阿托伐他汀对ApoE基因敲除小鼠血管外膜成纤维细胞表型的影响

目的:观察载脂蛋白E基因敲除(ApoE~(-/-))小鼠动脉粥样硬化斑块形成过程中血管外膜α-平滑肌肌动蛋白(α-SMA)和转化生长因子-β1(TGF-β1)的表达变化,同时探讨阿托伐他汀抗动脉粥样硬化的作用机制。方法:选择40只6周龄雄性ApoE~(-/-)小鼠随机分为模型组和阿托伐他汀干预组,给予高脂饲料喂养。阿托伐他汀干预组给予阿托伐他汀(20 mg·kg~(-1)·d~(-1))灌胃,模型组给予等量生理盐水灌胃。20只同龄C57BL/6小鼠给予普通饲料喂养作为正常对照组。各组小鼠喂养至10、15周龄,在各个时点处死动物,取升主动脉制备连续切片,通过Movat染色进行形态学观察,测量并计算血管外膜厚度及斑块相对面积;天狼星红染色检测胶原的表达;免疫组织化学染色检测不同时点血管外膜α-SMA及TGF-β1的表达变化。用实时荧光定量PCR检测胸主动脉外膜中TGF-β1 mRNA的表达水平,通过Western blot法检测主动脉外膜中TGF-β1蛋白的表达。结果:与模型组相比,阿托伐他汀干预组的斑块相对面积明显减小,血管外膜厚度及胶原合成明显下降;免疫组化结果显示15周龄模型组血管外膜α-SMA及TGF-β1的表达高于10周龄模型组;与模型组相比,阿托伐他汀干预组血管外膜α-SMA及TGF-β1的表达明显下降。各时点模型组的TGF-β1 mRNA和蛋白的表达明显高于对照组,给药干预后TGF-β1 mRNA和蛋白的表达明显降低。15周龄模型组血管外膜TGF-β1 mRNA和蛋白的表达高于10周龄模型组。结论:阿托伐他汀可能通过下调TGF-β1的表达调控血管外膜成纤维细胞表型的改变,进而延缓ApoE~(-/-)小鼠动脉粥样硬化的进程。     

C57BL/6小鼠胚胎成纤维细胞生物学特性及饲养层制备

背景：昆明小鼠胚胎成纤维细胞是目前最常用的饲养层细胞,C57BL/6小鼠胚胎成纤维细胞作为饲养层的研究鲜有报道。目的：体外分离和培养C57BL/6小鼠胚胎成纤维细胞,制备饲养层,力求扩大小鼠胚胎成纤维细胞的来源。方法：用不同浓度胰蛋白酶分步消化法体外分离和培养C57BL/6小鼠胚胎成纤维细胞,观察其生物学特性,研究其生长规律,并制备小鼠胚胎成纤维细胞饲养层,检测干细胞在所制备饲养层上的生长状态。结果与结论：不同浓度胰蛋白酶分步消化法制备的C57BL/6小鼠胚胎成纤维细胞生长状态好,获得的成纤维细胞数量多,增殖活跃。在细胞冻存后1,2周、1,3,6个月内复苏的细胞存活率差异无显著性意义。C57BL/6小鼠胚胎成纤维细胞在第2-5代增殖旺盛,第6代以后细胞增殖出现明显下降。种植到培养皿上的C57BL/6小鼠饲养层细胞在种植后3 d内活力高,种植4 d以后细胞活力急剧下降。所以C57BL/6小鼠胚胎成纤维细胞来源的饲养层的最佳使用时间为灭活后3 d内,C57BL/6小鼠胚胎成纤维细胞饲养层和昆明小鼠胚胎成纤维细胞饲养层一样,能很好地支持胚胎干细胞及诱导多能干细胞生长。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

FIZZ1在ApoE基因敲除小鼠动脉粥样硬化斑块内的表达

目的:探讨FIZZ1在ApoE基因敲除小鼠粥样斑块内的表达。方法:C57BL/6JApoE基因敲除鼠及C57BL/6J野生型小鼠各9只,分别喂食高脂饲料及普通饲料,24周后处死,自主动脉根部至腹主动脉离断整支血管,石蜡包埋后作连续切片,行HE染色及FIZZ1免疫组化,检测血管斑块内FIZZ1表达情况,RT-PCR检测斑块内FIZZ1mRNA表达。结果:ApoE基因敲除鼠高脂饲养24周后,主动脉根部动脉粥样硬化明显,斑块体积较大,免疫组化及其RT-PCR可见FIZZ1及其mRNA在粥样硬化斑块内明显表达,同龄野生型C57BL/6J鼠血管壁内未见FIZZ1及其mRNA表达。结论:FIZZ1能在动脉粥样硬化斑块内表达。     

