人钙结合蛋白S100A9基因克隆表达纯化及其对SH-SY5Y细胞作用的研究

钙结合蛋白S100A9在许多慢性疾病中聚集并可能在一些自身免疫性如老年痴呆中发挥了重要的作用。大量的报道也表明S100A9日益获得人们的注意。通过获取足量重组人S100A9蛋白,探索其对神经母细胞瘤细胞SH-SY5Y的作用及机制。运用全基因合成法合成人S100A9基因,构建人S100A9原核表达重组质粒p ET28a-S100A9,经单酶切法及PCR法鉴定重组质粒已构建成功。利用异丙基硫代-β-D-半乳糖苷(IPTG)在18℃、25℃、37℃分别诱导表达,经聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blotting鉴定,结果显示18℃时蛋白大量表达于上清中,在37℃时大量表达于包涵体。采用镍亲和层析法分别纯化两种来源重组蛋白,透析后低温冻干获得蛋白粉。使用CCK-8法检测上清来源和包涵体来源的S100A9蛋白对神经母细胞瘤细胞SH-SY5Y的增殖抑制作用。结果显示两种来源重组蛋白对SH-SY5Y细胞具有生长抑制作用,在浓度为0.05 mg/m L时即有明显抑制作用(p0.01),且二者作用无明显差异(p0.01)。采用AO/EB双重染色和流式细胞术初步检测S100A9对SH-SY5Y细胞的增殖抑制机理,结果显示该蛋白可促进SH-SY5Y细胞的凋亡。两种来源蛋白之间的比较也显示二者对于SH-SY5Y细胞作用无明显差异(p0.05)。本研究成功获得足量S100A9重组蛋白,且两种来源S100A9蛋白作用于SH-SY5Y细胞的生物学效应一致。     

低氧诱导因子-1α对SH-SY5Y细胞兴奋性毒性损伤的调节作用

探讨低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)在喹啉酸诱导人神经母细胞瘤SH-SY5Y细胞损伤中的作用。将体外培养的人神经母细胞瘤SH-SY5Y细胞分为对照组、喹啉酸低、中、高剂量组及HIF-1α抑制剂二甲氧基雌二醇(2-methoxyestradiol,2ME2)预处理喹啉酸高剂量组,采用噻唑蓝还原法和乳酸脱氢酶漏出率检测法测定细胞损伤程度,Hoechst 33342单荧光染色法观察细胞凋亡,免疫荧光染色法检测HIF-1α在细胞内的表达,免疫印迹法检测细胞HIF-1α、蛋白激酶B(protein kinase B,Akt)、磷酸化Akt(p-Akt)、Bcl-2和Bax的表达。结果显示,喹啉酸可剂量、时间依赖性地诱导SH-SY5Y细胞损伤,导致细胞凋亡。同时,喹啉酸可使SH-SY5Y细胞HIF-1α表达上调并发生核转位、p-Akt表达增加及Bax/Bcl-2表达比例增加,而2ME2可抑制喹啉酸诱导的SH-SY5Y细胞损伤及降低HIF-1α、p-Akt和Bax/Bcl-2的表达。由此说明,HIF-1α/Akt通路介导了喹啉酸诱导SHSY5Y的细胞凋亡。HIF-1α抑制剂(2ME2)能够减轻喹啉酸致SH-SY5Y细胞损伤程度,减少细胞凋亡。     

Doppel-induced cytotoxicity in human neuronal SH-SY5Y cells is antagonized by the prion protein

Doppel （Dpl） is a prion （PrP）-like protein due to the structural and biochemical similarities; however, the natural functions of Dpl and PrP remain unclear. In this study, a 531-bp human PRND gene sequence encoding Dpl protein was amplified from human peripheral blood leucocytes. Furl-length and various truncated human Dpl and PrP proteins were expressed and purified from Escherichia coil Supplement of the full-length Dpl onto human neuroblastoma cell SH-SY5Y induced remarkable cytotoxicity, and the region responsible for its cytotoxicity was mapped at the middle segment of Dpl [amino acids （aa） 81-122]. Interestingly, DpMnduced cytotoxicity was antagonized by the presence of full- length wild-type PrP. Analysis on fragments of PrP mutants showed that the N-terminal fragment （aa 23- 90） of PrP was responsible for the protective activity. A truncated PrP （PrPA32-121） with similar secondary structure as Dpl induced DpMike cytotoxicity on SH- SY5Y cells. Furthermore, binding of copper ion could enhance the antagonizing effect of PrP on Dpi-induced cytotoxicity. Apoptosis assays revealed that cytotoxicity induced by Dpl occurred through an apoptotic mechanism. These results suggested that the function of Dpl is antagonistic to PrP rather than synergistic.     

