非酒精性脂肪肝C57BL/6小鼠模型的建立

 BACKGROUND: The establishment of a safe, reliable and easily repeatable mouse model of nonalcoholic fatty liver disease is the prerequisite for the study of the diagnosis and treatment of the disease.     

人骨形成蛋白2噬菌粒表达载体的构建与鉴定

人骨形成蛋白２噬菌粒表达载体的构建与鉴定司晓辉杨连甲金岩陈梅红１（第四军医大学口腔医学院病理科１生化教研室，西安７１００３２）骨形成蛋白（ｂｏｎｅｍｏｒｐｈｏｇｅｎｅｔｉｃｐｒｏｔｅｉｎｓ，ＢＭＰ）具有广泛的生物学作用，特别是在形态发生和细胞分化两个...     

Id2基因过表达对C57BL/6小鼠免疫表型的影响

目的：建立分化抑制因子2(ID2)过表达和CD45.1,CD45.2嵌合体小鼠模型,探讨ID2过表达(ID2^o/o)对C57BL/6小鼠免疫表型的影响。方法：利用基因编辑技术获得ID2^o/o C57BL/6模型小鼠,采用PCR、琼脂糖凝胶电泳和Western blot对小鼠进行基因型鉴定。Western blot检测ID2^o/o和WT小鼠脾脏细胞中各种ID蛋白(ID1～ID4)表达情况。流式细胞术绝对定量方法检测WT和ID2^o/o小鼠脾脏和外周血细胞中各免疫细胞数量。构建骨髓1∶1竞争性嵌合体小鼠模型,以同样方法检测CD45.1⁺和CD45.2⁺白细胞及相应免疫细胞数量。结果：PCR和Western blot结果表明ID2^o/o小鼠构建成功。在ID2^o/o小鼠脾细胞中,4种ID蛋白的表达量均显著高于WT小鼠。流式细胞分析结果显示,Id2基因过表达使C57BL/6小鼠脾脏和外周血中的中性粒细胞、巨噬细胞、树突状细胞...     

人骨形成蛋白2基因克隆及真核表达载体的构建

在大肠杆菌中克隆人骨形成蛋白2基因并获得真核表达载体。由人成骨瘤细胞中提取总RNA，利用逆转录PCR方法扩增获得人骨形成蛋白2基因cDNA；将此基因片段重组到pGEM-T克隆载体中，转化到大肠杆菌DH5α后，蓝白斑筛选阳性克隆，利用限制性酶切、PCR扩增和核苷酸序列分析鉴定重组质粒；将pGEM-T克隆载体中人骨形成蛋白2基因重组到pcDNA3．1真核表达载体中，用限制性酶切和PCR扩增鉴定重组质粒。结果表明：重组在两种质粒中的基因片段为人骨形成蛋白2基因全编码序列。克隆获得人骨形成蛋白2基因．并得到此基因的真核表达载体，为人骨形成蛋白2的表达打下了基础。     

C57BL/6小鼠皮肤毛囊发育的实验研究

目的探讨胎鼠,乳鼠和成鼠毛囊发育的规律。方法石蜡和冰冻超薄切片,采用HE染色,AP活性染色,油红O染色,凋亡细胞鉴定等方法观察不同时期C57BL/6小鼠毛囊的发育情况。结果胚胎发育第15.5d(E15.5)开始出现毛囊,至E18.5胎鼠背部皮肤逐渐增厚,毛囊逐渐增多。乳鼠出生第1d毛囊大部分处于第4阶段之前,毛囊数量继续增加,至出生第4d皮肤基底层未见新生的毛囊,此时毛囊数量维持不变,出生第9d毛囊达到第8阶段。成鼠脱毛后第9d约有95%的毛囊进入生长Ⅳ期,第17d约有63.3%的毛囊进入退化期,第22d约有85%的毛囊处于退化期。结论小鼠毛囊的早期发育起始于E15.5,从E17.5到出生后3d毛囊数量呈快速增加趋势,因此,这一时期可以用于探讨毛囊再生机制等相关研究。     

