小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。     

心肌细胞特异性miR-30b转基因小鼠的建立及其功能研究

MicroRNAs(miRNAs)基因芯片结果显示,携带有MYH7基因突变的家族性肥厚性心肌病病人的心脏组织以及小鼠心力衰竭模型中miR-30b表达下调,提示miR-30b可能在心脏疾病发生发展过程中发挥了重要功能.为研究miR-30b在心脏组织中的功能,本实验室首先建立了在心肌细胞特异性启动子琢肌球蛋白重链(琢-MHC,5.5 kb)控制下过表达miR-30b的转基因小鼠.通过qRT-PCR方法,证实miR-30b表达水平在转基因小鼠心脏组织中明显升高(P0.05).miR-30b转基因小鼠心重/体重比和左心室/体重比无明显变化,心肌组织结构未见异常.目前,关于miR-30b在心肌梗死中的功能及相关机制未见报道.本文通过冠状动脉左前降支结扎法建立心肌缺血再灌注(ischemia-reperfusion,I/R)模型,以假手术组作为对照组.生化检测结果及TTC-Evans blue双染结果显示,I/R损伤后,与野生型小鼠相比,转基因小鼠LDH、CK-MB和cTn玉浓度显著减小(P0.05),并且心肌梗死面积明显减少(P0.05).超声心动图检测结果显示,转基因小鼠心功能显著改善.由此得出结论:miR-30b对缺血再灌注损伤的心肌具有保护作用,该研究成果可能为预防和治疗心肌梗死提供新策略.     

半滑舌鳎miR-200a和miR-200b前体克隆及其免疫应答分析

为了探究半滑舌鳎（Cynoglossus semilaevis）miR-200a和miR-200b在免疫应答中的作用,采用PCR方法克隆了半滑舌鳎miR-200家族的miR-200a和miR-200b的前体序列,长度分别为82和88 bp;用The mfold Web Server和Clustalx1.83软件对其前体序列进行了二级结构和同源性分析,miR-200a和miR-200b都具有典型的颈环结构,与其他物种具有较高的同源性。qRT-PCR分析结果显示,miR-200a和miR-200b在健康半滑舌鳎13种组织（肝脏、肠、脾脏、头肾、后肾、鳃、血液、脑、皮肤、肌肉、胃、心脏和卵巢）中均有表达,miR-200a在头肾中表达量最高,在血液中表达量最低,miR-200b在肝脏中表达量最高,在肌肉中表达量最低;miR-200a和miR-200b在鳗弧菌（Vibrio anguillarum）感染半滑舌鳎后不同时间点的4种免疫相关组织（肝脏、肠、脾脏和头肾）中的表达呈现出先上调后下降的规律,但表达达到峰值的时间点有所不同。miR-200a在肝脏和脾中的表达峰值出现在鳗弧菌感染后6h,在肠和头肾中则是鳗弧菌感染后12h,miR-200b在肠、脾和头肾中均在鳗弧菌感染后12h达到表达高峰;miR-200a和miR-200b在脂多糖（LPS）、肽聚糖（PGN）、葡聚糖（WGP）、聚肌胞苷酸（poly I:C）4种病原模拟物刺激后的半滑舌鳎肝脏细胞系中呈现出上调表达趋势,其中Poly I:C刺激半滑舌鳎肝脏细胞系后miR-200a上调表达趋势明显,6h的表达量为0h的9倍,在WGP刺激半滑舌鳎肝脏细胞后miR-200b上调表达趋势明显,2h的表达量为0的9倍。研究结果为揭示miRNA在半滑舌鳎免疫应答中的作用提供了科学依据。     

Mipu1基因在内毒素血症小鼠组织中的表达研究

Mipu1是本研究室首次克隆的一个核转录因子,其在内毒素血症中的表达改变情况目前尚不清楚.本研究采用real-time PCR方法检测了Mipu1基因在内毒素血症(12 mg/kg,2 h)小鼠心、肝、肺、脾、脑、肠和骨骼肌7个器官组织中的表达改变.结果发现,LPS(12 mg/kg,2 h)处理可促进小鼠肺和脾组织中Mipu1基因表达增高;但可抑制小鼠心、肝、脑、肠和骨骼肌组织中Mipu1基因的表达.Mipu1基因在内毒素血症小鼠各器官中表达的改变可能与其在内毒素血症中的生物学功能密切相关.     