ERK1/2信号通路介导PGI_2对γ-分泌酶亚基APH-1的调控作用

本文主要探讨环氧合酶-2的下游代谢产物PGI_2通过ERK1/2信号通路调控γ-分泌酶亚基APH-1蛋白表达的影响。应用免疫荧光技术检测PGI_2对人源和鼠源的神经母细胞瘤SH-SY5Y和鼠源神经母细胞瘤n2a细胞APH-1蛋白表达情况的调控作用,同时应用Western blot和Real-Time PCR的方法进一步检测PGI_2调控APH-1表达的具体机制,最后利用ELISA实验检测PGI_2对细胞内Aβ表达的调控作用。结果发现PGI_2处理SH-SY5Y和n2a细胞24 h后可通过ERK1/2信号通路增加APH-1蛋白表达,进而明显增加Aβ表达。以上结果说明COX-2代谢产物PGI_2可通过促进APH-1的表达,进一步增加γ-分泌酶的剪切活性,从而使Aβ产生增加,最终加速阿尔茨海默病的发病进程。     

Neuroligin对SH-SY5Y细胞增殖和凋亡的影响

目的:探讨Neuroligin(NLG)对人神经母细胞瘤SH-SY5Y增殖和凋亡的影响。方法:体外培养SH-SY5Y细胞24 h后,分别用浓度为50,100,200μmol/L的NLG siRNA转染SH-SY5Y细胞并共孵育24 h,然后采用噻唑蓝(MTT)法检测不同浓度NLG siRNA对SHSY5Y细胞增殖率的影响,以及Real-time PCR法检测SH-SY5Y细胞中凋亡基因Bax、Bcl-2、Bcl-x L和caspase-9基因表达水平的变化。结果:与空白对照组及siRNA阴性对照组相比,转染NLG siRNA后SH-SY5Y细胞增殖率显著下降,细胞凋亡相关基因被激活,导致细胞凋亡。结论:NLG对神经元细胞具有保护作用,抑制神经细胞凋亡。     

叠氮钠、过氧化氢对SH-SY5Y细胞内硫氧还蛋白还原酶的影响

过度氧化应激是诱发许多神经退变病的重要因素。叠氮钠(NaN3)是线粒体有氧呼吸链细胞色素c氧化酶(COX)的特异性抑制剂,过氧化氢(H2O2)释放氧自由基造成氧化损伤,两者都可以用于氧化应激情况下神经元损伤模型的建立。硫氧还蛋白还原酶(thioredoxin reductase,TR)特异性的还原氧化型的硫氧还蛋白(thioredoxin,TRx),调节细胞中氧化还原的平衡。现以不同浓度NaN3或H2O2,处理人神经母细胞瘤细胞(SH-SY5Y细胞),建立损伤模型。通过MTT法、形态学方法检测SH-SY5Y细胞损伤程度。同时,通过Western blot定量法、免疫细胞化学法,检测损伤的SH-SY5Y细胞中TR含量的改变,观察TR在胞内的分布。实验表明,NaN3、H2O2,均以浓度依赖方式损伤SH-SY5Y细胞;TR分布于SH-SY5Y细胞的胞浆,表明TR是一种分泌蛋白,损伤后分布无明显变化。但一定浓度的NaN3作用后3h,胞内TR水平显著降低,即神经系统内呼吸链受损可抑制TR的表达,为神经退变病的防治提供了新的思路。     

氯化钾或谷氨酸对人神经母细胞瘤SH-SY5Y细胞钙离子通透性的影响

用人神经母细胞瘤SH-SY5Y细胞系作为研究对象,通过测定细胞存活率(MTT法)和脂质过氧化代谢产物丙二醛(MDA),探讨了氯化钾(KCl)或谷氨酸分别对入神经母细胞瘤SH-SY5Y细胞的损伤作用。结果发现在含Ca     