C57BL/6小鼠不同组织器官中NKT细胞的比较

目的比较C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结中NKT细胞的含量、亚型和功能的特点。方法分离正常C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的淋巴细胞,利用细胞表面分子染色的方法,观察不同组织器官中CD3+NK1.1+NKT细胞及其亚型的含量;淋巴细胞经过PMA和离子霉素刺激后,应用细胞内细胞因子染色的方法,通过流式细胞仪观察NKT细胞IFN-γ、IL-4、IL-9和IL-17的产生情况。结果肝脏中NKT细胞的含量为(25.2±12)%,显著高于肺脏、脾脏和肠系膜淋巴结。肝脏、脾脏和肠系膜淋巴结中NKT细胞以CD4+细胞亚群为主,而肺脏中NKT细胞以CD4-CD8-亚群为主,同时肠系膜淋巴结的NKT细胞中存在CD4+CD8+亚群。不同组织器官中NKT细胞IFN-γ、IL-4、IL-9和IL-17产生的能力有差别。结论 C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的NKT细胞在含量、表型和功能方面可能存在明显的差异。     

C57/BL6小鼠小脑发生发育及衰老的形态学变化

目的:观察C57/BL6小鼠小脑发生发育及衰老的形态学变化规律.方法:应用石蜡连续切片、H-E染色结合体视学方法对胚龄(E)10、12、14、16、18、20 d和生后(P)1、3、7、14、21、28 d的仔鼠及生后2、3、6、15个月(M)的C57/BL6小鼠小脑的形态学变化进行系统观察和定量分析.结果:E12 d小脑原基出现.E16 d小脑半球、蚓部及皮质的外颗粒层出现.E18 d分子层的原基出现,小脑表面出现沟回结构.P1 d皮质分为外颗粒层、分子层、浦肯野细胞层和内颗粒层4层.P7 d皮、髓质分界清楚.P21d外颗粒层消失,皮质分为分子层、浦肯野细胞层和颗粒层3层.体视学分析显示,3个月之前,小脑总体积、皮质和髓质的体积、皮质各层（分子层、浦肯野细胞层和内颗粒细胞层/颗粒细胞层）的体积均逐渐增加,以P3～14 d增长最快;3个月之后趋稳定.E18 d～P14 d,外颗粒层体积先增大后减小,P21 d完全消失.结论:E12 d～P21 d是小鼠小脑发生发育的关键时期,细胞经历了增殖、分化和迁移,外颗粒层可能参与分子层和内颗粒层的形成.     

汉坦病毒感染C57BL/6小鼠组织中特异性抗原的检测

目的通过检测汉坦病毒感染C57BL/6小鼠组织中特异性病毒抗原,以建立汉坦病毒感染动物的评价体系。方法将汉坦病毒陈株按照原病毒液、10-1、10-2三个滴度经肌肉注射感染C57BL/6小鼠,在感染后的第3、6、9、12、15天,分别取小鼠的心、肝、脾、肺、肾、脑等组织研磨后制成病毒悬液,以ELISA法检测各组织中的病毒特异性抗原。结果 C57BL/6小鼠感染汉坦病毒后短期内在其肝脏和脾脏可以检测到特异性抗原,随着时间的延长,这些抗原逐步消失。结论上述结果为建立汉坦病毒感染动物的评价体系提供了一种参考。     

C57BL/6小鼠γδT细胞的组织器官分布和功能特点

目的 比较C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结中γδT细胞占所分离的淋巴细胞及其CD3+T细胞的百分比、表型和功能的特点.方法 分离正常C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的淋巴细胞,应用细胞表面分子染色的方法,使用流式细胞仪观察γδT细胞占从不同组织分离的淋巴细胞及CD3+T细胞的百分比及其表型的特点.细胞经PMA和离子霉素刺激后,应用细胞内细胞因子染色的方法,通过流式细胞仪观察γδT细胞产生IFN-γ、IL-4、IL-9和IL-17细胞因子的情况.结果 γδT细胞占分离淋巴细胞中的百分含量在肝脏明显高于肺脏、脾脏和肠系膜淋巴结(P＜0.05),而γδT细胞在肠系膜淋巴结CD3+T细胞的百分含量要显著的低于其他脏器(P＜0.05).不同组织器官中γδT细胞以CD4-CD8-表型为主,还存在少量CD8+ γδT细胞.在肠系膜淋巴结γδT细胞中CD4+细胞的量明显高于其他器官,且明显可见一群CD4+ CD8+细胞.不同组织中γδT细胞中IL-17+的细胞量要明显高于IFN-γ+和IL-4+细胞.γδT细胞基本不分泌IL-9.肺脏的γδT细胞分泌细胞因子的能力最强,其IL-17+细胞的量达到(26.6±12.1)％.IFN-γ+细胞的量在肝脏和肺脏中较高,分别为(1.36±0.37)％和(1.6±0.7)％.结论 C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结γδT细胞的含量、表型和功能方面存在显著性差异.     

