MS-222、丁香油、苯唑卡因对养殖美洲鲥幼鱼的麻醉效果

研究了MS-222、丁香油、苯唑卡因3种麻醉剂对美洲鲥Alosa sapidissima幼鱼的麻醉效果,并运用该3种麻醉剂的适宜剂量对美洲鲥幼鱼进行了运输试验。麻醉试验结果表明:在较高麻醉浓度(MS-222为75 mg/L以上,丁香油为20 mg/L以上,苯唑卡因为40 mg/L以上)下,鱼很快(30 min内)停止鳃盖张合运动,且停止鳃盖运动的鱼在空气中暴露一定时间(10 min内)后也能够复苏;在适宜的麻醉浓度(MS-222为20-30 mg/L,丁香油为8-10 mg/L;苯唑卡因为20-30 mg/L)下,鱼能够进入麻醉状态,且能保持很长时间(12 h);麻醉效果随着水温的升高而增强;在20 mg/L MS-222麻醉剂下,小规格鱼较大规格鱼更容易进入麻醉状态,而在10 mg/L丁香油和20 mg/L苯唑卡因麻醉剂下,小规格鱼却难进入麻醉状态。运输试验结果表明:麻醉运输组和对照组(非麻醉运输组)鱼血清中皮质醇的含量均显著高于基础组(运输前)(P<0.05);麻醉运输后鱼血清中皮质醇的含量虽均有一定程度的升高,但明显低于对照组,其中仅苯唑卡因麻醉运输组鱼血清中皮质醇的含量显著低于对照组(P<0.05)。试验结果表明,苯唑卡因更适合用于运输美洲鲥的麻醉。     

气相色谱-质谱法检测鱼肉中MS-222残留

本文建立了鱼肉中MS-222残留GC-MS检测方法.鱼肉样品经乙腈提取,氮吹浓缩,盐酸溶液引导MS-222电离,Waters Oasis MCX固相萃取柱净化后,气相色谱-四极杆质谱检测.方法检出限为2.5 μg·kg-1、定量限为5.0 μg·kg-1;0.0025～1.0 μg·mL-1范围内线性关系良好（R≥0.9996）;MS-222浓度范围在5.0～ 100.0 μg·kg-1的鱼肉加标样,日内和日间平均回收率为78.4％～91.2％,相对标准偏差为3.62％ ～9.49％.结果表明,该检测方法适用于低浓度水平鱼肉中MS-222残留检测.     

两种麻醉剂对罗非鱼的急性毒性及联合毒性研究

根据预试验结果确定最高安全浓度和最低全致死浓度,分别设置5个等对数浓度,进行MS-222和苯唑卡因及其1:1混合药物对罗非鱼(Oreochromis niloticus)的24h和96h毒性试验.结果:MS-222对罗非鱼的24h、96h半致死浓度及95%置信区间分别为88.84 mg/L(86.92～90.81 nag,/L)、76.18 mg/L(73.27～79.21 mg/L);苯唑卡因对罗非鱼的24h、96h半致死浓度及95%置信区间分别为44.49 mg/L(43.76～45.24 mg/L)、22.61 mg/L(20.29～25.20 mg/L);混合药物对罗非鱼的24h、96h半致死浓度及95%置信区间分别为63.35 mg/L(61.00～65.79 mg/L)、30.39 mg/L(29.48～31.34 mg/L).这表明:在同等剂量条件下,苯唑卡因对罗非鱼的毒性比MS-222强;将两种药物等比例混合后,在24h和96h毒性试验中,分别呈现出拮抗作用和协同作用.     

miR-423-5p在牛肌肉组织中表达及其靶基因预测

miR-423-5p在细胞中具有重要的生物学功能,如在肌细胞中能影响细胞的增殖.根据miRBase及NCBI网站显示的相关信息利用分析软件对miR-423在各物种之间的同源性进行了分析.取成年西门塔尔牛体内的骨骼肌、小肠、心脏组织利用茎环荧光定量PCR进行miR-423-5p表达量检测,将牛骨骼肌卫星细胞(MDSCs)...     

Ccr-lncRNA172145靶向miR-206在锦鲤体色调控中的作用初探

为了解非编码RNA在锦鲤体色分化变异中的分子调控机制,在课题组前期对锦鲤皮肤组织转录组测序的基础上,筛选到4条在3种皮肤(黑色、红色、白色)组织中显著差异表达的lncRNA;基于RNAhybrid和TargetScan靶基因预测软件,发现lncRNA172145与黑色素合成通路中miR-206之间存在靶向结合位点。借助CPC、CPAT及CNIT软件,对lncRNA172145编码能力进行分析,证实该序列为lncRNA,不具备编码蛋白的能力。然后,利用qRT-PCR技术对该序列时空表达水平进行了检测,发现在眼睛、黑色皮肤、鳍条及血液中表达量显著高于其他组织;自原肠胚时期表达量开始显著上升,高水平趋势一直持续到孵化后20 d。通过双荧光素酶报告实验,进一步证实lncRNA172145与miR-206之间存在靶向调控关系。最后,通过合成miR-206拮抗剂,对miR-206进行体内沉默,发现与注射阴性对照拮抗剂组和PBS组相比,miR-206拮抗剂组的lncRNA172145表达水平显著升高。研究表明,lncRNA172145可能通过靶向到miR-206,参与到黑色素合成通路的调控,这为后续...     

