肝硬化小鼠肠道菌群结构及血清炎性因子的变化简

目的：观察肝硬化小鼠肠道菌群结构及血清炎性因子水平的变化。方法：通过CCL灌胃构建小鼠的肝硬化模型;采用酶联免疫吸附法检测肝硬化进程中血清内毒素及炎性因子IL-1、IL-6、TNF-α水平的变化;采集肝硬化小鼠新鲜粪便,通过荧光定量PCR实验检测肠道目的菌群的改变。结果：肝硬化小鼠肠道中拟杆菌属和梭菌属细菌显著减少,韦荣球菌属、肠杆菌属和肠球菌属细菌显著增加;随着肝硬化进程的加重,小鼠血液中内毒素及炎性因子水平显著提高。结论：肝硬化导致小鼠肠道菌群失调,促进了血液中内毒素及炎性因子水平的提高,形成恶性循环。     

黏蛋白1基因敲除小鼠模型的构建

黏蛋白1(MUC1)属黏蛋白家族成员,分布于上皮细胞膜表面,由于在免疫炎症反应以及肿瘤发生中的重要作用而日益受到重视.为了进一步深入研究MUC1的生物学功能,构建了Muc1基因敲除小鼠模型.首先,根据小鼠Muc1基因组序列设计基因剔除策略,将2个loxP位点分别插在外显子2和3两侧,构建基因剔除载体Muc1-ABRLFn-pBR322.以电穿孔方法将载体导入胚胎干细胞(ES细胞),用G418和更昔洛韦进行正负筛选获得4个同源重组的ES细胞克隆.挑选其中一个阳性ES克隆行囊胚显微注射,获得16只嵌合率大于50%的雄鼠;其次,利用嵌合雄鼠与C57BL/6J野生型雌鼠交配后获得11只floxP杂合子小鼠(10雄1雌),通过杂合子小鼠回交,并进一步与EⅡa-Cre小鼠交配,最终成功得到Muc1全身敲除小鼠,其中纯合子小鼠未出现胚胎致死现象.初步表型观察未发现Muc1基因敲除相关器官组织结构的异常改变.本研究为MUC1的生物学功能的挖掘,尤其是MUC1在肿瘤发生转移中的作用机制的揭示提供了实验平台.     

Behavioral characterization of striatal‐enriched protein tyrosine phosphatase (STEP) knockout mice

Striatal‐enriched protein tyrosine phosphatase (STEP) has been described as a regulator of multiple kinases and glutamate receptor subunits critical for synaptic plasticity. Published behavioral and biochemical characterization from the founder line of STEP knockout (KO) mice revealed superior cognitive performance, with enhanced phosphorylation of substrates such as ERK, Fyn and GluN2B; suggesting that inhibitors of STEP may have potential as therapeutic agents for the treatment of neuropsychiatric disorders. The objectives of this work aimed to replicate and extend the previously reported behavioral consequences of STEP knockout. Consistent with previous reported data, STEP KO mice demonstrated exploratory activity levels and similar motor coordination relative to WT littermate controls as well as intact memory in a Y‐maze spatial novelty test. Interestingly, KO mice demonstrated deficits in pre‐pulse inhibition as well as reduced seizure threshold relative to WT controls. Immunohistochemical staining of brains revealed the expected gene‐dependent reduction in STEP protein confirming knockout in the mice. The present data confirm expression and localization of STEP and the absence in KO mice, and describe functional downstream implications of reducing STEP levels in vivo.     

Genetics of gene expression characterizes response to selective breeding for alcohol preference

Numerous selective breeding experiments have been performed with rodents, in an attempt to understand the genetic basis for innate differences in preference for alcohol consumption. Quantitative trait locus (QTL) analysis has been used to determine regions of the genome that are associated with the behavioral difference in alcohol preference/consumption. Recent work suggests that differences in gene expression represent a major genetic basis for complex traits. Therefore, the QTLs are likely to harbor regulatory regions (eQTLs) for the differentially expressed genes that are associated with the trait. In this study, we examined brain gene expression differences over generations of selection of the third replicate lines of high and low alcohol‐preferring (HAP3 and LAP3) mice, and determined regions of the genome that control the expression of these differentially expressed genes (_deeQTLs). We also determined eQTL regions (_rveQTLs) for genes that showed a decrease in variance of expression levels over the course of selection. We postulated that _deeQTLs that overlap with _rveQTLs, and also with phenotypic QTLs, represent genomic regions that are affected by the process of selection. These overlapping regions controlled the expression of candidate genes (that displayed differential expression and reduced variance of expression) for the predisposition to differences in alcohol consumption by the HAP3/LAP3 mice.     

