MMTV-Wnt-1转基因小鼠作为高发乳腺癌动物模型的观察

目的　观察MMTV Wnt 1转基因小鼠的乳腺癌发病情况及病理学变化规律。方法　观察MMTV Wnt 1转基因小鼠肿瘤发生情况 ,并采用原位移植将瘤组织置于裸鼠皮下 ,通过组织病理学切片来观察MMTV Wnt 1阳性转基因小鼠和移植鼠的病理学变化。结果　MMTV Wnt 1转基因小鼠最早从 7周龄开始出现乳腺瘤 ,发瘤鼠剖检可见脾、肝有不同程度的肿大 ,其他器官无明显病变 ;病理组织学检查发现发瘤鼠各脏器有不同程度的病变 ,但未出现肿瘤转移。将瘤组织移植裸鼠后 ,肿瘤可在裸鼠皮下生长 ,移植肿瘤病理学形态与原发瘤一致 ,未出现转移。结论　实验结果验证MMTV Wnt 1转基因小鼠可稳定自发乳腺肿瘤 ,可作为研究乳腺癌的良好的动物模型     

含体内激活启动子的重组减毒鼠伤寒沙门菌诱导转基因鼠细胞免疫应答

以庚型肝炎病毒 (hepatitisGvirus ,HGV)转基因小鼠为模型 ,探讨逆转免疫耐受的方法及与HGV致病性的关系。首先采用鼠伤寒沙门菌pagC基因的启动子 (PpagC)为转录调控元件 ,构建宿主体内表达HGVNS3抗原的重组减毒鼠伤寒沙门菌 ,并口服接种免疫HGV转基因小鼠。结果证明对诱导转基因小鼠血清HGVNS3抗体无明显影响 ,但对小鼠血清HGV抗原含量、肝组织HGV抗原及HGVmRNA表达量有明显抑制作用。体外培养脾细胞表现出针对HGVNS3抗原的细胞免疫反应 ,并检测到Th1 型细胞因子IFN γ。过继转移实验证明T细胞可能是通过IFN γ介导的机制抑制转基因鼠体内HGV的表达及复制。组织病理学检查显示 ,转基因小鼠肝组织见淋巴细胞浸润等轻度炎性变。T细胞免疫耐受消除的结果提示 ,这种宿主体内激活目的抗原表达的口服疫苗有望成为治疗病毒性肝炎的新方法。     

EB病毒BLLF-1基因转染小鼠T细胞淋巴瘤的研究

目的:研究EBV膜蛋白gp350/220的表达对共刺激分子ICOS的影响以及与T细胞淋巴瘤的关系。方法:繁殖饲养BLLF-1转基因昆明鼠以及正常昆明鼠,观察它们淋巴瘤发病率的差异。取发病的BLLF-1转基因昆明鼠脾脏淋巴细胞,用FITC标记的抗gp350/220单克隆抗体进行免疫荧光染色,检测gp350/220是否在该转基因昆明鼠淋巴细胞内表达及其表达部位。对发病转基因昆明鼠组织进行免疫组化染色,并与正常昆明鼠的进行对比分析。用RT-PCR方法检测转基因小鼠共刺激分子ICOS的表达变化。结果:BLLF-1转基因昆明鼠淋巴组织病理性改变与正常昆明鼠有显著差异,免疫荧光检测到该转基因小鼠淋巴细胞表达gp350/220于胞浆和胞膜上,病理学观察发现,发病小鼠淋巴结组织有反应性增生,脾脏淋巴瘤细胞浸润,免疫组化证明为T细胞淋巴瘤,转基因小鼠脾脏、肺脏及肿瘤中ICOS表达显著升高。结论:BLLF-1基因的表达,与该转基因小鼠发生T细胞淋巴瘤有关,并引起共刺激分子ICOS表达的变化,该转基因小鼠的建立,为我们进一步研究BLLF-1基因在T细胞淋巴瘤发病中的作用提供了良好的动物模型。     

HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

APPSWE转基因小鼠发育过程中海马CA1区神经细胞凋亡研究

目的探讨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、P00、P180）APPSWE转基因模型鼠与同时问点对照鼠,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茨海默病的发生、发展具有联系。     

