调控基因-Noggin的研究进展

Noggin基因最早是由California大学的Harland和William Simth于1992年从非洲爪蟾的胚胎中分离得到的,由于将其mRNA注射入爪蟾的胚胎可使头部明显增大而命名该基因为Noggin(美国俚语中为"头,脑袋"的意思)[1].现在已克隆出人的Noggin基因定位于染色体17q22,大鼠与小鼠的Noggin基因则定位于染色体11[2].Noggin基因在种属间高度保守,其编码产物为26kD蛋白,具有疏水性氨基酸末端,是一种分泌蛋白.Noggin基因在体内对多个系统的发育和/或重塑起重要的调控作用,现就其表达和功能作一简要综述.     

转染Noggin基因的骨髓基质细胞在体外分化为神经元样细胞的初步研究

目的构建Noggin基因的表达载体,初步探讨转染Noggin基因的骨髓基质细胞(BMSCs)在体外分化为神经细胞的情况。方法应用RT-PCR技术,从正常人胎脑组织中扩增出Noggin基因,利用基因工程技术构建载体pCS2 Tα1-Noggin。用Percoll分离液分离出大鼠BMSCs,借助脂质体将Noggin基因转入BMSCs。观察细胞形态的改变,并利用免疫组化鉴定分化细胞是否表达神经细胞特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)以及胶质纤维酸性蛋白(GFAP)。结果成功构建Noggin基因的真核表达载体pCS2 Tα1-Noggin。Percoll分离液分离出BMSCs,转染Noggin基因表达载体后,形似神经细胞;免疫组化检测到NSE、MAP-2表达,未检测到GFAP表达。结论转染Noggin基因的BMSCs在体外可以分化为神经元样细胞。     

Premature ovarian failure in a female with proximal symphalangism and Noggin mutation

OBJECTIVE: To report a case of premature ovarian failure (POF) and a mutation of the gene for Noggin (NOG). DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 33-year-old Japanese female with POF and proximal symphalangism. INTERVENTION(S): Direct sequence analysis of the NOG gene. MAIN OUTCOME MEASURE(S): Occurrence of POF. RESULT(S): A novel heterozygous G to A transition was identified at the nucleotide position 142 (142 G>A), which is predicted to cause an amino acid substitution of glutamic acid by lysine (E48K). CONCLUSION(S): Because NOG is expressed in the ovary and interacts with bone morphogenetic proteins, which play an important role in the ovarian function, a NOG mutation may constitute one of the multiple susceptibility genes for the development of POF.     

Noggin参与海人酸损伤后成年大鼠海马颗粒下区颗粒细胞增殖

目的 探讨侧脑室注射海人酸(KA)致大鼠海马损伤后Noggin的表达变化及其与颗粒细胞增殖的关系.方法 健康雄性SD大鼠32只采用随机数字表法分为实验组(16只)及对照组(16只).对照组又分为生理盐水对照组和空白对照组,各8只.实验组大鼠侧脑室注射KA,生理盐水对照组注射等剂量生理盐水.空白对照组不作处理.侧脑室注射KA 1周内,尼氏染色检测海马神经元的丢失.免疫荧光染色与原位杂交的方法检测海马齿状回BrdU标记细胞与Noggin mRNA阳性细胞的变化.结果 在侧脑室注射KA致海马损伤后1周,海马CA3、CA4区神经元丢失明显.与生理盐水对照组比较,实验组海马齿状回BrdU阳性细胞升高,差异有统计学意义(P=0.006),其中注射侧较对侧更为明显.海马Noggin mRNA阳性细胞在第3天时升高,第7天时下降.结论 侧脑室注射KA致海马损伤后.成年大鼠海马齿状回颗粒细胞异常增殖可能与Noggin表达波动有关.     

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 μg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.     

Noggin蛋白对D 半乳糖致衰老小鼠学习记忆及海马齿状回神经发生的影响

目的观察侧脑室注射Noggin蛋白后对D 半乳糖致衰老模型小鼠学习记忆行为能力与海马齿状回神经发生的影响。方法采用皮下注射D 半乳糖构建衰老小鼠模型后,连续7?d侧脑室注射Noggin蛋白,对照组同时注射等量生理盐水,造模42?d后对各组小鼠进行Y迷宫学习记忆能力测试,用BrdU标记海马齿状回增殖细胞。 结果Y迷宫检测结果表明,模型组小鼠学习记忆能力较正常对照组明显降低（P<0.05）,而模型治疗组小鼠学习记忆能力较模型对照组明显改善（P<0.05）;模型组与模型对照组小鼠海马齿状回BrdU阳性细胞数较正常对照组减少（P<0.05）,而模型治疗组BrdU阳性细胞数较模型组、模型对照组显著增多（P<0.05）。结论侧脑室给予Noggin蛋白可改善衰老模型小鼠的空间学习及记忆能力,可能与Noggin能保护海马神经元并促使齿状回神经干细胞增殖有关。        

In vivo gene delivery to proliferating cells in the striatum generated in response to a 6-hydroxydopamine lesion of the nigro-striatal dopamine pathway

