PS1/GFP融合蛋白对PS1的亚细胞定位的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系.构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上.     

PS1/GFP融合蛋白对PS1的亚细胞定位与功能的初步研究

PS1基因突变与早发家族性老年痴呆有密切联系。本文构建pEGFP-C1-PS1以及pEGFP-N2-PS1融合基因表达载体,于HEK293和CHO细胞系中表达PS1/GFP融合蛋白,以GFP绿色荧光作为PS1的亚细胞定位信号,通过SPOTII以及CONFOCAL显微镜进行观察,初步获得PS1全长蛋白在细胞中定位的部分信息,即本实验条件下,PS1定位于细胞核膜,细胞质内有不均匀的分布,少量存在于细胞-细胞接触处的细胞膜上。     

TUDCA, a Bile Acid, Attenuates Amyloid Precursor Protein Processing and Amyloid-β Deposition in APP/PS1 Mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid-β (Aβ) peptide in the hippocampus and frontal cortex of the brain, leading to progressive cognitive decline. The endogenous bile acid tauroursodeoxycholic acid (TUDCA) is a strong neuroprotective agent in several experimental models of disease, including neuronal exposure to Aβ. Nevertheless, the therapeutic role of TUDCA in AD pathology has not yet been ascertained. Here we report that feeding APP/PS1 double-transgenic mice with diet containing 0.4 % TUDCA for 6 months reduced accumulation of Aβ deposits in the brain, markedly ameliorating memory deficits. This was accompanied by reduced glial activation and neuronal integrity loss in TUDCA-fed APP/PS1 mice compared to untreated APP/PS1 mice. Furthermore, TUDCA regulated lipid-metabolism mediators involved in Aβ production and accumulation in the brains of transgenic mice. Overall amyloidogenic APP processing was reduced with TUDCA treatment, in association with, but not limited to, modulation of γ-secretase activity. Consequently, a significant decrease in Aβ(1-40) and Aβ(1-42) levels was observed in both hippocampus and frontal cortex of TUDCA-treated APP/PS1 mice, suggesting that chronic feeding of TUDCA interferes with Aβ production, possibly through the regulation of lipid-metabolism mediators associated with APP processing. These results highlight TUDCA as a potential therapeutic strategy for the prevention and treatment of AD.     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响。应用基因芯片技术对于pcDNA3.1()和pcDNA3.1()PS1TP1分别转染的HepG2细胞的基因表达谱进行分析。以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1()PS1TP1。以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1()的HepG2细胞进行DNA芯片分析并比较。在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调。PS1TP1基因的表达对于肝细胞基因表达谱有显著影响。DNA芯片技术是分析反式调节靶基因的有效技术途径。     

应用微阵列技术筛选PS1TP1基因转染细胞差异表达基因

阐明乙型肝炎病毒(HBV)前-S1蛋白反式激活蛋白1(PS1TP1)的表达对于肝细胞的基因表达谱的影响.应用基因芯片技术对于pcDNA3.1(-)和pcDNA3.1(-)-PS1TP1分别转染的HepG2细胞的基因表达谱进行分析.以肝癌细胞系HepG2基因作为模板,应用聚合酶链反应(PCR)技术扩增PS1TP1基因片段,以常规的分子生物学技术构建表达载体pcDNA3.1(-)-PS1TP1.以脂质体技术转染肝母细胞瘤细胞系HepG2,提取总RNA,逆转录为cDNA,与转染空白表达载体pcDNA3.1(-)的HepG2细胞进行DNA芯片分析并比较.在4096个基因表达谱的筛选中,发现有8个基因表达水平显著上调,14个基因表达水平显著下调.PS1TP1基因的表达对于肝细胞基因表达谱有显著影响.DNA芯片技术是分析反式调节靶基因的有效技术途径.     

Daddy, where did (PS)I come from?

