Protective effect of sodium butyrate on the cell culture model of Huntington disease

This study aimed to develop a cell culture model of Huntington disease and observe the effect of sodium butyrate on this cell culture model. Exon 1 of both a wild type and a mutant IT15 gene from the genomic DNA of a healthy adult and a patient with Huntington disease was amplified and cloned into the eukaryotic expression vector pEGFP-C1. Human neuroblastoma SH-SY5Y cells were transiently transfected with these recombinant plasmids in the absence and presence of sodium butyrate (0.1, 0.2, 0.5, 1.0 mmol/L). The MTT assay was used to measure cell viability. The results indicated that the N-terminal fragment of mutant huntingtin formed perinuclear and intranuclear aggregates and caused a decrease of SH-SY5Y cell viability. Sodium butyrate inhibited the decrease of SH-SY5Y cell viability caused by the N-terminal fragment of mutant huntingtin. This suggests that sodium butyrate has a protective effect on this cell culture model of Huntington disease.     

Protective effect of sodium butyrate on the cell culture model of Huntington disease

This study aimed to develop a cell culture model of Huntington disease and observe the effect of sodium butyrate on this cell culture model.Exon 1 of both a wild type and a mutant IT15 gene from the genomic DNA of a healthy adult and a patient with Hunt- ington disease was amplified and cloned into the eukaryotic expression vector pEGFP-C1.Human neuroblastoma SH-SY5Y cells were tran- siently transfected with these recombinant plasmids in the absence and presence of sodium butyrate(0.1,0.2,0.5,1.0 mmol/L).The MTT assay was used to measure cell viability.The results indicated that the N-terminal fragment of mutant huntingtin formed perinuclear and intranuclear aggregates and caused a decrease of SH-SY5Y cell viability.Sodium butyrate inhibited the decrease of SH-SY5Y cell via- bility caused by the N-terminal fragment of mutant huntingtin.This suggests that sodium butyrate has a protective effect on this cell cul- ture model of Huntington disease.     

Molecular characterization of human reticulon 3 as a potential marker during the differentiation of human neuroblastoma SH-SY5Y cells

Neuroendocrine-specific protein (NSP) -reticulons are endoplasmic reticulum-associated protein complexes, which are localized in the endoplasmic reticulum (ER) and identified as markers for neuroendocrine differentiation. In the present study, human reticulon 3 gene (hRTN3) was cloned and its expression pattern in a variety of tissues was investigated. Truncated hRTN3s corresponding to the C-terminal (hRTN3-C) domain were expressed and purified. hRTN3 mRNA was down-regulated during the differentiation of human neuroblastoma cell line SH-SY5Y induced by all-trans-retinoic acid (RA), which suggests that, like other members of the reticulon family, hRTN3 is a potential marker for neuroendocrine differentiation.     

高糖诱导ADTIQ的生成和多巴胺的代谢失衡

 2 型糖尿病患者比正常人更容易患帕金森病(PD),而新内源性神经毒素1-乙酰基-6, 7-二羟基-1, 2, 3,4-四氢异喹啉(ADTIQ)可能成为2 型糖尿病并发PD 的一个关键因素,ADTIQ 是由丙酮醛与多巴胺反应合成,但是其在多巴胺能神经元中的生成条件,以及在糖尿病情况下ADTIQ 的生成是否影响多巴胺代谢尚不清楚。本研究以SH-SY5Y 细胞为模型,研究ADTIQ 在细胞模型中的生成条件,发现ADTIQ 是在葡萄糖代谢旺盛的条件下,丙酮醛内源性产生与神经元内的多巴胺反应生成。研究揭示,高糖条件下,SH-SY5Y 细胞和2 型糖尿病的大鼠模型中外周多巴胺的质量浓度减少,而与多巴胺合成和转运相关的酪氨酸羟化酶和多巴胺转运体合成增加。可见,高糖能诱导SH-SY5Y 细胞内ADTIQ 的生成和多巴胺代谢的失衡,ADTIQ 的生成可能成为糖尿病并发PD 的关键因素。     

ADTIQ导致SH-SY5Y细胞凋亡和多巴胺转运体表达降低

新内源性神经毒素1-乙酰基-6,7-二羟基-1,2,3,4-四氢异喹啉(ADTIQ)生成与糖代谢密切相关,可能成为糖尿病并发PD的一个关键因素,但是其神经毒性还不清楚.针对ADTIQ的神经毒性,该研究采用SH-SY5Y细胞为模型,MTT法,Annexin V/PI双染法,Caspase 3/7活性检测细胞存活率和细胞凋亡,Western-blot检测多巴胺转运体(DAT)的表达量.结果表明,随着ADTIQ浓度增大,细胞的存活率逐渐下降,凋亡率增加,同时ADTIQ导致DAT表达降低.研究提示ADTIQ导致多巴胺神经元的凋亡与PD的发生息息相关,糖尿病引起ADTIQ增加,可能成为高糖损伤多巴胺神经元的一个重要原因.     