The degeneration of neurons in the mammalian brain is commonly associated with the division of cells located in the damaged area. The aim of the present study has been to characterise the phenotype of newly born cells in the striatum of adult rats following 6-hydroxydopamine lesion of the nigro-striatal pathway. Newborn cells were identified through labelling with either bromodeoxyuridine or retrovirus encoding green fluorescence protein. We report here that the overwhelming majority of these cells have glial characteristics. In order to promote the generation of new neurons we retrovirally introduced either the noggin or neurogenin2 genes into newborn cells following the 6-hydroxydopamine lesion. Transduction with neurogenin2 resulted in the production of cells resembling neuroblasts, however these cells did not appear to survive. Noggin transduction did not result in the generation of new neurons, but interestingly, greatly increased the number of oligodendrocytes generated from newborn cells.     

Noggin对成骨的作用研究进展

Noggin是对机体多个系统的生长发育和重塑起重要调节作用的一种糖蛋白.其对神经系统和脑发育的影响已经为人们所熟知,近年来随着研究的不断深入,noggin对骨骼系统生长发育的显著作用逐渐被人们所认识:包括调节胚胎的形态发生,抑制体外成骨细胞增值与分化,抑制体内骨形成,调节颅缝融合和关节的正常形成以及辅助治疗骨肿瘤疾病等,是调节骨生长发育的重要细胞因子.其作用机制主要是通过与BMPs相互影响,调节BMPs信号通路的过程,间接调节骨形成.     

Effect of bone morphogenetic protein-2 on proliferation and apoptosis of gastric cancer cells

Objective: To investigate the effects of bone morphogenetic protein-2 (BMP-2) on the proliferation, differentiation and apoptosis of normal human gastric mucosal cells and gastric cancer cells.Methods: Poorly differentiated gastric cancer BGC823 cells, moderately differentiated gastric cancer cells and normal human gastric mucosal epithelial GES-1 cells were independently treated with recombinant human BMP-2 or its inhibitor Noggin. MTT assay was performed to detect the proliferation, flow cytometry done to measure the cell cycle and apoptosis and immunohistochemistry carried out to determine the expression of cyclin-dependent kinase 4 (CDK4).Results: BMP-2 exerted inhibitory effect on the growth of all types of cells and the inhibition become more evident with the increase of BMP-2 dose. After treatment with 200 ng/ml BMP-2, cancer cells arrested in G1 phase and those in S phase reduced. Gastric cancer cells had higher CDK4 expression than GES-1 cells. BMP-2 decreased CDK-4 expression in cancer cells but had no influence in GES-1 cells. Noggin conferred promotive effect on the growth of 3 types of cells. In 2 types of cancer cells, treatment with 2000 ng/ml Noggin significantly increased the proportion of cells in S phase but reduced that in G1 phase. However, Noggin did not affect the cell cycle of GES-1 cells. The CDK4 expression was markedly increased in 2 types of cancer cells but that of GES-1 remained unchanged after treatment with 2000 ng/ml Noggin.Conclusions: BMP-2 may inhibit the proliferation of both normal and malignant gastric epithelial cells, down-regulate CDK4 expression in gastric cancer cells and arrest gastric cancer cells in G1-phase in cell cycle. Through antagonizing BMP-2, Noggin, may accelerate the proliferation of gastric cancer cells. Thus, the abnormality of BMP signaling pathway may play an important role in the pathogenesis of gastric cancer.     

毛囊真皮鞘细胞中过表达Noggin对皮肤及毛囊生长的作用

背景:Noggin蛋白是一个抑制BMP信号通路的重要分子,可以与BMP2/4结合形成复合物,从而阻断BMP信号,影响生物有机体的正常发育过程。研究认为真皮鞘细胞是一类能够长期自我更新的真皮干细胞,参与毛囊再生过程中真皮毛乳头和真皮鞘的形成。在毛囊发育过程中,真皮毛乳头中Noggin参与毛囊的起始,Noggin的缺失会导致毛囊数量的减少和毛囊生长缓慢,但人们对于Noggin蛋白在毛囊真皮鞘中的作用所知甚少。目的:研究Noggin蛋白在毛囊真皮鞘中的生物学功能。方法:利用真皮鞘特异的αSMA-Cre ERT2工具鼠在真皮鞘中特异过表达Noggin蛋白,分别在出生后8,9 d对实验组αSMA-CreER;pMES-Noggin小鼠和对照组αSMA-CreER小鼠注射4-羟基他莫昔芬,在出生后21,23,28 d获取皮肤组织,苏木精-伊红染色观察毛囊生长情况和皮下脂肪层厚度,免疫组化染色分析表型。结果与结论:通过苏木精-伊红染色结果发现毛囊生长并没有受到影响,但毛囊皮下脂肪组织生长发育受到严重影响,小鼠皮下脂肪层变薄。免疫组化结果说明BMP信号降低可能是导致毛囊脂肪层变薄的原因。这一发现拓展了人们对于真皮鞘细胞和Noggin蛋白的认识,也为人们更加准确理解毛囊再生和组织生长机制提供了重要依据。