巨细胞病毒感染C57BL/6小鼠诱导NK细胞免疫应答的模型研究

目的 研究小鼠巨细胞病毒(murine cytomegalovirus,MCMV)感染C57BL/6小鼠诱导自然杀伤(natural killer,NK)细胞免疫应答的最佳剂量和最佳时间。 方法 分别根据剂量和时间效应进行分组,剂量效应：无特定病原体( specific pathogen free,SPF)级8周龄C57BL/6雌鼠15只,根据MCMV滴度分为四组,未感染的0个蚀斑形成单位(plaque forming unit,PFU)组(3只)、2.5×10⁴PFU组(4只)、5.0×10⁴PFU组(4只)和10.0×10⁴PFU组(4只),随后常规饲养7天;时间效应：将8周龄C57BL/6雌鼠14只随机分为四组,分别为0天组(4只)、3天组(4只)、7天组(4只)、14天组(2只),建模0天腹腔注射5.0×10⁴PFU的MCMV感染各组小鼠。在感染后对应天数处死各组小鼠,取小鼠脾脏制成单细胞悬液,并用流式细胞术分析各种组NK细胞比例、Ly49H及颗粒酶B(granzyme B,GZMB)、NK细胞溶酶体相关膜蛋白-1(CD107a)和γ-干扰素(interferon-γ,IFN-γ)的表达。 结果 MCMV感染7天后,与0 PFU组相比,2.5×10⁴PFU组、5.0×10⁴PFU组和10.0×10⁴PFU组脾脏NK细胞占淋巴细胞的比例明显下降{(1.90±0.32)%比[(0.91±0.14)%,(0.62±0.16)%,(0.85±0.26)%],F＝21.271,P<0.05},CD27^＋CD11b^＋NK细胞比例显著下降{(22.40±4.55)%比[(13.20±1.29)%,(10.78±1.21)%,(11.38±1.76)%],F＝17.272,P<0.05},CD11b^＋NK细胞比例显著增加{(59.87±5.33)%比[(71.08±1.82)%,(74.25±1.95)%,(70.90±2.49)%],F＝14.641,P<0.05)},CD43^＋KLRG1^＋NK细胞占NK细胞的比例增加{(39.40±5.73)%比[(77.00±0.67)%,( 81.23±2.21)%,( 76.63±7.36)%],F＝55.282,P<0.05)},Ly49H^＋NK细胞亚群数量显著增加{(42.07±1.21)%比[(63.50±1.30)%,(63.58±3.71)%,(61.68±4.84)%],F＝32.273,P<0.05)},组间差异具有统计学意义。在NK细胞功能方面,MCMV感染7天后,与0 PFU组相比,2.5×10⁴PFU组、5.0×10⁴PFU组和10.0×10⁴PFU GZMB的表达显著增加{(287.00±30.79)%比[(384.25±63.91)%,(529.75±66.08)%,(466.50±83.38)%],F＝8.730,P<0.05},IFN-γ分泌减少{(36.00±5.33)%比[(4.88±3.35)%,(3.03±1.56)%,(3.61±2.18)%],F＝87.663,P<0.05}。时间效应的比较结果显示,MCMV感染3天后,NK细胞CD107a和颗粒酶B表达与0天相比显著升高[(22.98±4.58)%比(6.32±0.75)%,(5969.25±1159.86)%比(788.50±88.17)%,F值分别为41.072和67.448,P值均<0.05],差异具有统计学意义。 结论 本研究表明,MCMV感染C57BL/6小鼠模型可选择的最佳感染剂量为5×10⁴PFU,最佳建模时间为感染后3天。     

Porphyromonas gingivalis accelerates atherosclerosis in C57BL/6 mice fed a high-fat diet

Objective: Porphyromonas gingivalis has been shown to accelerate atherosclerotic lesion development in atherosclerotic apo E-deficient mice. Here, we investigated whether repeated P. gingivalis injection affected the inflammatory and atherosclerotic responses of C57BL/6 mice fed a high-fat diet (HFD).

Materials and methods: Eight-week-old C57BL/6 mice fed either HFD or a regular chow diet (RD) were inoculated intravenously with P. gingivalis or phosphate-buffered saline three times per week for 10 weeks and sacrificed at 19 weeks of age. Atheromatous lesions in the proximal aorta of each animal were analyzed histomorphometrically, and the serum cytokine and C-reactive protein (CRP) levels were determined.

