淀粉样前体蛋白在痴呆模型大鼠背海马结构内的表达

研究用ＡｌＣｌ３灌胃，建立鼠老年性痴呆模型。用免疫细胞化学ＡＢＣ法，观察了淀粉样前体蛋白在背海马结构的表达。结果显示在痴呆模型鼠背海马结构双侧各部均可见较强的淀粉样前体蛋白表达，且与对照组间有显著性差异。光镜结果，阳性反应产物主要位于神经元核周质和突起及其分支。细胞大小不等，多数淀粉样前体蛋白样免疫反应阳性神经元胞体为梭形和锥形。背海马结构内淀粉样前体蛋白样免疫反应神经元主要分布在ＣＡＩ、ＣＡ２、     

β-淀粉样前体蛋白转基因小鼠发育过程中海马 CAI区神经细胞凋亡变化

目的:探讨瑞典家系β-淀粉样前体蛋白(APPSWE)转基因小鼠发育过程中海马 Cal 区神经细胞凋亡规律.方法:取不同发育时间(P0、P7、P14、P30、P90、P180)APPSWE 转基因模型鼠与同时间点对照鼠,Nissl 染色观察海马结构和锥体细胞形态,免疫组织化学方法观察海马细胞内 Caspase-3 表达变化,RT-PCR 检测 Caspase-3 mRNA 表达变化.结果:随着小鼠的生长发育,P14 时间点以后,模型组 Cal 区神经元 Caspase-3 阳性细胞密度比对照组高;RT-PCR 检测结果与Caspase-3 免疫组织化学结果基本一致.结论:APPSWE 转基因小鼠发育中的海马神经细胞过度凋亡可能与阿尔茨海默病的发生、发展具有联系.     

β-淀粉样前体蛋白转基因小鼠发育过程中海马CA1区神经细胞凋亡变化

目的：探讨瑞典家系β-淀粉样前体蛋白（APPSWE）转基因小鼠发育过程中海马CA1区神经细胞凋亡规律。方法：取不同发育时间（P0、P7、P14、P30、P90、P180）APPSWE转基因模型鼠与同时间点对照鼠，Nissl染色观察海马结构和锥体细胞形态，免疫组织化学方法观察海马细胞内Caspase-3表达变化，RT-PCR检测Caspase-3mRNA表达变化。结果：随着小鼠的生长发育，P14时间点以后，模型组CA1区神经元Caspase-3阳性细胞密度比对照组高；RT-PCR检测结果与Caspase-3免疫组织化学结果基本一致。结论：APPSWE转基因小鼠发育中的海马神经细胞过度凋亡可能与阿尔茨海默病的发生、发展具有联系。     

嗅鞘细胞移植入淀粉样前体蛋白转基因小鼠脑内降低β淀粉样蛋白沉积

目的 观察嗅鞘细胞移植入淀粉样前体蛋白(APP)转基因小鼠脑内后的存活和迁移状况及其对模型β淀粉样蛋白沉积的作用,以进一步探索嗅鞘细胞移植对阿尔茨海默病的治疗效果.方法 取自发绿色荧光蛋白的新生3d龄增强型绿色荧光蛋白(EGFP)转基因小鼠,体外分离培养嗅球嗅鞘细胞,在小鼠脑立体定位仪的固定下,参照AP小鼠脑立体定位图谱,将收集的嗅鞘细胞移植入APP转基因小鼠脑内.移植1个月后,取脑组织冷冻切片,在双光子共焦显微镜下观察细胞存活和迁移状况并采集图像.移植1个月后,行荧光免疫组织化学和免疫印迹法检测淀粉样蛋白沉积,每组4只.结果 嗅鞘细胞移植入APP转基因小鼠脑内存活良好,并以移植点为中心向外迁移,部分绿色荧光细胞呈现成血管现象,移植之后APP转基因小鼠脑内的β淀粉样蛋白沉积减少,APP表达明显减少(P<0.05).结论 嗅鞘细胞对减少APP转基因小鼠脑内淀粉样蛋白沉积有一定帮助.     