4,4'-二甲氧基查耳酮改善高脂饲养小鼠的胰岛素敏感性

目的 观察黄酮类药物4,4'-二甲氧基查耳酮(DMC)在高脂饲养的小鼠中胰岛素敏感性的变化,并探讨其作用机制.方法 将高脂喂养的野生型C57/BL 6小鼠分为对照组和实验组每隔1 d按3μg/kg腹腔分别注射药物DMC和溶剂DMSO.每周检测记录两组小鼠进食和体质量,在第6周检测小鼠糖耐量(GTT)、胰岛素耐量(ITT...     

B细胞连接蛋白的表达抑制小鼠实验性自身免疫性脑脊髓炎

目的研究B细胞连接蛋白BLNK(B-cell linker protein)的表达对小鼠实验性自身免疫性脑脊髓炎(experimentalautoimmune encephalomyelitis,EAE)的影响并初步探讨其可能的机制。方法 MOG35-55多肽免疫BLNK缺陷(BLNK-deficient,BLNK-/-)鼠及C57BL/6鼠制备EAE小鼠模型,观察实验动物的临床症状及中枢神经系统的病理学变化;应用ELISA方法检测血清中的MOG特异性IgG和IgM抗体水平;采用流式细胞术检测脾脏中调节性T细胞的变化;分选的脾脏B淋巴细胞采用RT-PCR技术评价B细胞所分泌的细胞因子的变化。结果 BLNK-/-鼠临床症状评分明显高于C57BL/6鼠(P0.01);HE染色及LFB染色结果显示,BLNK-/-鼠与C57BL/C鼠比较炎症感染灶及脱髓鞘病灶明显增多。BLNK-/-鼠血清中的MOG特异性IgG和IgM抗体水平显著低于C57BL/C鼠(P0.005)。BLNK-/-鼠与C57BL/6鼠相比,脾脏中调节性T细胞百分比及B细胞所分泌的IL-10的表达明显减少,而IL-2的表达显著增高(P0.05);IFN-γ的表达水平在2组小鼠之间无统计学差异(P0.05)。结论 BLNK的表达抑制EAE,其机制可能是通过对B细胞分泌的Th1/Th2细胞因子的调节来实现。     

肿瘤疫苗Ad-hDCT抑制黑色素瘤B16细胞在C57BL/6小鼠脑内增殖

目的 观察肿瘤疫苗Ad-hDCT预防接种对移植到C57BL/6小鼠脑实质内的黑色素瘤B16细胞增殖的抑制作用.方法 40只C57BL/6小鼠随机分为2组:Ad-BHG对照组(20只)和Ad-hDCT免疫接种组(20只).借助小鼠脑立体定位仪行B16细胞脑内注射;采用大体观察、流式细胞术、石蜡切片HE染色及冷冻切片CD...     

Api6在高脂高胆固醇饮食引起肺部炎症中所发挥作用的研究*

 目的：研究凋亡抑制因子6（Api6）在高脂高胆固醇饮食所致C57BL/6J小鼠肺部炎症反应中的作用。方法：6～8周龄的C57BL/6J雄性小鼠喂养于SPF环境中,随机分成2组,分别给予普通饮食和高脂高胆固醇饮食喂养。喂养16周后收集肺组织并采用免疫组织化学和ELISA法鉴定肺组织的炎症状态。实时定量PCR和Western blotting鉴定Api6 mRNA与蛋白的表达水平,流式细胞术检测小鼠支气管肺泡灌洗液细胞凋亡情况。体外培养巨噬细胞RAW264.7,流式细胞术检测Api6对氧化型低密度脂蛋白（oxLDL）引起的细胞凋亡的影响。结果：高脂高胆固醇饮食喂养小鼠16周后,C57BL/6J小鼠肺组织出现以巨噬细胞蓄积以及肿瘤坏死因子α和单核细胞趋化蛋白1升高为主的炎症反应。与普通饮食组相比,高脂高胆固醇饮食喂养小鼠肺组织的Api6 mRNA和蛋白表达水平都显著上调（P<0.01）,同时支气管肺泡灌洗液中的巨噬细胞凋亡水平明显下降（P<0.01）。体外实验证实500 μg/L的重组Api6处理RAW264.7细胞可显著抑制oxLDL引起的细胞凋亡（P<0.05）。结论：高脂高胆固醇饮食可致C57BL/6J小鼠肺组织巨噬细胞蓄积,其机制可能与Api6抑制巨噬细胞的凋亡有关。     