鱼藤酮诱导SH-SY5Y细胞对过氧化氢损伤易感性增高与硫氧还蛋白下调有关

帕金森病(PD)的发病机制与线粒体呼吸链复合物I(complex I)活性降低以及氧化应激损伤密切相关.使用complex I特异性抑制物鱼藤酮,损伤人神经母细胞瘤SH-SY5Y细胞后,给予H2O2造成氧化应激损伤,以研究细胞抗氧化损伤能力变化的可能机制.结果表明,鱼藤酮处理后,细胞对H2O2所致氧化损伤的易感性增高,且细胞形态及细胞存活率的改变与鱼藤酮浓度呈量效关系.与此同时,细胞内抗氧化蛋白之一,硫氧还蛋白(thioredoxin)水平在细胞损伤时明显下降.以上结果表明,线粒体complex I抑制对细胞氧化应激易感性的影响可能与胞内硫氧还蛋白水平降低有关,提示硫氧还蛋白在诊断神经元损伤和神经保护中有一定的运用前景.     

Sphingosine-1-phosphate induces Ca2+ mobilization via TRPC6 channels in SH-SY5Y cells and hippocampal neurons

Sphingosine-1-phosphate (S1P) is a widely expressed biologically active sphingolipid that plays an important role in cell differentiation, migration, proliferation, metabolism and apoptosis. S1P activates various signaling pathways, some of which evoke Ca2+ signals in the cytosol. Few studies have focused on the mechanism by which S1P evokes Ca2+ signals in neurons. Here, we show that S1P evokes global Ca2+ signals in SH-SY5Y cells and hippocampal neurons. Removal of extracellular calcium largely abolished the S1P-induced increase in intracellular Ca2+, suggesting that the influx of extracellular Ca2+ is the major contributor to this process. Moreover, we found that S1P-induced Ca2+ mobilization is independent of G protein-coupled S1P receptors. The TRPC6 inhibitor SAR7334 suppressed S1P-induced calcium signals, indicating that the TRPC6 channel acts as the downstream effector of S1P. Using patch-clamp recording, we showed that S1P activates TRPC6 currents. Two Src tyrosine kinase inhibitors, Src-I1 and PP2, dramatically inhibited the activation of TRPC6 by S1P. Taken together, our data suggest that S1P activates TRPC6 channels in a Src-dependent way to induce Ca2+ mobilization in SH-SY5Y cells and hippocampal neurons.     

SH-SY5Y与U87细胞共培养条件性培养基可降低N-甲基-salsolinol介导THP-1细胞的凋亡

 神经免疫在帕金森病(PD)的致病机理中发挥重要的作用,PD 患者的外周血淋巴细胞的数量发生了变化,提示外周免疫系统在PD 的发生发展中发挥一定的作用。但是外周单核细胞(PBMC)在其中发挥的具体作用尚不清楚。外源性神经毒素(MPTP)类似物,内源性神经毒素(NMSal)可能是导致PD 发生的一种因素。研究采用NMSal 损伤的SH-SY5Y与U87 细胞共培养的条件性培养基培养外周单核细胞THP-1,探讨NMSal 损伤的多巴胺能神经元细胞对外周单核细胞的影响。结果表明,该条件性培养基可以降低NMSal 毒性诱导的THP-1 细胞的凋亡、氧化应激水平(MDA 和H₂O₂)、线粒体的损伤和凋亡相关蛋白FADD、Bax 和caspase3 的表达和活化水平。PD 病人中损伤的多巴胺能神经元与星形胶质细胞的相互作用可能会影响PBMC,进而影响PD 病情的进展。     

Genome-wide distribution of histone H3 acetylation in all- trans retinoic acid induced neuronal differentiation of SH-SY5Y cells