山楂叶金丝桃苷对高糖诱导SH-SY5Y细胞损伤的保护作用及机制

为研究金丝桃苷对高糖诱导的人神经母细胞瘤(SH-SY5Y)细胞氧化损伤的保护作用及机制,用含100mmo L/L葡萄糖和分别为20、50、100μmo L/L金丝桃苷的培养基共同孵育SH-SY5Y细胞36 h,检测细胞活力、细胞培养液中乳酸脱氢酶(LDH)水平及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,细胞内活性氧(ROS)水平、丙二醛(MDA)、还原型谷胱甘肽(GSH)含量和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性及SIRT1和NF-кB基因的mRNA水平和蛋白含量。结果显示金丝桃苷可提高高糖诱导后SH-SY5Y细胞的存活率,抑制细胞LDH释放,清除ROS,降低MDA含量与caspase-3活性,增强SOD、CAT活性和GSH含量;同时,金丝桃苷还能提高SIRT1基因的mR-NA表达及蛋白含量,降低NF-кB基因的mRNA水平和蛋白含量。结果表明金丝桃苷能通过激活SIRT1基因,抑制NF-кB基因保护高糖所致SH-SY5Y细胞的氧化损伤。     

色钉菇粗多糖对MPP~+损伤的SH-SY5Y细胞的保护作用

本文旨在研究色钉菇(Chroogomphus rutilus)粗多糖对多巴胺能神经元的保护作用。用浓度为200、400和800μg/mL的色钉菇粗多糖对SH-SY5Y细胞进行预处理后,加入1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium,MPP+)处理48h。采用MTT法检测MPP+损伤的SH-SY5Y细胞的存活率,用Annexin V-FITC染色和流式细胞术检测细胞凋亡率。结果显示,400μg/mL和800μg/mL的色钉菇粗多糖预处理可显著提高MPP+损伤的SH-SY5Y细胞存活率,降低细胞凋亡率。为了排除色钉菇粗多糖通过直接结合来降低MPP+实际浓度的可能性,将多糖洗净后,再予以细胞MPP+处理,MTT结果显示800μg/mL的色钉菇粗多糖预处理仍可提高细胞存活率。以上结果提示,色钉菇粗多糖对SH-SY5Y细胞起到有效的保护作用,其保护作用的大部分与多糖和MPP+的直接结合无明显关系,而是源于多糖对细胞的直接作用,因此色钉菇多糖有开发成防治帕金森病药物的应用前景。     

PXR激活在拮抗SH-SY5Y细胞凋亡中的作用

人神经母细胞瘤细胞SH-SY5Y细胞可以表达神经元特异性的酪氨酸羟化酶、多巴胺-β-羟化酶以及多巴胺转运体等,因此可用于建立帕金森病的体外模型。虽然帕金森综合症发病的确切机制至今尚不清楚,但众多的病理学资料证实该病患者存在中脑黑质多巴胺能神经元的凋亡。自由基、兴奋性     

Transient expressions of doppel and its structural analog prionΔ32-121 in SH-SY5Y cells caused cytotoxicity possibly by triggering similar apoptosis pathway

Doppel (Dpl) is a recently identified prion (PrP)-like protein due to the structural and biochemical similarities, however, its natural function and pathogenic role in neurodegenerative diseases remains unclear. To investigate the possible pathogenic pathway of Dpl and its structural analog for cell apoptosis, mammalian expressing recombinant plasmids containing human PRND gene encoding the full-length Dpl and a truncated human PRNP gene deleting the sequences encoding the peptide from aa 32 to 121 (PrPΔ32-121) were generated. MTT assays showed the cell viabilities of the human neuroblastoma cell line SH-SY5Y receiving Dpl and PrPΔ32-121 expressing plasmids were remarkably lower. Obvious apoptosis phenomena were observed to be associated with the cells transient expressing Dpl and PrPΔ32-121, including reduced mitochondrial transmembrane potential (ψm), decreased pro-caspase-3 quantity, more numbers of annexin V- and annexin V/PI-double-stained cells and depressed Bcl-2 level. Moreover, we also found that the Dpl- and PrPΔ32-121-induced cytotoxicities and relevant apoptotic events in SH-SY5Y cells could be fully antagonized by co-expression of the human full-length PrP. These data highly indicate that cytotoxicity induced by the expression of Dpl and truncated PrP in neural derived cells are closely related with the apoptosis process, probably triggering the mitochondrial pathway. It also implies that the cell-benefit activity of the full-length PrP may result from its anti-apoptosis capacity.     