γ射线对C57BL/6J和C3H/HeN小鼠肺成纤维细胞生物学行为影响的比较研究

目的比较C57BL/6J和C3H/HeN两种小鼠肺成纤维细胞以不同剂量的60Coγ射线照射后生物学行为的异同。方法原代分离培养C57BL/6J和C3H/HeN两种小鼠肺成纤维细胞(LF),应用2、4、6和8Gy的60Coγ射线照射后,通过MTT比色法、流式细胞术、AgNOR染色和免疫荧光细胞化学染色法,检测照射后两种LF的增殖活力、细胞周期以及a-平滑肌肌动蛋白(a-SMA)、基质金属蛋白酶-1(MMP-1)和金属蛋白酶组织抑制剂-1(TIMP-1)表达的变化。结果以2~8Gy照射后,C57BL/6J和C3H/HeN小鼠的LF增殖活力与正常对照组相比较未见明显增强。照射后,C57BL/6J小鼠的LF非整倍体增多,a-SMA高表达,MMP-1的表达呈弱阳性,TIMP-1的表达呈增强趋势。照射后,C3H/HeN小鼠的LF出现G2-M期阻滞,a-SMA的表达减弱至消失,MMP-1和TIMP-1的表达均呈增强趋势。结论以60Coγ射线照射后,C57BL/6J和C3H/HeN两种小鼠的LF生物学行为不同,C57BL/6J小鼠的LF呈“活化”状态,为该种小鼠易发生肺纤维化的细胞学基础提供了实验依据。     

胞质转导肽-HBcAg18-27-Tapasin诱导C57BL/6小鼠T淋巴细胞分泌Th1型细胞因子及HBV特异性CTL的表达

目的 观察融合蛋白胞质转导肽(CTP)-HBcAg18-27-Tapasin诱导C57BL/6小鼠T淋巴细胞分泌Th1型细胞因子及HBV特异性细胞毒T淋巴细胞(CTL)的表达.方法 C57BL/6小鼠随机分为实验组CTP-HBcAg18-27-Tapasin、对照组CTPHBcAg18-27、HBcAg18-27-Tapasin及空白组(生理盐水).经肌肉免疫小鼠,ELISA检测T淋巴细胞分泌细胞因子;流式细胞术(FCM)检测T淋巴细胞内的细胞因子;CCK-8法检测T淋巴细胞增殖活性.结果 实验组能有效刺激小鼠T细胞分泌Th1型细胞因子;FCM检测实验组融合蛋白诱导的CTL水平明显高于其他组;且实验组T淋巴细胞增殖活性明显高于对照组及空白组.结论 CTP-HBcAg18-27-Tapasin融合蛋白免疫C57BL/6小鼠后,能提高T淋巴细胞增殖活性,能有效刺激T淋巴细胞分泌Th1型细胞因子及增加CTLs的表达.     

趋化因子CCL2在实验性自身免疫性脑脊髓炎中的作用

目的:探讨趋化因子CCL2在实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)中的作用及其可能机制。方法:选取CCL2~(-/-)敲除小鼠和C57BL/6小鼠,利用髓鞘少突细胞糖蛋白(Myelin oligodendrocyte glycoprotein,MOG33-35)建立EAE小鼠模型,观察小鼠发病情况,并根据Konot神经功能评分标准对其进行评分;待模型组小鼠发病评分普遍为4~5分时,收集CCL2~(-/-)、C57BL/6模型组和C57BL/6空白组小鼠(n=10)脊髓组织进行HE染色观察和免疫组化检测,分析小鼠脊髓组织的病理变化和CD4~+、CD8~+T淋巴细胞数量改变。结果:CCL2~(-/-)、C57BL/6模型组小鼠发病率分别为0%、100%;其中,1只CCL2~(-/-)模型组小鼠在免疫后第21天出现轻微拖尾随后恢复正常。免疫后第21天时,CCL2~(-/-)模型组小鼠的神经功能评分明显低于C57BL/6模型组(P0.05)。HE染色结果显示,C57BL/6模型小鼠的脊髓组织中脱髓鞘现象明显,其炎性细胞数目显著高于CCL2~(-/-)组(P0.05);同时,免疫组化结果表明,CCL2~(-/-)小鼠脊髓组织中CD4~+T淋巴细胞、CD8~+T淋巴细胞均分别低于C57BL/6小鼠对照组(P0.05),尤其CD8~+细胞量改变存在显著差异(P0.05)。结论:趋化因子CCL2可能通过抑制T淋巴细胞的浸润抑制EAE的发生发展。     

Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: a novel hypothesis that complements the "Response-to-Injury Hypothesis" and the "Inflammation Hypothesis"

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This article introduces the hypothesis that the activation of the adventitia, specifically the fibroblasts, contributes to the formation of intimal atherosclerotic lesions. The evidence for this hypothesis includes: (a) the early proliferative changes seen in fibroblasts found in the adventitia; (b) the increase and the alteration of extracellular matrix deposition in the adventitia; (c) fibroblast differentiation into myofibroblasts and migration into the intima; and (d) fibroblast synthesis and release of cytokines that have potent effects on neighboring smooth muscle and endothelial cells prior to intimal lesion formation. In conclusion, the activation of adventitial fibroblasts is a key regulator of vascular function and structure from the "outside-in" and contributes to the development of atherosclerotic lesions. The outer location of the adventitia makes it a suitable location for drug delivery and gene therapy aimed at preventing and treating atherosclerosis.     

Deficiency of Vlgr1 resulted in deafness and susceptibility to audiogenic seizures while the degree of hearing impairment was not correlated with seizure severity in C57BL/6- and 129-backcrossed lines of Vlgr1 knockout mice

Vlgr1 (very large G-protein coupled receptor 1) knockout mice against hybrid backgrounds of the 129/Ola and C57BL/6 mouse strains show hearing deficit and high susceptibility to audiogenic seizures. The present study examined how hearing impairment and susceptibility to audiogenic seizures in Vlgr1-deficient mice change according to the genetic background of 129 and C57BL/6 mouse strains, which are popular strains for genetic studies. C57BL/6 mice have normal hearing ability during adolescence and are resistant to audiogenic seizures, and the 129S1/SvImJ substrain does not have a severe hearing deficit or convulsions as a result of audiogenic seizures; therefore, these strains were chosen for the present backcross study. C57BL/6-backcrossed Vlgr1 knockout mice and 129 (129S1/SvImJ)-backcrossed Vlgr1 knockout mice were established and their phenotypes investigated. Vlgr1 knockout mice showed hearing loss and high susceptibility to audiogenic seizures regardless of their genetic backgrounds. 129-backcrossed Vlgr1 knockout mice exhibited 10–20 dB more severe hearing loss than C57BL/6-backcrossed Vlgr1 knockout mice. In general, 129-backcrossed Vlgr1 knockout mice showed a higher incidence of wild running than C57BL/6-backcrossed Vlgr1 knockout mice, and this incidence became smaller as they matured. However, C57BL/6-backcrossed Vlgr1 knockout mice showed a significantly higher mortality rate as a result of auditory stimulation 3 weeks postnatally than 129-backcrossed mice.     

Metabolic and structural bone disturbances induced by hyperlipidic diet in mice treated with simvastatin

Simvastatin can modulate lipid and bone metabolism. However, information related to the interaction between diet and simvastatin on bone structure and biomechanics is scarce. Thus, this study evaluated the effects of simvastatin on femoral biomechanics and cortical/trabecular bone structure in wild‐type mice nourished with a hyperlipidic diet. Three‐month‐old male wild‐type mice (C57BL6 strain) were divided into four groups: (1) group W, nourished with a standard diet; (2) group WH, fed a hyperlipidic diet; (3) group WS, nourished with a standard diet plus oral simvastatin (20 mg/kg/day); and (4) group WHS, fed a hyperlipidic diet plus oral simvastatin (20 mg/kg/day). All animals received only their specific diet and water for 60 days. Blood samples were collected for the analysis of calcium, triglycerides, total cholesterol (TC) and fraction serum levels. Diet manipulation was able to induce a dyslipidaemic status in mice, characterized by triglyceride and TC rise in WH animals. Simvastatin prevented hypercholesterolaemia and reduced TC and LDL serum levels, but did not prevent hypertriglyceridaemia and HDL serum levels in the WHS group. In the WH mice the hyperlipidaemia was associated with reduction in trabecular bone thickness, femur structural and material property alterations. Simvastatin prevented these morphological alterations and minimized femur biomechanical changes in WHS mice. Taken together, the results indicated that the hyperlipidic diet intake acts as a risk factor for bone integrity, generating bones with reduced resistance and more susceptible to fractures, an effect attenuated by simvastatin that is potentially related to the modulatory action of this drug on lipid and bone metabolism.     