The reacton centre I (RCI)-type photosystems from plants, cyano-, helio- and green sulphur bacteria are compared and the essential properties of an archetypal RCI are deduced. Species containing RCI-type photosystems most probably cluster together on a common branch of the phylogenetic tree. The predicted branching order is green sulphur, helio- and cyanobacteria. Striking similarities between RCI- and RCII-type photosystems recently became apparent in the three-dimensional structures of photosystem I (PSI), PSII and RCII. The phylogenetic relationship between all presently known photosystems is analysed suggesting (a) RCI as the ancestral photosystem and (b) the descendence of PSII from RCI via gene duplication and gene splitting. An evolutionary model trying to rationalise available data is presented.     

Alzheimer病患者APP及PS1基因表达量的研究

为了检测Alzheimer病（Alzheimer’s disease,AD）患者外周血中淀粉样前体蛋白（Amyloid Precursor Protein, APP）基因及早老素1（Presenilin 1, PS1）基因的表达情况,进而探讨APP及PS1基因的表达与AD的相关性,采用SYBRGreenⅠ的方法对45例AD患者、25例血管性痴呆（vascular dementia, VD）患者及60名正常对照组样本的mRNA进行绝对定量,检测得到APP基因及PS1基因在对照组中的表达水平分别为0.026±0.005 amol/μg cDNA和0.026±0.004 amol/μg cDNA;在AD患者组中的表达量分别为0.044±0.006 amol/μg cDNA和0.051±0.011 amol/μg cDNA;,在VD患者组中的表达水平分别为0.072±0.013 amol/μg cDNA和0.039±0.005 amol/μg cDNA 。经显著性检验,AD患者组APP基因的表达水平上调,t=2.639, P<0.01;PS1基因的表达水平同样呈上调趋势,t=2.173,P<0.05,差异均具有统计学意义。VD患者组APP基因的表达水平上调,t=3.028,P<0.01;PS1基因的表达水平也同样呈上调趋势,t=2.012,P<0.05,均有显著性差异。因此,APP及PS1基因的表达水平的增高并不一定与AD发生特异性关联,而可能与多种导致痴呆的脑部病变发生关联。     

鼠PS1-GFP融合蛋白表达载体的构建研究

Presenilin1(PS1)的突变是早发家族性老年痴呆发生的重要因素之一。目前对于PS1结构和功能的研究表明,PS1作为一种多次跨膜并具有γ-secretase活性的蛋白酶,很可能参与众多的细胞信号传导通路,影响细胞分化和调亡、组织及个体的发育等等。但对于PS1的精细结构和功能目前了解甚少,为探讨PS1的精细结构和功能,本研究构建了鼠PS1-GFP融合蛋白表达载体。方法:我们设计了扩增PS1的cDNA全长的引物,以C57BL/6小鼠9天胚的RNA为模板,利用RT-PCR的方法从C57BL/6小鼠9天胚中获得PS1的cDNA,经测序确认后,将其克隆到pMD18-TVector中。设计引物:上游5’-GCCGAATTCTATGACAGAGATACCTGCACC-3’;下游5’-GAAGGATCCGATATAAAACTGATGGAATGC-3’,上游含有EcoRI酶切位点,下游含有BamHI酶切位点。利用高保真DNA聚合酶通过PCR方法将pMD18-T-PS1质粒中的PS1基因亚克隆到pEGFP-C1载体中,获得pEGFP-C1-PS1融合蛋白表达载体,然后利用EcoRI和BamHI从pEGFP-C1-PS1融合蛋白中切下PS1基因,从pMD18-T-PS1质粒中切下载体部分,经过连接,获得中间载体,利用该重组载体中的EcoRI和SalI酶切位点,酶切连接入pEGFP-N2载体中,获得pEGFP-N2-PS1融合蛋白表达载体,经测序鉴定后备用。     