Time-course changes in the expression levels of miR-122, -155, and -21 as markers of liver cell damage,inflammation, and regeneration in acetaminophen-induced liver injury in rats

Drug-induced liver injury (DILI) is a significant threat to patient health and a major concern during drug development. Recently, multiple circulating microRNAs (miRNAs) have been reported to be potential biomarkers for DILI. To adapt and validate miRNAs for clinical use, we investigated the time-course changes in miR-122 expression levels in an acetaminophen-induced liver injury model in rats. In addition, miR-155 and miR-21 were evaluated as makers of inflammation and regeneration, respectively, to characterize liver status. Our results revealed that miR-122 is an early and sensitive biomarker of hepatocellular injury at a stage when alanine transaminase, aspartate transaminase, and total bilirubin were not detectable. However, no significant differences in the expression levels of other miRNAs (miR-155 and -21) were observed between treatment and vehicle groups. Collectively, these time-course changes in the expression levels of miRNAs may be useful as markers for clinical decision-making, in the diagnosis and treatment of DILI.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

miR-125a/125b基因家族进化分析及其在猪睾丸组织中的表达变化

采用生物学信息方法在miRBase中搜索动物miR-125a/125b基因家族序列,利用Ensembl数据库信息确定miR-125a/125b在基因组中的位置,采用MEGA 5.0构建miR-125a/125b家族的系统进化树,并利用qRT-PCR检测miR-125a和miR-125b在沙子岭猪睾丸组织不同发育时期(D1、D30、D60、D90、D120、D150、D180)的表达变化。结果在miRBase数据库检索29个物种共58条miR-125家族序列,分布广泛,且有高度的保守性,除一个位于内含子区,两个位于外显子区,其余miR-125a/125b位于基因间隔区,在进化过程中,同种物种的进化方向具有高度的一致性,进化速度也有所不同。qRT-PCR检测结果显示,miR-125a/125b在沙子岭猪睾丸组织中不同发育时期的具有相同的趋势,即出生后其表达水平缓慢升高,保育期开始降低,性成熟期又开始呈现升高趋势,随着体成熟表达量开始降低。本研究结果为进一步探明miR-125a/125b基因家族在猪睾丸组织中的功能及作用机制奠定基础。     

MiR‐222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS1 gene

Apoptosis of granulosa cells affects follicular atresia and reproduction and is regulated by miRNAs and the expression of certain genes. For the present study, we investigated the regulatory relationship between microRNA‐222 (miR‐222) and THBS1 in porcine follicular granulosa cells (pGCs) and its effects on apoptosis to provide empirical data for developing methods to improve pig fecundity. Results revealed that miR‐222 promotes the proliferation of pGCs. MiRNA mimics and luciferase reporter assays revealed that miR‐222 functions as an anti‐apoptotic factor in pGCs. MiR‐222 mimics in pGCs result in the upregulation of the anti‐apoptotic BCL‐2 gene, down‐regulation of the proapoptotic caspase‐3 gene, and inhibition of apoptosis. MiR‐222 inhibitors reduced BCL‐2 and had no significant effect on caspase‐3. MiR‐222 mimics promoted estrogen levels. Inhibition of THBS1 inhibited pGC apoptosis. Transfection of THBS1‐siRNA reduced the proapoptotic BAX gene. MiR‐222 can directly target the 3′‐untranslated region of the THBS1 gene. MiR‐222 mimics suppressed THBS1 mRNA and proteins, but these were upregulated by the miR‐222 inhibitor. Transfection of THBS1‐siRNA resulted in the inhibition of the miR‐222 inhibitor, which suggests that miR‐222 inhibits pGC apoptosis by targeting THBS1. These findings suggest that miR‐222 and THBS1 play important roles in follicular atresia, ovarian development, and female reproduction.     

猪miR-23a靶向调控Smad3基因表达的研究

为了确定miR-23a是否靶向调控Smad3基因,试验利用NotⅠ和XhoⅠ酶构建包含Smad3-3′-UTR的野生型(psiCHECKTM-2-W-Smad3-3′-UTR)和突变型双荧光酶报告载体(psiCHECKTM-2-M-Smad3-3′-UTR),并在PK-15细胞中转染miR-23amimics、miR-23ainhibitor及其阴性对照,采用双荧光酶检测试剂盒检测荧光素酶活性,用实时荧光定量PCR和Western blotting法分别检测Smad3基因的mRNA和蛋白表达水平。结果表明,将含Smad3-3′-UTR的野生型和突变型双荧光酶报告载体与miR-23amimic共转染PK-15细胞,野生型报告质粒表达的荧光素酶活性显著低于其阴性对照组(P<0.05);转染miR-23amimics能显著下调Smad3基因mRNA及其蛋白表达水平(P<0.05);而转染miR-23ainhibitor组与miR-23ainhibitor阴性对照组相比,Smad3基因蛋白表达差异不显著(P>0.05)。综合上述结果可知,猪miR-23a可靶向作用于Smad3基因。