Doppel蛋白及其对动物生殖的影响

Doppel(简称Dpl)是新发现的一种糖基磷脂酰肌醇锚定(GPI)结构糖蛋白, 在结构上与朊蛋白(Prion protein, PrP)相似, 其编码基因位于朊蛋白编码基因(Prion protein gene, PRNP)下游, 但在生理功能上两者差异较大。Dpl蛋白在成年动物体内的表达主要集中在睾丸组织, 对雄性动物精子完整性、活力以及维持正常受精能力等生殖功能具有重要作用。以下主要综述了Dpl蛋白的生物学特征、生理功能以及对雄性动物生殖调控的影响, 旨在为Dpl蛋白的功能研究和雄性动物生殖调控研究提供理论参考。     

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.     

The prion-like protein Doppel fails to interact with itself, the prion protein and the 37 kDa/67 kDa laminin receptor in the yeast two-hybrid system

The prion-like protein termed Doppel (Dpl) shows approx. 25% sequence identity with all known prion proteins (PrP). We recently showed that the cellular PrP is dimeric under native conditions, a finding which was confirmed by the investigation of its crystal structure. Human PrP further interacts with its cellular receptor, the 37 kDa/67 kDa laminin receptor (LRP/LR). Here we report that human Doppel fails to interact with (i). itself, (ii). the human 37 kDa/67 kDa LRP/LR, and (iii). the human cellular prion protein (huPrP) in the yeast two-hybrid system. Our findings suggest that Dpl and PrP are not related or are only marginally related with respect to their ligand binding behaviour.     

The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa

The prion-like Doppel protein (Dpl) has many biochemical and structural properties in common with the cellular prion protein (PrP(c)), and the physiological role of neither protein is known. Experimental data suggest either direct or indirect interaction between the two proteins. In this study, we investigated the expression pattern and biochemical characteristics of Dpl in human tissues and in Chinese hamster ovary cells transfected with wild-type or variant human Dpl gene constructs. Human Dpl appears to be a glycosylphosphatidylinositol-anchored glycoprotein with N- and O-linked sugars. It was found on Sertoli cells in the testis, on the flagella of epididymal and mature spermatozoa, and in seminal plasma. Dpl coexists only with N-terminally truncated isoforms of PrP(c) on mature spermatozoa. The localization of human Dpl on both Sertoli cells (somatic cells) and spermatozoa (germinal cells) strongly suggests that this protein may play a major role in human male fertility. Finally, our data indicate that spermatozoa are thus an interesting model for studies of the potential interaction between Dpl and PrP(c).     

Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility.     

Control of the expression of human neuropeptide Y by leptin: in vitro studies

Neuropeptide Y (NPY) participates in the regulation of reproduction and food intake. The adipose-secreted hormone, leptin, has also been involved in these processes, and has been shown to exert its effects in part by controlling NPY synthesis and release at the hypothalamic level. In the present study, we utilized the SH-SY5Y human neuroblastoma cell line, to study the leptin-NPY interrelationships. SH-SY5Y cells were found to express leptin receptors (RT-PCR and Western blot analyses). A 24-h treatment with leptin at different concentrations did not affect NPY gene expression, but resulted in a stimulation of NPY release. This stimulated secretion was blocked by the combined treatment with leptin and the muscarinic agonist carbachol or the phorbol ester TPA. Leptin and carbachol also caused an increased intracellular content of NPY. In conclusion, the SH-SY5Y human neuroblastoma cell line appears to be a suitable in vitro model for studying the pharmacological effects of leptin on the biosynthesis and secretion of NPY.     

Tissue- and cell type-specific modification of prion protein (PrP)-like protein Doppel, which affects PrP endoproteolysis

A prion protein (PrP)-like protein, Doppel (Dpl) is a homologue of cellular PrP (PrP^C). Immunoblotting revealed heterogeneous glycosylation patterns of Dpl and PrP^C in several cell lines and tissues, including brain and testis. To investigate whether the glycosylation and modification of Dpl and PrP^C could influence each other, PrP gene (Prnp)-deficient neuronal cells, transfected with Prnp and/or the Dpl gene (Prnd), were analyzed by deglycosylation with peptide N-glycosidase F. The modification of Dpl was not influenced by PrP^C, whereas an N-terminally truncated fragment of PrP^C was reduced by Dpl expression. These results indicated that Dpl was glycosylated in a cell type- and tissue-specific manner regardless of PrP^C, while PrP^C endoproteolysis was modulated by Dpl expression.