miR-106b转基因小鼠的建立

目的:建立miR-106b转基因小鼠模型,探讨其在阿尔茨海默病(Alzheimer’s disease, AD)发病中的作用。方法:构建miR-106b表达载体,显微注射法建立miR-106b转基因小鼠。PCR鉴定转基因小鼠的基因型,real time RT-PCR检测miR-106b转基因小鼠脑组织中miR-106b的表达情况,Western blot检测miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达。结果:构建了高表达miR-106b转基因小鼠;与对照相比,miR-106b转基因小鼠脑组织中TGFBR2蛋白的表达升高。结论:miR-106b转基因小鼠的建立为研究该microRNA在AD发病中的作用提供了工具。     

microRNA-483和microRNA-486在克隆和转fat-1基因牛组织中的表达

利用荧光定量PCR比较正常受精牛、克隆牛和转基因牛的心、肝、脾、肺、肾、胎盘、子叶、子宫内膜中miR-483和miR-486的表达水平。结果显示,miR-483和miR-486在正常受精牛、克隆牛和转fat-1基因牛的组织中均有表达,其中在心脏中表达量显著高于其他组织。而转fat-1基因牛心脏中miR-483和miR-486表达量均低于正常牛。miR-483和miR-486在不同组织中表达量存在一定差异,在心脏中呈现高表达,提示miR-483和miR-486表达降低可能与心肌肥大、心肌梗死等病理生理过程有关。     

心肌细胞过表达miR-27b导致小鼠发生心肌纤维化和线粒体损伤

以往的miRNA芯片研究结果显示, miR-27b在人类心脏疾病标本和压力负荷引起的小鼠心肌肥厚模型中表达水平明显升高, 提示其在心脏疾病发生过程中发挥了重要功能。为研究miR-27b在心脏组织中的功能, 文章建立了在心肌细胞特异性 a-肌球蛋白重链(a-MHC)启动子(5.5 kb)控制下过表达miR-27b的转基因小鼠。通过Real-time PCR检测, 发现miR-27b前体和成熟体表达水平在转基因小鼠心脏组织中明显升高。miR-27b转基因小鼠不仅出现心肌肥厚, 还表现出明显的心肌纤维化。进一步研究表明心肌纤维化的关键调节分子金属基质蛋白酶13(MMP13)是miR-27b的靶分子, 在miR-27b转基因小鼠中MMP13显著下调, 胶原分子I和 III则显著上调。此外, 还发现miR-27b转基因小鼠会出现心脏超微结构的损伤。以上研究结果表明, miR-27b可能通过抑制MMP13促进心肌纤维化。     

心肌细胞过表达miR-27b导致小鼠发生心肌纤维化和线粒体损伤

以往的miRNA芯片研究结果显示, miR-27b在人类心脏疾病标本和压力负荷引起的小鼠心肌肥厚模型中表达水平明显升高, 提示其在心脏疾病发生过程中发挥了重要功能。为研究miR-27b在心脏组织中的功能, 文章建立了在心肌细胞特异性 a-肌球蛋白重链(a-MHC)启动子(5.5 kb)控制下过表达miR-27b的转基因小鼠。通过Real-time PCR检测, 发现miR-27b前体和成熟体表达水平在转基因小鼠心脏组织中明显升高。miR-27b转基因小鼠不仅出现心肌肥厚, 还表现出明显的心肌纤维化。进一步研究表明心肌纤维化的关键调节分子金属基质蛋白酶13(MMP13)是miR-27b的靶分子, 在miR-27b转基因小鼠中MMP13显著下调, 胶原分子I和 III则显著上调。此外, 还发现miR-27b转基因小鼠会出现心脏超微结构的损伤。以上研究结果表明, miR-27b可能通过抑制MMP13促进心肌纤维化。     