With chromatin immunoprecipitation (ChIP) and promoter DNA microarray analyses (ChIP-on-chip), we analyzed the variations of acetylation on histone H3 in all-trans retinoic acid (RA) induced neuronal cell differentiation. Neuroblastoma SH-SY5Y cells were treated with RA for 24 h and the acetylation on histone H3 in the promoter region of the genes was detected. Results showed that, after treatment, the level of acetylation on histone H3 elevated in 597 genes in the genome, and reduced in the other 647 genes compared with those of the control. In summary, we have successfully adopted a high throughput technique to detect and analyze variations of acetylation of histone H3 in human genome at the early phage of RA induced neuronal differentiation of the SH-SY5Y cells. Supported by National Natural Science Foundation of China (Grant Nos. 90408007 and 30721063) and National Key Basic Research and Development Program of China (Grant No. 2004CB518605)     

Neuronal growth inhibitory factor inhibits pheochromo-cytoma PC12 in vitro

Neuronal growth inhibitory factor (GIF),named Metaliothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF's neuronal growth inhibitory activity.``     

Neuronal growth inhibitory factor inhibits pheochromocytoma PC12 in vitro

Neuronal growth inhibitory factor (GIF), named Metallothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF’s neuronal growth inhibitory activity.     

Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma

Proto-oncogenes may be important in the cellular processes central for the growth and differentiation of normal cells. N-myc is a DNA sequence which shares limited homology to the proto-oncogene c-myc and has been found to be amplified in both primary tissue and cell lines from neuroblastoma, a childhood tumour of neuroectodermal origin. Differentiation of this embryonal tumour is of clinical importance, since occasional tumours have been noted to differentiate in vivo to benign ganglioneuroma. In vitro, many human neuroblastoma cell lines can be induced to differentiate morphologically and biochemically by a variety of agents. Retinoic acid (RA), an analogue of vitamin A, has been shown to inhibit neuroblastoma cell growth and clonability in soft agar, and to induce extensive neurite outgrowth. Therefore we examined the relationship of N-myc expression to the in vitro differentiation of these cells. We report here that in the case of RA-induced differentiation, a decreased level of expression is detected within 6 h of treatment and precedes both cell-cycle changes and morphological differentiation.     

绿色荧光蛋白标记的NTE活性域表达载体的构建及表达 

利用含有特定限制性内切酶识别位点的引物，通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列，经T载体克隆测序正确后，双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFPN3中，通过酶切反应鉴定，构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNESTEGFP。采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SHSY5Y中，用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中，而且没有导致内质网膜的聚集，表明表达载体成功构建和表达。     

绿色荧光蛋白标记的NTE活性域表达载体的构建及表达

利用含有特定限制性内切酶识别位点的引物,通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列,经T载体克隆测序正确后,双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFP-N3中,通过酶切反应鉴定,构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNEST-EGFP.采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SH-SY5Y中,用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中,而且没有导致内质网膜的聚集,表明表达载体成功构建和表达.     

Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.     

Induction of c-fos during myelomonocytic differentiation and macrophage proliferation

Previous studies have suggested a role for c-fos in cellular differentiation in fetal membranes, haematopoietic cells and teratocarcinoma stem cells. In other cell types, such as fibroblasts, c-fos expression is normally very low, but is rapidly induced by peptide growth factors, implicating c-fos in growth control mechanisms. Here, we show that the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced macrophage-like differentiation of HL60 human promyelocytic precursor cells is accompanied by the induction of both c-fos mRNA and protein within 15 min after treatment, suggesting a functional role for c-fos in this differentiation system. In quiescent terminally differentiated macrophages, expression of c-fos is inducible by the macrophage-specific growth factor colony-stimulating factor-1 (CSF-1). The kinetics of c-fos induction, however, are entirely different from those in growth factor-stimulated fibroblasts, supporting the view that the c-fos gene product may serve different functions in different cell types.     

nm23-H1基因与人早幼粒白血病细胞HL-60增殖的相关性分析

目的探讨nm23-H1基因的表达与白血病细胞HL-60增殖之间的关系。方法：以25ng／mL阿糖胞苷处理HL-60细胞，MTT法测定细胞生长抑制率，NBT还原比色法判断细胞分化状况，RT-PCR检测nm23-H1基因表达的变化；构建nm23-H1基因的真核表达质粒pEGFP-N1-nm23-H1，转染HL-60细胞，通过细胞生长曲线和血清依赖性实验检测nm23-H1基因的过表达对HL-60细胞生长的影响。结果：小剂量Ara-C对HL-60细胞的生长呈时间依赖性抑制，作用4d后细胞NBT还原能力增强且nm23-H1基因的表达下调；转染nm23-H1基因的HL-60细胞生长加快、血清依赖性下降。结论：Ara-C对HL-60细胞增殖的抑制作用与下调nm23-H1基因的表达有-定关系；nm23-H1基因在HL-60细胞中的过表达有促细胞增殖的作用，即增高了HL-60细胞的恶性程度。