β-淀粉样前体蛋白分子生物学研究进展

β-淀粉样前体蛋白基因定位于21号染色体长臂,具“管家基因”结构特点。在组织中广泛表达但有一定的特异性。其近侧启动子区(-96～+101)为转录活性所必需,研究发现β-淀粉样前体蛋白基因表达调控由多种顺式元件和反式因子的相互作用所决定。β-淀粉样前体蛋白呈细胞表面受体构象,具有多种生物学功能。业已证明其分子生物学功能的改变与Alzheimer's病的发生、发展有着十分密切的关系。     

高表达人淀粉样前体蛋白降低SH-SY5Y细胞M胆碱受体结合

目的观察阿尔茨海默病致病相关基因淀粉样前体蛋白(APP)基因的高表达致高度生成,对人神经母细胞瘤SH-SY5Y细胞的胆碱乙酰转移酶(ChAT)活性及M和N胆碱受体功能的影响,探讨β-淀粉样肽(Aβ)生成增加对细胞胆碱功能的作用。方法采用Lipofectamine 2000试剂盒将携带野生型人APP基因的pCMV695质粒转染至人神经母细胞瘤SH-SY5Y细胞中。以ELISA方法测定Aβ生成量。取稳定高表达Aβ细胞的克隆,用放射免疫法测定ChAT的活性;放射配基结合测定M、N乙酰胆碱受体结合。结果与未转染APP的SH-SY5Y细胞相比,当Aβ生成量为对照的2~2.6倍时,SH-SY5Y-APP细胞中未观察到明确的细胞毒性形态学改变;ChAT活性无明显变化,但细胞M受体结合降低18.5%~21.8%(P<0.05);N受体结合未出现明显变化。结论单纯Aβ产生增加就可通过影响M受体结合功能而引起脑内胆碱能功能障碍。     

雌激素减少去卵巢兔高胆固醇诱导的淀粉样前体蛋白信使RNA过表达并抑制前额叶皮层Aβ表达(英文)

长期以来循环胆固醇增高被认为与冠心病相关,现在认为与阿尔茨海默病(Alzheimer’s disease,AD)也有关联。雌激素是一种神经保护剂,为了证明胆固醇促进APP mRNA表达和雌激素的抑制作用及其与AD的关系,我们测定了接受胆固醇和/或雌激素兔血清脂质,显示了其APP mRNA和Aβ表达。与先前报道相似,胆固醇饮食增高血清TC,TG,LDL-C和HDL-C。雌激素可逆转胆固醇饮食诱导的血清TC,TG和LDL-C增高,但降HDL-C作用不明显。高胆固醇饮食去卵巢兔APP mRNA过度表达。高胆固醇饮食同时补充雌激素去卵巢兔与正常兔间无显著差异。高胆固醇去卵巢兔Aβ刚果红染色阳性。我们的结果提示,补充雌激素降低胆固醇可能有效地预防AD。     

胆碱能前体细胞移植入β-淀粉样前体蛋白转基因小鼠脑内的存活和迁移

目的 观察新生小鼠海马原代培养的胆碱能神经前体细胞移植人淀粉样前体蛋白(amyloid precusor protein,APP)转基因小鼠脑内以后的存活和迁移状况,以探索胆碱能神经前体细胞对阿尔茨海默病的治疗前景.方法 取新生24h以内增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠,以原代细胞分离培养方法获得海马胆碱能神经前体细胞,以前体细胞特异性标记抗巢蛋白(Nestin)和胆碱能细胞特异性标记胆碱乙酰基转移酶(cholineacetyltransferase, ChAT)做细胞免疫组化鉴定细胞特性.细胞培养成功后,收集细胞,在小鼠脑立体定位仪的固定下,参照小鼠脑立体定位图谱对APP转基因小鼠施行脑内注射.移植1周以后灌注取移植鼠脑组织行冰冻切片,在共聚焦显微镜下通过外源细胞的绿色自发荧光观察移植细胞的存活和迁移情况.结果 在移植位点,外源性绿色荧光细胞存活良好并以移植点为中心向外迁移,且呈继续迁移的趋势.结论 新生小鼠海马原代细胞培养以后呈现神经前体细胞的特性并且胆碱能阳性,移植入APP转基因小鼠脑内以后,这些胆碱能阳性的神经前体细胞存活良好并呈现较强迁移能力,为进一步探索该细胞对阿尔茨海默病的治疗可能性提供了依据.     