全组织提取物检测A-to-I RNA编辑酶活性

目的:建立一种简便、快速、可靠的检测A-to-IRNA编辑酶活性的方法。方法与结果:一步法制备的C57BL/6小鼠十种组织的全组织提取物,在各提取物中检测到A-to-IRNA编辑酶的非特异性编辑活性,不同组织中A-to-IRNA编辑酶活性的强度依次为脑>肺>胸腺>脾>淋巴结>肝>肾>睾丸>心脏>骨骼肌,编辑活性与加入反应体系中的蛋白量成正相关。结论:一步法制备的全组织提取物可用于检测A-to-IRNA编辑酶活性,该方法操作简便、可控、省时。     

贵州白山羊DRA和DRB1基因的表达差异分析

本研究旨在利用实时荧光定量PCR技术检测贵州白山羊DRA和DRB1基因在心、肝、肾以及免疫组织共9种动物组织中的相对表达差异。分析数据得到,贵州白山羊DRA、DRB1基因在9种组织中均有表达,在脾组织中两个基因的表达量均明显高于其他组织,在心、肝、肾和颌下腺四中组织中DRA和DRB1基因的表达量均明显低于其他组织,其中在心和肝两种组织中表达量最低。结果表明,在5种免疫组织中除颌下腺外,DRA和DRB1基因的表达量都较高且都在脾脏组织中高表达。     

中华姬鼠与大林姬鼠的同工酶差异

中华姬鼠（Ａｐｏｄｅｍｕｓｄｒａｃｏ）和大林姬鼠（Ａｐｏｄｅｍｕｓｐｅｎｉｎｓｕｌａｅ）是形态学上十分相似的两种鼠类。为了对两种姬鼠的分类提供生物化学方面的依据,采用聚丙烯酰胺凝胶等电聚焦电泳方法比较和分析了两种姬鼠的ＬＤＨ同工酶、ＥＳＴ同工酶和ＳＯＤ同工酶的差异。结果表明,两种姬鼠的ＬＤＨ同工酶酶谱基本相似,而ＥＳＴ同工酶和ＳＯＤ同工酶酶谱则存在明显的种间差异。根据ＥＳＴ同工酶Ａ２带的有无和ＳＯＤ同工酶主带等电点的差别,能将两种姬鼠很容易区分开来。     

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn^−/− mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn^−/− mice. Increased stability of AMPKγ in Crbn^−/− MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4^CRBN directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4^CRBN regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.     

Protective role of estrogen-induced miRNA-29 expression in carbon tetrachloride-induced mouse liver injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.     

MiR-92d-3p suppresses the progression of diabetic nephropathy renal fibrosis by inhibiting the C3/HMGB1/TGF-β1 pathway

The pathogenesis of diabetic nephropathy (DN) has not been fully elucidated. MicroRNAs (miRNAs) play an important role in the onset and development of DN renal fibrosis. Thus, the present study aimed to investigate the effect of miR-92d-3p on the progression of DN renal fibrosis. We used qRT-PCR to detect the expression levels of miR-92d-3p in the kidneys of patients with DN. Then, after transfecting lentiviruses containing miR-92d-3p into the kidneys of a DN mouse model and HK-2 cell line, we used qRT-PCR to detect the expression levels of miR-92d-3p, C3, HMGB1, TGF-β1, α-SMA, E-cadherin, and Col I. The expression levels of interleukin (IL) 1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the HK-2 cells were detected through enzyme-linked immunosorbent assay (ELISA), and Western blotting and immunofluorescence were used in detecting the expression levels of fibronectin, α-SMA, E-cadherin, and vimentin. Results showed that the expression levels of miR-92d-3p in the kidney tissues of patients with DN and DN animal model mice decreased, and C3 stimulated HK-2 cells to produce inflammatory cytokines. The C3/HMGB1/TGF-β1 pathway was activated, and epithelial-to-interstitial transition (EMT) was induced in the HK-2 cells after human recombinant C3 and TGF-β1 protein were added. miR-92d-3p inhibited inflammatory factor production by C3 in the HK-2 cells and the activation of the C3/HMGB1/TGF-β1 pathway and EMT by C3 and TGF-β1. miR-92d-3p suppressed the progression of DN renal fibrosis by inhibiting the activation of the C3/HMGB1/TGF-β1 pathway and EMT.     