Results: Long-term HFD feeding as compared to RD feeding led to a slight increase in atheromatous lesions in the aortic sinus as well as increases in the levels of serum monocyte chemoattractant protein 1. Further, P. gingivalis injection significantly enhanced the formation of atherosclerotic plaque, and increased CRP and inflammatory cytokine levels, in mice fed the HFD, although no further increase in LDL was observed.

Conclusion: These results suggest that bacteremia-induced by repeated injection with P. gingivalis accelerates atherosclerosis in normal C57BL/6 mice by initiating inflammation, and is therefore implicated in chronic infection-related pathogenicity.     

昆明种小鼠与C57BL/6J小鼠磷酸二酯酶β亚单位编码基因pde6b序列特点的比较分析

目的:分段克隆昆明种小鼠磷酸二酯酶(PDE) β亚单位编码基因pde6b CDS序列全长,分析比较昆明种小鼠与C57BL/6J小鼠PDE β亚单位编码基因pde6b序列的差异.方法:设计覆盖pde6b CDS区的引物序列,通过逆转录-聚合酶链式反应扩增并克隆入载体pMD18-T中,转化大肠杆菌,酶切鉴定后测序.通过生物信息检索、序列拼接并应用生物信息学相关软件进行序列分析.结果:克隆了野生型昆明种小鼠的pde6b CDS全长.昆明种小鼠与C57BL/6J小鼠pde6b CDS区(NM_008806)相比较存在如下不同:昆明种小鼠第706位碱基为A,而数据库中序列NM_008806为G;第1149位碱基前者为T,后者为C.昆明种小鼠与C57BL/6J小鼠pde6b编码的蛋白序列差异并不明显,仅有第236位氨基酸残基为甘氨酸(G)突变成丝氨酸(S),为相对保守性替换关系.结论:克隆了昆明种小鼠pde6b CDS 区全长序列;pde6b基因在不同种属小鼠之间编码序列存在差异,表现出多样性特点,但蛋白序列保守性较高.     

Differences in resistance of C57BL/6 and C57BL/10 mice to infection by Mycobacterium avium are independent of gamma interferon

After infection with a low-virulence strain of Mycobacterium avium, C57BL/6 and C57BL/10 mice had clear differences in the control of the infection in their livers and spleens. This difference in susceptibility was not associated with differences in the H-2 complex. It was dependent on the activity of CD4(+) T cells but unrelated to the ability of these cells to secrete gamma interferon or to the development of delayed-type hypersensitivity responses at 3 weeks of infection. It was associated with lower total numbers of CD4(+) cells present in infected spleens and was related to an earlier induction of protective T cells, as measured by adoptive-transfer assays. These data further strengthen the notion of gamma-interferon-independent mechanisms of protection against mycobacteria.     

Sex-differences in age-related cognitive decline in C57BL/6J mice associated with increased brain microtubule-associated protein 2 and synaptophysin immunoreactivity

Understanding cognitive aging is becoming more important as the elderly population grows. Here, the effects of age and sex on learning and memory performance were compared in female and male young (3-4 months old) middle-aged (10-12 months old) and old (18-20 months old) wild-type C57BL/6J mice. Old males and females performed worse than young or middle-aged mice in novel location, but not novel object recognition tasks. Old mice, of both sexes, also showed impaired spatial water maze performance during training compared with young or middle-aged mice, however only old females failed to show robust spatial bias during probe trials. While there was no age-difference in passive avoidance performance for males, females showed an age-related decline. There was no difference in cognitive performance between young and middle-age mice of either sex on any task. Cognitive performance was associated with alterations in immunoreactivity of microtubule-associated protein 2-positive dendrites and synaptophysin-positive pre-synaptic terminals in hippocampal CA1, CA3, and dentate, entorhinal cortex, and central nucleus of amygdala. Overall, microtubule-associated protein 2 immunoreactivity was increased in old females compared with both young and middle-age females with no significant difference in males. In contrast, synaptophysin immunoreactivity increased from young to middle-age in females, and from middle-age to old in males; females had higher levels of synaptophysin immunoreactivity than males in middle-age only. Elevated levels of microtubule-associated protein 2 and synaptophysin may constitute a compensatory response to age-related functional decline in mice.     