Dietary antioxidants decrease serum soluble adhesion molecule (sVCAM-1, sICAM-1) but not chemokine (JE/MCP-1, KC) concentrations, and reduce atherosclerosis in C57BL but not apoE*3 Leiden mice fed an atherogenic diet

Dietary antioxidants are reported to suppress cellular expression of chemokines and adhesion molecules that recruit monocytes to the artery wall during atherosclerosis. In the present study we measured the effect of feeding apoE*3 Leiden mice or their non-transgenic (C57BL) littermates with atherogenic diets either deficient in, or supplemented with, dietary antioxidants (vitamin E, vitamin C and beta-carotene) for 12 weeks, on serum levels of CC (JE/MCP-1) and CXC (KC) chemokines and soluble adhesion molecules (sVCAM-1, sICAM-1) and atherosclerotic lesion size. ApoE*3 Leiden mice developed gross hypercholesterolaemia, and markedly accelerated (10-20 fold; P < 0.0001) atherogenesis, compared with non-transgenic animals. Antioxidant consumption reduced lesion area in non-transgenic, but not apoE*3 Leiden, mice. Serum sVCAM-1 and sICAM-1 levels were significantly (P<0.0001) increased (sVCAM-1 up to 3.9 fold; sICAM-1 up to 2.4 fold) by 4-8 weeks in all groups, and then declined. The initial increase in the concentration of adhesion molecules was reduced by 38%-61% (P < 0.05) by antioxidant consumption, particularly in non-transgenic mice. By contrast, serum chemokine levels tended to increase more rapidly from baseline in apoE*3 Leiden mice, compared with non-transgenic animals, but were unaffected by dietary antioxidants. We conclude that dietary antioxidants reduce circulating soluble adhesion molecules and atherosclerosis in C57BL mice.     

An immunohistochemical analysis of fibroblasts in giant cell arteritis

BackgroundGiant cell arteritis (GCA) is a systemic vasculitis of large and medium vessels characterized by an inflammatory arterial infiltrate. GCA begins in the adventitia and leads to vascular remodeling by promoting proliferation of myofibroblasts in the intima. The morphology of the fibroblasts in the adventitia in GCA is unclear. Access to temporal artery biopsies allows morphological studies and evaluation of the microenvironment of the arterial wall. We evaluated the distribution of vascular fibroblasts and of markers of their activation in GCA.MethodsFormalin-fixed paraffin-embedded tissue sections from 29 patients with GCA and 36 controls were examined. Immunohistochemistry was performed for CD90, vimentin, desmin, alpha-smooth muscle actin (ASMA), prolyl-4-hydroxylase (P4H), and myosin to evaluate the distribution of fibroblasts within the intima, media, and adventitia.ResultsTemporal arteries from patients with GCA showed increased levels of CD90, vimentin, and ASMA in the adventitia and intima compared to the controls. Desmin was expressed only in the media in both groups. P4H was expressed similarly in the adventitia and intima in the two groups. Adventitial and intimal CD90⁺ cells co-expressed P4H, ASMA, and myosin at a high level in GCA.ConclusionThe results suggest a role for adventitial fibroblasts in GCA. Inhibiting the differentiation of adventitial fibroblasts to myofibroblasts has therapeutic potential for GCA.     

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.     

Electrocardiographic characterization of stress-induced myocardial infarction in atherosclerotic mice