Therapeutic inducers of the HSP70/HSP110 protect mice against traumatic brain injury

Traumatic brain injury (TBI) induces severe harm and disability in many accident victims and combat‐related activities. The heat‐shock proteins Hsp70/Hsp110 protect cells against death and ischemic damage. In this study, we used mice deficient in Hsp110 or Hsp70 to examine their potential requirement following TBI. Data indicate that loss of Hsp110 or Hsp70 increases brain injury and death of neurons. One of the mechanisms underlying the increased cell death observed in the absence of Hsp110 and Hsp70 following TBI is the increased expression of reactive oxygen species‐induced p53 target genes Pig1, Pig8, and Pig12. To examine whether drugs that increase the levels of Hsp70/Hsp110 can protect cells against TBI, we subjected mice to TBI and administered Celastrol or BGP‐15. In contrast to Hsp110‐ or Hsp70i‐deficient mice that were not protected following TBI and Celastrol treatment, there was a significant improvement of wild‐type mice following administration of these drugs during the first week following TBI. In addition, assessment of neurological injury shows significant improvement in contextual and cued fear conditioning tests and beam balance in wild‐type mice that were treated with Celastrol or BGP‐15 following TBI compared to TBI‐treated mice. These studies indicate a significant role of Hsp70/Hsp110 in neuronal survival following TBI and the beneficial effects of Hsp70/Hsp110 inducers toward reducing the pathological consequences of TBI.

     

Diet‐induced obesity accelerates the onset of terminal phenotypes in α‐synuclein transgenic mice

Parkinson's disease (PD) and diabetes belong to the most common neurodegenerative and metabolic syndromes, respectively. Epidemiological links between these two frequent disorders are controversial. The neuropathological hallmarks of PD are protein aggregates composed of amyloid‐like fibrillar and serine‐129 phosphorylated (pS129) α‐synuclein (AS). To study if diet‐induced obesity could be an environmental risk factor for PD‐related α‐synucleinopathy, transgenic (TG) mice, expressing the human mutant A30P AS in brain neurons, were subjected after weaning to a lifelong high fat diet (HFD). The TG mice became obese and glucose‐intolerant, as did the wild‐type controls. Upon aging, HFD significantly accelerated the onset of the lethal locomotor phenotype. Coinciding with the premature movement phenotype and death, HFD accelerated the age of onset of brainstem α‐synucleinopathy as detected by immunostaining with antibodies against pathology‐associated pS129. Amyloid‐like neuropathology was confirmed by thioflavin S staining. Accelerated onset of neurodegeneration was indicated by Gallyas silver‐positive neuronal dystrophy as well as astrogliosis. Phosphorylation of the activation sites of the pro‐survival signaling intermediate Akt was reduced in younger TG mice after HFD. Thus, diet‐induced obesity may be an environmental risk factor for the development of α‐synucleinopathies. The molecular and cellular mechanisms remain to be further elucidated.

     

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: A novel approach to generate tumor cell specific human antibodies

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ^null (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4⁺ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.     

Age‐related deterioration of motor function in male and female 5xFAD mice from 3 to 16 months of age

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to age‐related cognitive and sensori‐motor dysfunction. There is an increased understanding that motor dysfunction contributes to overall AD severity, and a need to ameliorate these impairments. The 5xFAD mouse develops the neuropathology, cognitive and motor impairments observed in AD, and thus may be a valuable animal model to study motor deficits in AD. Therefore, we assessed age‐related changes in motor ability of male and female 5xFAD mice from 3 to 16 months of age, using a battery of behavioral tests. At 9‐10 months, 5xFAD mice showed reduced body weight, reduced rearing in the open‐field and impaired performance on the rotarod compared to wild‐type controls. At 12‐13 months, 5xFAD mice showed reduced locomotor activity on the open‐field, and impaired balance on the balance beam. At 15‐16 months, impairments were also seen in grip strength. Although sex differences were observed at specific ages, the development of motor dysfunction was similar in male and female mice. Given the 5xFAD mouse is commonly on a C57BL/6 × SJL hybrid background, a subset of mice may be homozygous recessive for the Dysf ^im mutant allele, which leads to muscular weakness in SJL mice and may exacerbate motor dysfunction. We found small effects of Dysf ^im on motor function, suggesting that Dysf ^im contributes little to motor dysfunction in 5xFAD mice. We conclude that the 5xFAD mouse may be a useful model to study mechanisms that produce motor dysfunction in AD, and to assess the efficacy of therapeutics on ameliorating motor impairment.     

Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.