水溶性phellinus多糖PS1的分离提取及免疫活性研究

目的：从Phellinus pini子实体中提取分离到一种水溶性多糖纽分PS1,并对其免疫活性进行初步研究。方法：先进行热水提取,DEAE-纤维素离子交换树脂,sepharose CL-4B凝胶树脂分离纯化PS1,并利用GC色谱、FT-1R光谱进行组成成分分析,然后通过刚果红吞噬实验、脾脏B淋巴细胞增殖实验及免疫器官指数评价其免疫活性。结果：PS1为一种β型杂多糖,总糖含量70．36％,蛋白质含量4．69％,分子量为230kDa。糖类构成有鼠李糖、阿拉伯糖、木糖、甘露糖、半乳糖、葡萄糖,其摩尔比为2．14：2．36：0．44：4．28：5．17：85,61。多糖PS1能显著提高正常小鼠的脾指数和胸腺指数（P〈0．01）,免疫力低下小鼠的肝指数、胸腺指数也有提高,但效果不明显．PS1均能提高正常和免疫低下小鼠的巨噬细胞吞噬能力。此外。PS1能显著促进脾细胞体外增殖能力（P〈0．01）。结论：多糖PS1能显著提高机体的免疫能力。可作为一种生物效应调节剂应用于医药或保健食品领域。     

PS1基因突变敲入小鼠B免疫记忆细胞功能变化

探讨PS1基因突变敲入小鼠B免疫记忆细胞功能变化。OVA免疫PS1基因突变敲入小鼠,通过ELISA抗体检测观察B细胞免疫记忆功能;分离小鼠脾淋巴细胞,检测脾淋巴细胞增殖实验、B细胞表面抗原表达情况。痘苗病毒免疫正常和基因突变敲入小鼠两次,在免疫后的一年进行痘病毒腹腔攻毒实验,观察PS1基因突变敲入小鼠存活情况。通过分离中枢和外周淋巴细胞进行体外功能检测表明,PS1基因突变敲入小鼠细胞增殖功能明显降低(P0.0001);B记忆细胞表面蛋白表达有差异(P=0.045);痘苗攻毒实验结果表明,PS1基因突变敲入小鼠死亡率明显增高(P0.0001)。研究结果表明,PS1基因在调控B记忆细胞功能方面发挥重要作用,但是通过调控T细胞功能而发挥间接还是直接针对B细胞发挥调控作用,还需要进一步研究结果确认。     

Development of Photosystem II in dark grown Chlamydomonas reinhardtii. A light-dependent conversion of PS IIβ, QB-nonreducing centers to the PS IIα, QB-reducing form

The green alga Chlamydomonas reinhardtii is a facultative heterotroph and, when cultured in the presence of acetate, will synthesize chlorophyll (Chl) and photosystem (PS) components in the dark. Analysis of the thylakoid membrane composition and function in dark grown C. reinhardtii revealed that photochemically competent PS II complexes were synthesized and assembled in the thylakoid membrane. These PS II centers were impaired in the electron-transport reaction from the primary-quinone electron acceptor, Q_A, to the secondary-quinone electron acceptor, Q_B (Q_B-nonreducing centers). Both complements of the PS II Chl a–b light harvesting antenna (LHC II-inner and LHC II-peripheral) were synthesized and assembled in the thylakoid membrane of dark grown C. reinhardtii cells. However, the LHC II-peripheral was energetically uncoupled from the PS II reaction center. Thus, PS II units in dark grown cells had a -type Chl antenna size with only 130 Chl (a and b) molecules (by definition, PS II units lack LHC II-peripheral). Illumination of dark grown C. reinhardtii caused pronounced changes in the organization and function of PS II. With a half-time of about 30 min, PS II centers were converted froma Q_B-nonreducing form in the dark, to a Q_B-reducing form in the light. Concomitant with this change, PS II units were energetically coupled with the LHC II-peripheral complement in the thylakoid membrane and were converted to a PS II form. The functional antenna of the latter contained more than 250 Chl(a+b) molecules. The results are discussed in terms of a light-dependent activation of the Q_A-Q_B electron-transfer reaction which is followed by association of the PS II unit with a LHC II-peripheral antenna and by inclusion of the mature form of PS II (PS II) in the membrane of the grana partition region.Abbreviations Chl chlorophyll - PS photosystem - Q_A primary quinone electron acceptor of PS II - Q_B secondary quinone electron acceptor of PS II - LHC light harvesting complex - F₀ non-variable fluorescence yield - F_plf intermediate fluorescence yield plateau leyel - F_max maximum fluorescence yield - F_i initial fluorescence yield increase from F₀ to F_pl (F_pl–F₀) - F_v total variable fluorescence yield (F_m–F0) - DCMU dichlorophenyl-dimethylurea     