淀粉样前体蛋白基因的分子遗传学和分子生物学

淀粉样前体蛋白 (amyloid precusor protein,APP)是 Aβ(β- am yloid peptide,Aβ)的前体蛋白。后者是阿尔茨海默疾病 (Alzheimer's disease,AD)最主要的病理标志。 APP突变后增加神经毒性蛋白 Aβ42 / 43的表达量 ,从而导致家族性 AD(FAD)的发生。本文综述了 APP的基因结构、功能、突变体的类型及基因突变的病理学后果 ,同时追踪了如下两方面的研究进展 :1Aβ产生的途径 ;2 APP代谢的调控机制     

阿尔茨海默病与β-淀粉样蛋白相关性研究现状

阿尔茨海默病(AD)是一种中枢神经系统变性病,常伴随有认知障碍、记忆力减退、人格改变等。AD的病因和发病机制尚不明确,由β淀粉样蛋白沉积(amyloid-β,Aβ)所积聚的细胞外老年斑(SP),tau蛋白过度磷酸化产生的神经细胞内神经原纤维缠结(NFT)和神经元丢失伴胶质细胞增生是AD发生的病理学基础。本文综述归纳了AD与Aβ相关性的最新进展情况。     

双重引物PCR鉴定APPSWE转基因小鼠

本实验旨在探讨一种简单易行,能有效识别APPSWE转基因小鼠基因检测中假阴性结果的方法。在PCR体系中设计两对引物,一对为APP基因特异性引物,另一对为根据其内源性管家基因β-actin设置的参照引物。利用同一个PCR反应体系,对两对引物进行扩增。结果显示,双重引物PCR扩增的特异性片段与单引物扩增片段完全吻合,利用该法检出的转基因阳性率高于单引物PCR检出的转基因阳性率。本方法简单易行,能够有效识别以往利用单一PCR检测出现的假阴性结果,并且具有很好的特异性和灵敏性,值得在转基因小鼠鉴定实验中推广。     

APPSWE转基因鼠的繁育及子代鉴定的研究

目的:探讨老年性痴呆(AD)转基因模型鼠(TgAPPSWE2576杂合子鼠,Tg2576)的优化繁育方法,鉴定APPSWE基因阳性的子代鼠,为下一步AD研究奠定基础。方法:(1)用3种不同的交配方式观察子代鼠的存活率及APPSWE基因阳性的比率;(2)从子鼠鼠尾中提取基因组DNA,用PCR方法扩增APPSWE基因片段,电泳后观察结果;(3)PCR产物装入pGEM-T easy载体中,测序证实其基因序列。结果:雄性和雌性Tg2576互交,所产4窝子鼠,2 d内全部死亡。雄性Tg2576和雌性C57BL交配所产子鼠存活率81%,APPSWE基因的阳性率为43.3%。雌性Tg2576和雄性C57BL交配存活率82.4%,APPSWE基因的阳性率21.9%。经秩和检验两种繁育方式所产子鼠的存活率无显著差异(P＞0.05),APPSWE基因的阳性率有显著差异(P＜0.05)。琼脂糖电泳显示PCR产物分子量为428 bp,与目的基因片段相对分子质量大小一致;DNA序列测定证实PCR产物基因序列和APP片段基因序列一致,并存在APPSWE(K670N,M671L)突变。结论:雄性Tg2576和雌性C57BL品系小鼠交配是繁育APPSWE基因阳性子鼠的较好方法;本实验所用PCR方法能够精确鉴定APPSWE基因阳性子鼠,为AD的实验研究提供了较理想的动物模型。     

Human CD8{alpha} expression in NK cells but not cytotoxic T cells of transgenic mice

