NIRF可抑制乙型肝炎病毒在水动力法转染HBV小鼠模型中的复制及表达

目前对NIRF(Np95/ICBP-90 like RING finger protein)的研究正朝着细胞癌变进程以及表观遗传学的方向发展,但在体内NIRF对乙型肝炎病毒(HBV)的复制及表达的影响,目前尚不明确.通过高压水动力法转染HBV小鼠模型,不同时间点收集血液和肝组织标本,荧光定量PCR检测血清及肝组织中病毒载量,WesternBlot检测肝组织HBc(hepatitis B virus core protein)表达,ELISA检测血清HBeAg表达,并通过免疫组化染色检测HBsAg、HBcAg在肝组织中的定位及表达.小鼠转染pAAV-HBV1.3和NIRF以后,血清及肝组织病毒载量降低(n=3,P<0.05),HBc蛋白及HBV相关抗原的表达受到抑制,说明在水动力法转染HBV小鼠模型中NIRF对HBV的复制及表达起到抑制作用,期待能为后续的HBV致病机理及治疗药物的研究与开发提供支持与帮助.     

西藏土壤中耐辐射阿氏芽胞杆菌T61的分离和鉴定

【目的】对分离自西藏土样的菌株T61进行分离、鉴定和UV辐射抗性分析。【方法】对菌株T61进行形态和生理生化鉴定;对16S r RNA基因进行克隆和测序,构建系统进化树;测定脂肪酸成分和GC含量,将T61与最相近种进行DNA-DNA杂交;测定T61的UV辐射抗性曲线。【结果】T61细胞杆状,长度约为2μm,直径约为1μm,革兰氏阳性,可产生内生孢子。G+C含量为38.02%。脂肪酸主要成分是C14:0 iso、C15:0 iso和C15:0 anteiso。16S r RNA基因与阿氏芽胞杆菌B8W22T和巨大芽胞杆菌IAM13418T相似度最高,分别达到99.93%和99.53%。DNA-DNA杂交分析表明,T61与阿氏芽胞杆菌B8W22T的相似度为81.4%,而与巨大芽胞杆菌IAM13418T的相似度只有50.3%。UV辐射抗性分析显示,T61 D10为100 J/m2,远高于辐射敏感的大肠杆菌K12和枯草芽孢杆菌等菌株。【结论】菌株T61是一株阿氏芽胞杆菌,命名为Bacillus aryabhattai T61,其对UV辐射具有较强的抗性。     

消毒剂对环境菌株抑制作用的研究

目的 研究企业常用消毒剂对于洁净室环境监测分离的菌株样本的抑制作用。方法 通过VITEK2-COMPACT全自动细菌鉴定及药敏分析系统鉴定收集到的环境菌株。对3种消毒剂(碘伏、无水乙醇和苯扎溴铵)进行梯度稀释,利用打孔法研究3种消毒剂在不同含量下对环境菌株的抑制作用。结果 共检出革兰阳性菌8种、革兰阴性菌2种、酵母菌1种、芽孢杆菌2种;苯扎溴铵对革兰阳性菌的抑菌能力都较强,随着含量的降低,抑菌作用逐渐减弱;碘伏对革兰阴性菌及酵母菌的抑制作用都非常强,随着含量降低,抑菌作用逐渐降低。3种消毒剂对2种芽孢杆菌的抑制作用均有限。结论 用1.00%苯扎溴铵和0.50%的碘伏抑菌作用都非常强。另外,应配合使用杀孢子剂,避免芽孢杆菌孢子在空气中传播。     