Aβ在APP／PS1双转基因与APP／PS1／Tau三转基因阿尔茨海默病模型小鼠脑海马区分布的比较研究

目的：比较研究Aβ免疫阳性产物在老年APP／PSI双转基因（2xTg）与APP／PSI／Tau三转基因（3xTg）阿尔茨海默病模型小鼠海马的分布。方法：采用15月龄2xTg与同龄3xTg,分别进行Aβ单克隆抗体6E10免疫组化检测AB阳性神经元及斑块。结果：在海马区2xTg组Aβ沉积多发生于细胞外,形成大量Aβ阳性斑,而3xTg组则主要沉积于细胞内。结论：这种Aβ分布的差异可能与3xTg模型早期即有神经元丢失有关。     

Cellular FLICE-like Inhibitory Protein (c-FLIP) and PS1-associated Protein (PSAP) Mediate Presenilin 1-induced γ-Secretase-dependent and -independent Apoptosis,Respectively

Presenilin 1 (PS1) has been implicated in apoptosis; however, its mechanism remains elusive. We report that PS1-induced apoptosis was associated with cellular FLICE-like inhibitory protein (c-FLIP) turnover and that γ-secretase inhibitor blocked c-FLIP turnover and also partially blocked PS1-induced apoptosis. A complete inhibition of PS1-induced apoptosis was achieved by knockdown of PS1-associated protein (PSAP), a mitochondrial proapoptotic protein that forms a complex with Bax upon induction of apoptosis, in the presence of γ-secretase inhibitor. PS1-induced apoptosis was partially inhibited by knockdown of caspase-8, Fas-associated protein with death domain (FADD), or Bid. However, knockdown of Bax or overexpression of Bcl-2 resulted in complete inhibition of PS1-induced apoptosis. These data suggest that PS1 induces apoptosis through two pathways: the γ-secretase-dependent pathway mediated by turnover of c-FLIP and the γ-secretase-independent pathway mediated by PSAP-Bax complex formation. These two pathways converge on Bax to activate mitochondria-dependent apoptosis. These findings provide new insight into the mechanisms by which PS1 is involved in apoptosis and the mechanism by which PS1 exerts its pathogenic effects. In addition, our results suggest that PS2 induces apoptosis through a pathway that is different from that of PS1.     

APP/PS1双转基因小鼠的繁育、鉴定及评价

目的：评价APP/PS1双转基因小鼠基因表达及认知行为能力的变化,为AD的相关研究提供有效的动物模型。方法：采用雄、雌鼠1：1合笼配对的方式,令APP/PS1双转基因小鼠自然交配进行繁育。PCR鉴定APP/PS1双转基因鼠仔鼠的基因型后,选择APP/PS1阳性小鼠作为模型（AD）组,同批APP/PS1阴性为对照（CT）组,每组8只小鼠。以Morris水迷宫实验检测仔鼠的空间学习记忆能力,以HE染色、刚果红染色观察仔鼠脑片组织病理学改变。结果：①APP/PS1双转基因鼠仔鼠基因经PCR扩增,出现约360 bp的目的基因条带,表明成功繁育出转入APP/PS1基因的仔鼠;②Morris水迷宫实验结果显示,与7月龄阴性小鼠（CT组）比较,同月龄的双转基因AD组小鼠的空间学习记忆能力明显降低（P<0.05）;③HE染色结果显示,AD组小鼠海马结构及细胞形态出现明显异常;刚果红染色结果显示,AD组小鼠脑片组织出现β淀粉样蛋白斑块沉积。结论：APP/PS1双转基因小鼠较好地模拟了AD的病理变化及行为学特征,可作为研究AD发病机制及开发AD防治药物的实验工具。     