In our previous work, DNase hypersensitivity mapping was usedto identify an enhancer within the human CD8 (hCD8) gene whichallowed T cell-specific expression of a reporter construct intransiently transfected cell lines. To study the role of thisintronic enhancer in vivo, transgenic mice were made using humanCD8 genomic constructs. We found that while a 14 kb wild-typehuman CD8a (WThCD8) genomic construct did not lead to expressionin mature peripheral CD8⁺ T cells, this transgene was consistentlyexpressed in small populations of T cells and B cells, and ina subset of mouse NK cells. While murine CD8 is not normallyexpressed on resting NK cells, expression of the human CD8 transgeneon mouse NK cells is appropriate since CD8 is expressed on asubset of human NK cells. Deletion of the intronic enhancerresulted in a complete loss of transgene expression in mostlines and a loss of expression only in NK cells in one line.Our results indicate, firstly, that cis-acting sequences withinthe 14 kb genomic fragment are sufficient for NK cell-specificexpression. In addition, our results suggest that the enhancermay have dual roles in regulation of transgene expression. Itmay enhance general expression of the transgene and may alsobe required for NK cell-specific expression.     

No evidence for a point mutation at codon 713 and 717 of amyloid precursor protein gene in Japanese schizophrenics

Summary A point mutation at codon 717 of amyloid precursor protein (APP) gene has been demonstrated to play an important pathogenic role in some cases of familial Alzheimer's disease (FAD). Recently, a single case of chronic schizophrenia with a point mutation at codon 713 of APP gene which sits very close to the mutation in FAD was reported. We screened for these two kinds of mutations in 39 schizophrenic patients using polymerase chain reaction (PCR) and restriction enzyme technique. A mutation of codon 713 creates aMaeIII restriction site and that of codon 717 creates aBclI site. Enzyme digestion with amplified PCR product revealed no restriction site in all subjects. None of our subjects had either of these two kinds of mutations. Our findings support the hypothesis that the case of a mutation at codon 713 of APP gene is a natural non-pathogenic variant and, as well as a mutation at codon 717, has no relation with the genetic predisposition to schizophrenia.     

Functional expression of a human TCR{beta} gene in transgenic mice

A functionally rearranged TCRß (Tcrb) gene was isolatedfrom a cloned human T helper cell recognizing the CS.T3 epitopeof Plasmodium falciparum with HLA-DR2. Transgenic mice weregenerated by co-injection of the human gene together with themouse Tcrb enhancer. Analysis of transgenic mice shows thatthe functional Tcrb gene of xenogenic, i.e. human, origin exertsallelic exclusion of endogenous Tcrb genes. Cytofluorometricanalysis revealed expression of the human TCRß chainon virtually all thymocytes and peripheral T cells togetherwith endogenous TCRß chains and CD3 components. Nosurface expression of mouse TCRß chain or rearrangementof endogenous Tcr genes was detectable. Expression of the hybridreceptor causes a reduction in the number of thymocytes anda bias for CD4⁺CD8^– T cells in the thymus as comparedwith non-transgenic littermates. Peripheral transgenic T cellsmount a normal prollferative response against allogenelc targetsin mixed lymphocyte reactions. These results show that a hybridmouse/human TCR is able to pass positive and negative selectionin the thymus, and is functional in transgenic mice.     

Aging and excitotoxic stress exacerbate neural circuit reorganization in amyloid precursor protein intracellular domain transgenic mice

The cleavage of amyloid precursor protein (APP) by presenilins simultaneously generates amyloid-β (Aβ) and APP intracellular Domain (AICD) peptides. Aβ plays a pivotal role in Alzheimer's disease (AD) pathology and recently AICD was also shown to contribute to AD. Transgenic mice overexpressing AICD show age-dependent tau phosphorylation and aggregation, memory deficits, and neurodegeneration. Moreover, these mice show aberrant electrical activity and silent seizures beginning at 3–4 months of age. Here we show that AICD mice also displayed abnormal mossy fiber sprouting beginning about the same time and that this sprouting intensified as the animals aged. Expression of neuropeptide Y was increased in mossy fiber terminals in aged but not young AICD mice. Importantly, young AICD mice injected with kainic acid showed similar pathology to that observed in aged AICD mice. These data show that elevated levels of AICD render neurons hypersensitive to stress and induce hippocampal circuit reorganization, which can further exacerbate hyperexcitability. These results further demonstrate that AICD, in addition to Aβ, can play a significant role in AD pathogenesis.