Systolic blood pressure during the formation of a social dominance hierarchy in C57BL/6j mice

Housing male mice in social groups typically results in social dominance hierarchies and elevated systolic blood pressures. An exception to this pattern occurs in the C57BL/6j inbred mouse strain. C57BL/6j male mice, when handled prior to the experiment, tend to display neither social dominance hierarchies nor systolic hypertension. In the present experiment, we found that when C57BL/6j males compete for social dominance, as indicated by wounding, they also show elevations in systolic pressure. The association of social hierarchy and systolic pressure elevation can be found not only in aggressive mouse strains, but also within the more pacific C57BL/6j strain when it is exposed to competition.     

Deformed optic nerves in coisogenic albino and pigmented C57BL/6J-c2J mice

Summary The incidence of deformed optic nerves (DON) in C57BL/6J-C^2J mice is similar to that of a pigmented substrain of Long-Evans rats and much less than that of an albino substrain of Sprague Dawley rats. C57BL/6J-c^2J albinos have no more DON than do coisogenic black mice. This suggests that albinism is not a factor in DON and that observed DON in Sprague Dawley rats are strain or substrain characteristics.     

Comparison of C57BL/6 and DBA/2 mice in food motivation and satiety

Demand functions describe the relationship between the consumption of a commodity and its mean or unit price. In the first experiment, we analyzed food demand in two strains of mice (C57BL/6 and DBA/2) that differ on several behavioral dimensions, but have not been examined extensively for differences in feeding and meal patterns. Mice worked for food pellets in a continuous access closed economy in which total intake and meal patterns could be measured. A series of fixed (FUP), variable (VUP), and progressive (PUP) unit price schedules were imposed. Under all schedules, DBA/2 mice consumed significantly more food than C57BL/6, a difference that was not attributable to disparity in body weight or weight gain. The higher intake of DBA/2 mice was due predominantly to larger meal size compared with C57BL/6, with no strain difference in meal frequency. In a second experiment, strain differences in meal size were not found to correlate with anorectic sensitivity to cholecystokinin (CCK) administration, or with c-Fos expression induced by CCK in PVN, AP and NTS. Thus, DBA/2 mice were motivated to sustain a higher daily food intake and meal size than C57BL/6 under the range of demand costs employed in the present work, but this strain difference is unlikely to be due to CCK action or responsiveness.     

Macrophages are mediators of gastritis in acute Helicobacter pylori infection in C57BL/6 mice

Helicobacter pylori is the etiological agent of human chronic gastritis, a condition seen as a precursor to the development of gastrointestinal ulcers or gastric cancer. This study utilized the murine model of chronic H. pylori infection to characterize the role of macrophages in the induction of specific immune responses and gastritis and in the control of the bacterial burden following H. pylori infection and vaccination. Drug-loaded liposomes were injected intravenously to deplete macrophages from C57BL/6 mice, and effective removal of CD11b+ cells from the spleens and stomachs of mice was confirmed by immunofluorescence microscopy. Transient elimination of macrophages from C57BL/6 mice during the early period of infection reduced the gastric pathology induced by H. pylori SS1 but did not affect the bacterial load in the stomach. These data suggest that macrophages are important to the severity of gastric inflammation during H. pylori infection.     

Experimental myelitis caused by herpes simplex virus type 2 in C57BL/6N and BALB/cN mice

Intraperitoneal and intracranial inoculation of herpes simplex virus type 2 (HSV 2) into BALB/cN and C57BL/6N mice was carried out to induce experimental myelitis. The myelitis was clearly observed in C57BL/6N mice following intraperitoneal inoculation. Within 24 hours before death, the mice showed urinary and rectal incontinence and paraplegia of the hind legs. Randomly distributed, severe necrosis was demonstrated in the spinal cord, mainly at the lower cord. In BALB/cN mice the clinical symptoms were not clearly observed, as the mice died shortly after their onset. Although spinal cord necrosis was more prominent in C57BL/6N mice than BALB/cN mice, brain necrosis was only found in the latter, and not in the former. Both strains of mouse showed marked nuclear pyknosis of the nerve cells and slight nuclear pyknosis of the astrocytes in the brain where HSV 2 antigen was demonstrated immunohistochemically. The antigen was also detected in the necrotic spinal cord. In contrast, intracranial inoculation of the virus into both strains did not cause myelitis. Spinal cord necrosis was not demonstrated and virus DNA was not detected, by PCR, in spinal cord samples. In the brain, however, the virus was demonstrated by both PCR and immunohistochemistry.