早老蛋白与淀粉样沉淀前体蛋白的相互作用及A_β的形成

Aβ(βAmyloid)在脑内的沉积是Alzheimer病的病理现象。对Alzheimer病的研究揭示Aβ的生成与早老蛋白和淀粉样沉淀前体蛋白相互作用密切相关。早老蛋白和淀粉样沉淀前体蛋白基因的突变均能改变淀粉样沉淀前体蛋白的正常切割,使得Aβ的生成量增加。转基因小鼠模型的建立为早老蛋白与淀粉样沉淀前体蛋白相互作用及Aβ生成机理的研究奠定了基础。     

脱钙对鼠颅骨标本中肥大细胞组织化学与免疫组织化学染色的影响

目的:探讨脱钙对鼠颅骨标本中肥大细胞组织化学与免疫组织化学染色影响.方法:用不同脱钙液处理小鼠颅骨标本,组织切片后进行苏木素一伊红染色、甲苯胺蓝染色、醛品红染色以及免疫组织化学染色.结果:经过不同脱钙液处理后的颅骨组织切片组织结构保存完好,肥大细胞的组织化学染色(甲苯胺蓝和醛品红染色),及肥大细胞中胰蛋白酶的免疫组织化学染色清晰.结论:不同脱钙液(8%盐酸脱钙液、EDTA脱钙液、混合酸脱钙液)处理不影响鼻腔粘膜中肥大细胞的组织化学和免疫组织化学染色.     

他莫昔芬诱导的Cre重组酶在hGfapCreERT2转基因鼠小脑中的表达研究

目的探讨他莫昔芬诱导的hGfapCreERT2转基因鼠小脑中表达Cre重组酶的细胞类型。方法 hGfapCre-ERT2/Rosa26R转基因小鼠在胚胎晚期和出生早期用他莫昔芬诱导Cre重组酶表达,对小脑组织切片行X-gal染色,然后用细胞种类特异性抗体进行免疫组织化学染色,并和X-gal染色双重标记。结果在出生后第7天(P7)、第14天(P14)和第60天(P60),X-gal阳性染色和胶质细胞抗体Blbp阳性染色共标记,和神经元抗体Neun、浦肯野细胞抗体Calbindin及少突胶质细胞前体细胞抗体NG2不共标。结论自胚胎晚期第17.5天(E17.5)后用他莫昔芬诱导hGfapCreERT2转基因鼠,发现Cre重组酶特异性在小脑星形胶质细胞中表达,不在神经元、浦肯野细胞、少突胶质细胞前体细胞中表达。     

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。     

H102对APP转基因小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响

目的:研究H102对APP695转基因模型小鼠脑内淀粉样蛋白和淀粉样蛋白前体蛋白表达的影响方法:9月龄转基因小鼠随机分为模型组和药物注射组,正常对照组采用月龄和性别与之相匹配的C57BL/6J小鼠。药物注射组给予侧脑室注射H102,每只每次3μl,连续10d;模型组和正常对照组给予等体积NS。应用免疫组织化学结合刚果红组织学染色,普通光学显微镜观察海马和颞叶皮层蛋白表达的变化。免疫印迹法检测小鼠大脑皮层APP蛋白的表达。结果:Aβ和APP免疫组化染色结果显示对照组海马CA1区神经元胞浆着色呈阴性或弱阳性,模型组较对照组阳性细胞增多,表达增强,胞浆着色明显加深。药物注射组同模型组相比,胞浆着色变淡,表达减弱。刚果红染色观察转基因小鼠模型组和H102注射组大脑颞叶皮层和海马的淀粉样斑块,可见H102注射组淀粉样斑块数较模型组明显减少。正常对照组未见阳性淀粉样斑块。免疫印迹检测显示模型组APP蛋白表达明显增加,给药组与模型组相比具有统计学意义。结论:APP695转基因小鼠大脑CA1区Aβ蛋白和APP蛋白表达增加,H102能够明显抑制该转基因小鼠Aβ蛋白和APP蛋白表达。     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

Neuroprotective Effects of (-)-Epigallocatechin-3-gallate in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

