,观察组空腹血糖(FBG)、空腹胰岛素(FINS)、胰岛素抵抗(HOMA-IR)、血清卵泡刺激素(FSH)、黄体生成素(LH)、睾酮(T)水平水平均显著低于对照组(P0.05),卵泡液BMP-15、GDF-9水平显著高于对照组,IGF-1水平明显低于对照组(P0.05),总有效率显著高于对照组(P0.05)。结论:百令胶囊质量多囊卵巢综合征效果显著,可有效调节内分泌,并改善卵泡液BMP-15、GDF-9、IGF-1的水平。     

骨形成蛋白2/珍珠层粉/壳聚糖支架制备及生物性能研究

目的:制备骨形成蛋白2/珍珠层粉/壳聚糖复合多孔支架,观察支架生物性能。方法:采用冷冻干燥法制备骨形成蛋白2/珍珠层粉/壳聚糖多孔支架。用光学显微镜和扫描电子显微镜观察支架表面形貌及孔径大小,用比重瓶法检测支架孔隙率,热重分析探讨支架的热稳定性,用微力试验机进行压缩性能测试,并将支架与兔骨髓间充质干细胞共培养检测细胞黏附性能,将支架埋置大鼠皮下观察其炎症反应。结果与结论:制备的骨形成蛋白2/珍珠层粉/壳聚糖支架孔径大小为100~300μm,孔隙率为91.64%,压缩应力达3.37MPa,与细胞共培养贴附较好,有良好的组织相容性,提示该支架可做为组织工程支架材料应用于临床上骨组织缺损的修复。     

Inhibition of Phosphatidylinositol 3-Kinase Activity Blocks Cellular Differentiation Mediated by Glial Cell Line-Derived Neurotrophic Factor in Dopaminergic Neurons

Abstract: Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 µ M 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 n M wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.     

Glial Cell Line-Derived Neurotrophic Factor Exerts Neurotrophic Effects on Dopaminergic Neurons In Vitro and Promotes Their Survival and Regrowth After Damage by 1-Methyl-4-Phenylpyridinium

Abstract: The effect of glial cell line-derived neurotrophic factor (GDNF) on the growth of mesencephalic dopaminergic neurons and on their survival following exposure to the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) was examined in vitro. In cultures developing under normal conditions, GDNF at 1 ng/ml optimally improved the survival and stimulated the growth of dopaminergic neurons without affecting glial growth. In cultures treated with MPP⁺, GDNF could not prevent toxicity to dopaminergic neurons. The uptake of [³H]dopamine and the number of tyrosine hydroxylase-positive neurons were similarly reduced by MPP⁺ in the presence or absence of GDNF. However, after removal of MPP⁺, GDNF protected dopaminergic neurons from the continuous cell death and stimulated the regrowth of dopaminergic fibers damaged by MPP⁺. We conclude that GDNF supports the growth of normally developing dopaminergic neurons and stimulates their survival and recovery after damage. These findings suggest that GDNF could be useful in the development of therapeutic approaches to Parkinson's disease, which is characterized by dopaminergic cell loss.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.     

重组人骨形态形成蛋白2在家蚕幼虫中表达及产物纯化

将编码人ＢＭＰ２ｃＤＮＡ基因插入昆虫杆状病毒转移载体ｐＢａｃＰＡＫ１,与修饰的家蚕核形多角体病毒Ｂｍ－ＢａｃＰＡＫＤＮＡ共转染家蚕细胞,通过同源重组得到含有在多角体蛋白基因启动子控制下的ＢＭＰ２ｃＤＮＡ基因的重组病毒Ｂｍ－ＢａｃＰＡＫ－ＢＭＰ２。用重组病毒感染家蚕幼虫,第五天ＢＭＰ２表达率最高,每毫升蚕血淋巴中约１０μｇ表达产物;表达产物在在体内被加工成Ｃ－端１６ｋＤ片段,以二硫键连结成分子量为３０ｋＤ的同源二聚体;经纯化获得９０％以上纯度的成熟ＢＭＰ２,与骨基质胶原结合后植入大鼠皮下,７天后在局部诱导生成软骨组织。     

Construction of recombinant adenovirus vector containing hBMP2 and hVEGF165 genes and its expression in rabbit Bone marrow mesenchymal stem cells

To construct an adenovirus vector co-expressing human bone morphogenetic protein (hBMP2) and human vascular endothelial growth factor (hVEGF165) as well as green fluorescence protein (GFP) as a marker, with which the intracellular expression of the inserted genes could be identified in Bone marrow mesenchymal stem cells (BM-MSCs). BMP2 and VEGF165 genes were PCR amplified from a cDNA library and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. The virus solution (Ad-BMP2-VEGF165) was generated by co-transfecting HEK293 cells with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1, 3Cre. The virus solution was further purified and virus titer was determined accordingly. The expression of the target genes was subsequently detected and quantified in rabbit BM-MSCs by using real time PCR, ELISA and Western blotting. The recombinant adenovirus vector containing BMP2 and VEGF165 (Ad-BMP2-VEGF165) was successfully constructed, which was confirmed by Sanger sequencing, colony PCR, as well as visually detection of GFP, and the titer of the adenovirus was 1 × 10¹⁰ PFU/mL, and the proteins level of BMP2 and VEGF165 secreted in the supernatant are significantly higher than the control. Recombinant adenovirus vector containing hBMP2 and hVEGF165 genes was successfully constructed. The transfection rate of BM-MSCs by the adenovirus was high (95% at 100 MOI) and the BMP2 and VEGF165 genes was highly expressed in the cells. The present study provides a method to efficiently express the target genes in BM-MSCs and an vector for further research of bone defect repair using dual genes of BMP2 and VEGF165.     

Expression of the SM-20 Gene Promotes Death in Nerve Growth Factor-Dependent Sympathetic Neurons

Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM-20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM-20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3-kinase inhibitor LY294002. In addition, SM-20 protein synthesis is elevated in NGF-deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM-20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM-20 may function to regulate cell death in neurons.     

Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨

目的:探讨Notch信号对骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导间充质干细胞成骨分化的影响以及作用机制。方法:(1)DAPT或Ad-dominant-negative mutants of Notch1(Addn Notch1)和BMP4-CM处理小鼠胚胎成纤维细胞,检测早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP);(2)茜素红S染色实验检测晚期成骨钙盐沉积情况;(3)半定量反转录聚合酶链反应(RT-PCR)检测成骨分化相关基因ALP,Runx2,Col1a1的表达;(4)免疫细胞化学检测p-Smad1/5/8的表达;(5)结晶紫染色和流式细胞术检测细胞的增殖及周期改变。结果:(1)DAPT抑制BMP4诱导的早期成骨分化,且呈浓度依赖性;(2)Delta-like 1(DLL1)促进BMP4诱导的成骨分化,DAPT和dn Notch1抑制BMP4诱导的成骨分化;(3)DLL1促进BMP4诱导的成骨相关基因ALP,Runx2,Col1a1的表达,DAPT抑制这些基因的表达;(4)DLL1促进BMP4诱导的细胞核内p-Smad1/5/8的表达,而DAPT抑制其表达;(5)DLL1促进BMP4诱导的细胞增殖,而DAPT抑制BMP4诱导的细胞增殖。结论:Notch信号通过BMP/Smads信号通路促进BMP4诱导的MSCs成骨分化,在此过程中也有促细胞增殖的作用。     

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。