不同强度运动训练对心肌凋亡途径微小RNA及其靶蛋白的影响

目的：考察不同负荷运动训练对小鼠心肌凋亡相关miR-1,miR-21和靶蛋白的影响,探讨运动干预心肌凋亡的可能机制。方法：选取21只C57BL/6小鼠,随机分为3组（n=7）：安静组（SE组）、训练1组（ET1组）、训练2组（ET2）。SE组不进行训练,ET1组完成8周递增负荷游泳训练,5天/周,1次/天,第1周30 min/count,每周增加10 min,第7、8周时间维持在90 min;ET2组在ET1组方案基础上增加负荷,前5周与ET1相同,后3周每天训练2次。TUNEL检测考察心肌凋亡水平,Western blot和RT-PCR分别测定蛋白和miRs的变化。结果：ET1组游泳训练对小鼠心肌凋亡影响不明显,miR-1表达无显著变化,但其靶蛋白Bcl-2表达显著增高（P<0.01）,miR-21及其靶蛋白PDCD4表达均无显著变化。ET2组游泳训练显著降低心肌凋亡水平及miR-1表达（P<0.01）、提高Bcl-2表达（P<0.05）;同时显著提高miR-21表达（P<0.05）,但对PDCD4表达无明显影响。结论：ET1组训练对心肌凋亡干预不明显,ET2组运动训练可降低心肌凋亡水平,miR-1及靶蛋白Bcl-2变化可能是机制之一,PDCD4对运动训练不敏感,miR-21可能与其它靶蛋白参与运动干预心肌凋亡的分子机制。     

Cyclosporine blocks the induction of class I and class II MHC products in mouse kidney by graft-vs-host disease

The ability of cyclosporine to prevent the increase in Ia and H-2K expression that occurs in mice with graft-vs-host disease (GVHD) was examined by means of absorption, indirect immunofluorescent staining (IIF), and indirect immunoperoxidase staining (IIP). Acute GVHD was induced in irradiated C3H/HeJ mice (H-2k) by injections of bone marrow and spleen cells from C57BL/6J mice (H-2b). Ten days after induction of acute GVHD, the spleens of mice not receiving cyclosporine expressed only donor Ia, reflecting their reconstitution by donor cells. The kidneys of such mice had a 10-fold increase in host Ia and H-2K expression, as previously reported. Treatment with cyclosporine reduced the amount of donor Ia and H-2K in spleens, and prevented the enhanced expression of recipient Ia and H-2K in kidneys in a dose-dependent manner. IIF or IIP staining showed that the principal change was in kidney tubules, where the induction of Ia and H-2K expression was greatly diminished. Cyclosporine administered to normal mice did not alter Ia expression except at high doses, at which it decreased Ia expression in kidneys and in spleens. The results suggest that prevention of enhanced MHC product expression could be part of the immunosuppressive actions of cyclosporine.     

MiR-15b can target insulin receptor to regulate hepatic insulin signaling in mice

Now diabetes is growing to be a health problems globally. However, its specific pathogenesis still needs further exploration. Here we showed that miR-15b was upregulated in the palmitate-induced HepG2 cells and livers of hyperglycemic mice. At the same time, we confirmed that the insulin receptor was a direct target of miR-15b. Then we found that the manipulation of miR-15b expression level could affect the insulin signaling pathway of HepG2 cells and the inhibition of miR-15b in liver of ob/ob mice can improve insulin sensitivity of mice. Furthermore, our study demonstrated that palmitate could upregulate the expression of miR-15b by activating PPARα. Our findings established PPARα-responsive miR-15b as a critical regulator of hepatic insulin signaling, thus serving as a new potential therapeutic target for diabetes.