The purpose of this study is to evaluate neuroprotective effects of (-)-Epigallocatechin-3-gallate (EGCG) in a transgenic mouse model of Amyotrophic lateral sclerosis (ALS). SOD1-G93A transgenic mice and wild-type mice were randomly divided into EGCG-treated groups (10 mg/kg, p.o) and vehicle-treated control groups. Rotarod measurement was performed to assess the motor function of mice starting at the age of 70 days. Nissl staining to examine the number of motor neurons and CD11b immunohistochemical staining to evaluate activation of microglia in the lumbar spinal cords were conducted at the age of 120 days. In addition, for further observation of regulation of cell signaling pathways by EGCG, we used immunohistochemical analysis for nuclear factor kappa B (NF-κB) and cleaved caspase-3 as well as western blot analysis to determine the expression of nitric oxide synthase (iNOS) and NF-κB in the spinal cord. This study demonstrated that oral administration of EGCG beginning from a pre-symptomatic stage significantly delayed the onset of disease, and extended life span. Furthermore, EGCG-treated transgenic mice showed increased number of motor neurons, diminished microglial activation, reduced immunohistochemical reaction of NF-κB and cleaved caspase-3 as well as reduced protein level of iNOS and NF-κB in the spinal cords. In conclusion, this study provides further evidences that EGCG has multifunctional therapeutic effects in the mouse model of ALS.     

大鼠实验性帕金森病的基因治疗

将稳定转染了大鼠酪氨酶羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因的大鼠成肌细胞移植于帕金森病大鼠模型的纹状体,进行基因治疗研究。ＲＴ－ＰＣＲ和免疫组织化学检测都证明转基因细胞可在纹状体内存活并表达ＴＨ,动物的不对称旋转行为明显改善,而且疗效可维持半年以上。     

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein- and 4-hydroxynonenal-adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10-month-old transgenic pigs in which almost all rods had died, compared to faint staining in two 10-month-old control pig retinas. In 22- and 24-month-old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.     

特殊染色及免疫组化双重染色在恒河猴糖尿病模型中的应用

目的在链脲佐菌素（STZ）诱导恒河猴糖尿病动物模型基础上,用特殊染色、免疫组化双染方法显示胰腺等组织中的特征性病变。方法健康恒河猴5只,小剂量（30mg／kg）多次静脉注射STZ,濒死状态时将动物安乐死。取胰腺、心脏、肾脏、脾脏、肝脏、眼球、脑等器官制成石蜡切片,用HE、PAS、Masson、天狼星红和甲苯胺蓝等方法进行特殊染色,用免疫组化双重染色法同时显示胰岛A、B细胞。结果模型动物的胰岛萎缩,数量减少。Masson染色见外分泌部间质内纤维增生。局灶性慢性肾炎,病变区肾间质纤维组织增生,部分肾小球及肾小管萎缩。天狼星红染色见脾脏中央动脉管壁增厚。免疫组化双染见胰岛A胰高血糖素表达增多,胞浆呈棕褐色。B胰岛素表达减少,胞浆粉红色。结论HE染色结合特殊染色和免疫组化双重染色可较好地对STZ诱导糖尿病动物模型进行组织学评价。     

Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain

In the current protocol, we describe the Congo red staining method and a method for separately quantifying vascular and parenchymal amyloid deposits in brain tissue sections. Congo red staining detects amyloid deposits in brain tissue of amyloid precursor protein transgenic mice and human Alzheimer's tissue. It detects compacted amyloid in a beta-sheet secondary structure and labels amyloid in both the brain parenchyma (amyloid plaques) and blood vessels. Congophilic amyloid in blood vessels is called cerebral amyloid angiopathy (CAA). To date, analysis of CAA has largely used a severity rating scale, including both qualitative and quantitative characteristics. Here, we describe a simple method for quantifying total Congophilic staining and resolution of this staining into the parenchymal and vascular components based on morphological criteria. It is becoming increasingly important to separately quantify various components of the Alzheimer's pathology, given the advancement of amyloid-lowering therapies into clinical trials. The entire procedure for the Congo red staining can be performed at room temperature (20-25 degrees C) in a fume hood. The staining protocol should take 1 h 30 min including time for coverslipping slides. Time required for image analysis depends greatly on the number of samples being analyzed and the software being used. In our hands, 30 images can be collected per hour and quantified in a further 2 h.     

Number of Abeta inoculations in APP+PS1 transgenic mice influences antibody titers, microglial activation, and congophilic plaque levels.

There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimer's disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.     

苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。