APP/PS1转基因老年性痴呆模型小鼠肠道甲醛浓度异常升高

【目的】肠道菌群通过"微生物-肠道-脑轴"影响中枢神经系统的功能,同时也与老年性痴呆的发生发展相关,特别是盲肠内微生物菌群的变化更为显著。肠道菌群可以产生和代谢甲醛,而肠道能够迅速吸收甲醛;体内甲醛含量与老年性痴呆病人的认知损害程度呈正相关。因此,本文比较了7月龄APP/PS1转基因老年性痴呆模型小鼠(简称APP/PS1转基因小鼠)与同月龄C57BL/6J野生型小鼠(简称C57BL/6J小鼠)肠道菌群产生甲醛的情况。【方法】取APP/PS1转基因小鼠(n=8)与C57BL/6J小鼠(n=9)的不同肠段(十二指肠、小肠、盲肠、结肠),采用2,4-Dinitrophenylhydrazi zne(DNPH)显色偶联高效液相色谱(HPLC coupled with DNPH)测定肠道消化物和肠壁组织的甲醛。【结果】APP/PS1转基因小鼠盲肠消化物内的甲醛含量,较C57BL/6J小鼠存在显著升高(P=0.036);而两者小肠和结肠消化物甲醛含量无显著差别。在两种小鼠之间,小肠壁内甲醛存在差异(P=0.052),而盲肠和结肠壁甲醛含量无显著差异(P0.05)。【结论】肠道菌群是小鼠体内甲醛的主要来源之一,无论肠道消化物,还是肠道壁组织均为盲肠的甲醛含量最高。这些结果表明,APP/PS1转基因小鼠肠道菌群存在甲醛代谢失调,从而导致其肠道消化物的甲醛含量升高。     

siRNA against presenilin 1 (PS1) down regulates amyloid β42 production in IMR-32 cells

Background  

One of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.     

APP/PS1转基因小鼠海马结构UCH-L1的表达及亚甲蓝对其改变的影响

目的探讨亚甲蓝(MB)对APP/PS1转基因小鼠记忆相关蛋白泛素羧基末端水解酶1(carboxyl-terminalhydrolase-L1,UCH-L1)在海马结构的表达及记忆改善的影响。方法 3月龄APP/PS1转基因小鼠及相同品系的野生小鼠分为3组,每组10只:治疗组,APP/PS1小鼠口服亚甲蓝(25mg/kg/d)4个月;模型组,APP/PS1小鼠无药物干预;对照组为正常野生小鼠。待三组小鼠均为7月龄时,跳台实验测试三组小鼠的学习记忆能力;Western blot及免疫荧光技术检测海马结构UCH-L1的含量变化。结果亚甲蓝可以减少小鼠跳台试验错误次数,延长小鼠跳台试验的潜伏期(P<0.01)。亚甲蓝治疗组海马结构的可溶性UCH-L1含量明显增多(P<0.01)。结论亚甲蓝可能是通过上调海马结构可溶性UCH-L1的表达改善APP/PS1小鼠的学习记忆能力。     

游泳运动对APP/PS1转基因小鼠学习记忆能力的影响

探讨游泳运动对APP/PS1转基因小鼠学习记忆能力的影响。选择11月龄的雄性APP/PS1转基因小鼠,随机平均分为对照组和游泳组,对照组常规饲养而游泳组进行1个月的游泳运动训练。分别采用刚果红染色、Tunel检测、Western Blot和Morris水迷宫等实验方法观察小鼠大脑皮层Aβ斑形成、神经元凋亡、线粒体生成相关蛋白表达和学习记忆能力的变化情况。结果发现,游泳运动可以减少APP/PS1转基因小鼠大脑皮层Aβ斑的形成、抑制神经元凋亡、促进线粒体生成、增强小鼠的学习记忆能力。由此可见,游泳运动可作为一项防治阿尔茨海默病的行为治疗候选方案。