AIM: We have previously shown that mental and hypoxic stress can trigger the development of myocardial infarction (MI) in atherosclerotic apoE(-/-) x LDLR(-/-) mice. The purpose of the present study was to characterize the interval between stress and MI and determine whether electrophysiological changes precede the precipitation of an infarct by assessing telemetry recordings of the electrocardiogram. METHODS: Isoflurane anaesthetized apoE(-/-) x LDLR(-/-) (n = 16) and C57BL/6J (n = 8) mice were exposed to systemic hypoxia by reducing the inhaled oxygen concentration to 10% for 10 min. Mental stress was induced in eight conscious apoE(-/-) x LDLR(-/-) and eight C57BL/6J mice by blowing air into the cage. Physiological parameters were recorded every 30 min for 2-6 days by implanted transmitters. RESULTS: During stress all mice developed transient ischaemic STU-area changes, which returned to normal at the end of stress. During the recovery phase (6 days) 50% (4/8) of the mentally stressed apoE(-/-) x LDLR(-/-) mice developed increased STU-area variability (P < 0.05) followed by dramatic STU-area elevations and spontaneous death at approximately 12-24 h. In hypoxia-exposed apoE(-/-) x LDLR(-/-) mice 56% (9/16) developed MI as determined by elevated serum levels of the infarction marker troponin T which correlated with increased variability in the STU-area (P < 0.05). CONCLUSION: This is the first mouse model showing that increased STU-area variability is indicative of MI development in atherosclerotic mice following ischaemic stress. Furthermore, our findings suggest a two-phase pathway for the infarction development: an initial phase comprising a transient ischaemic response which triggers a delayed second phase of ischaemia and MI.     

Amyloid A protein amyloidosis induced in apolipoprotein-E-deficient mice.

Apolipoprotein E (apoE) is a constituent of lipoproteins other than low-density lipoprotein, and it principally acts in the transport and metabolism of plasma cholesterol and triglyceride. ApoE is a minor constituent of various kinds of amyloidoses and may play a role as a pathological chaperone for fibrillogenesis of amyloid fibril protein with the amyloid P component and proteoglycans. In this study, we examined the role of apoE in amyloidogenesis in vivo in apoE-deficient mutant mice with amyloid A protein (AA) amyloidosis induced by inflammatory stimulation. Amyloid deposition was seen in six of nine C57BL/6J control mice and in six of eight apoE-deficient mutant mice after the intraperitoneal and subcutaneous injections of the mixture of complete Freund's adjuvant and Mycobacterium butyricum. Moreover, amyloid deposition in apoE-deficient mice as well as C57BL/6J control mice started 48 or 72 hours after injection of amyloid-enhancing factor and silver nitrate, although the amount of amyloid deposit in C57BL/6J control mice was slightly larger than that in apoE-deficient mice. These amyloid deposits reacted with anti-mouse AA antibody were seen in the perifollicular area of the spleen. Immunoreactivity of apoE was seen irregularly in the amyloid deposits of C57BL/6J control mice but not in the amyloid deposit of apoE-deficient mice. From these results, we concluded that apoE is not always necessary for amyloid deposition and that the existence of apoE might slightly accelerate AA amyloid deposition in the earliest phase of AA amyloid deposition.     

miR-1、miR-16和miR-208前体在乳鼠心肌细胞和心肌成纤维细胞中的表达

目的检测心肌特异的miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中的表达。方法利用出生1～3d的Sprague-Dawley（SD）大鼠和C57BL/6小鼠,采用2.5%胰酶消化和差速贴壁法分离心肌细胞和心肌成纤维细胞。提取原代的乳小鼠、乳大鼠心肌细胞及P1代的心肌成纤维细胞总RNA,以β-actin为内参照,逆转录酶-聚合酶链式反应（RT-PCR）分别检测miR-1、miR-16和miR-208的前体表达水平。用FluorChem8900软件分析琼脂糖凝胶电泳后PCR产物条带的灰度值,计算出表达的目的miRNA前体与β-actin的比值。结果有效分离并培养了乳C57BL/6小鼠、乳SD大鼠心肌细胞及心肌成纤维细胞。RT-PCR结果显示,在乳SD大鼠心肌细胞及心肌成纤维细胞中miR-1和miR-16均有相近水平的表达;心肌细胞中miR-208的表达显著低于miR-208b（P〈0.01）,miR-208b在心肌细胞中的表达显著高于其在心肌成纤维细胞中的表达（P〈0.05）。乳C57BL/6小鼠心肌细胞中miR-1a-1的表达显著低于miR-1a-2（P〈0.01）,而miR-1a-2在心肌细胞和心肌成纤维细胞中的表达水平一致;miR-16-1在心肌细胞和心肌成纤维细胞中表达水平相近,而miR-16-2在两种细胞中均不表达;心肌细胞中miR-208a和miR-208b表达水平差别不明显,心肌成纤维细胞中只有miR-208a表达,并且在水平上低于其在心肌细胞中的表达（P〈0.05）。结论 miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中呈细胞特异性的表达。