矮化香蕉及其野生型GA3ox基因的结构特点和表达分析

香蕉的矮化突变是香蕉无性繁殖后代最常见的表型变异之一,但其变异的分子调控机理目前尚未研究清楚; 而内源赤霉素是影响植物株高的重要激素之一,GA3-氧化酶是赤霉素生物合成后期的关键酶。为探究GA3-氧化酶编码基因对香蕉矮化的分子调控机理,该研究以威廉斯B6矮化突变体及其野生型亲本为材料,通过RT-PCR技术克隆得到矮化香蕉及其野生型亲本GA3ox基因的全长cDNA序列,并对其推测的氨基酸序列进行比对分析,同时利用qRT-PCR技术对GA3ox基因在不同组织中的表达水平差异进行分析。结果表明:(1)矮化香蕉GA3ox-A和野生型香蕉GA3ox-G的ORF长度均为864 bp,均编码287个氨基酸,经序列比对分析发现两条氨基酸序列之间存在5个位点的差异,从而产生具有不同性质的蛋白质。(2)氨基酸序列同源性分析表明,矮化香蕉GA3ox的氨基酸序列与油棕、海枣、椰子的同源性最高。(3)qRT-PCR显示,GA3ox基因在矮化香蕉叶片和茎秆中的表达水平整体上低于野生型,其中GA3ox在野生型茎秆中的表达水平是矮化植株的2.2～32倍。综上推测,GA3ox基因可能对香蕉茎杆的矮化变异具有重要的调控作用。该研究结果为揭示香蕉矮化突变的分子机制与筛选优良矮化香蕉株系奠定了基础。     

microRNA-448 inhibits stemness maintenance and self-renewal of hepatocellular carcinoma stem cells through the MAGEA6-mediated AMPK signaling pathway

Hepatocellular carcinoma (HCC) occurs mainly in patients with chronic liver disease and cirrhosis. Increasing evidence has identified the involvement of microRNAs (miRNAs) acting as essential regulators in the progression of HCC. As predicted by microarray analysis, miR-448 might potentially affect HCC progression by regulating the melanoma-associated antigen (MAGEA). Therefore, the present investigation focused on exploring whether or not miR-448 and MAGEA6 were involved in the self-renewal and stemness maintenance of HCC stem cells. The interaction among miR-448, MAGEA6, and the AMPK signaling pathway was evaluated. It was noted that miR-448 targeted and downregulated MAGEA6, thus activating the AMP-activated protein kinase (AMPK) signaling pathway in HCC. Furthermore, for the purpose of exploring the functional relevance of MAGEA6 and miR-448 on the sphere formation, colony formation, and invasion and migration of HCC stem cells, the CD133⁺CD44 ⁺ HCC stem cells were sorted and treated with the mimic or inhibitor of miR-448, small interfering RNA (siRNA) against MAGEA6 or an AMPK activator AICAR. MAGEA6 silencing or miR-448 overexpression was demonstrated to inhibit the abilities of sphere formation, colony formation, cell migration, and invasion of HCC cells. Afterwards, a rescue experiment was conducted and revealed that MAGEA6 silencing reversed the effects of miR-448 inhibitor on stemness maintenance and self-renewal of HCC stem cells. Finally, after the in vivo experiment was carried out, miR-448 was observed to restrain the tumor formation and stemness in vivo. Altogether, miR-448 activates the AMPK signaling pathway by downregulating MAGEA6, thus inhibiting the stemness maintenance and self-renewal of HCC stem cells, which identifies miR-448 as a new therapeutic strategy for HCC.     

基于网络药理学探讨“黄芪-白术-熟地黄”组方防治肾病综合征的作用机制

本研究旨在通过网络药理学方法和分子对接技术探讨黄芪-白术-熟地黄组方(HBS)治疗肾病综合征的作用机制.通过多个数据库获取肾病综合征基因并进行功能模块分解,找出肾病综合征基因参与的主要生物学过程.通过文献以及数据库查找HBS活性成分和基因靶点,筛选出HBS治疗肾病综合征的有效靶点.通过有效靶点的KEGG和GO富集分析,...     

Effects of caffeine on in vivo and in vitro oocyte maturation in mice

The objective was to investigate, using a mouse model, the effects of caffeine on the number of ovulated oocytes, the rate of oocyte maturation, the susceptibility of oocytes to activating stimuli, spindle morphology, and distribution of cortical granules (CGs). Mice were given caffeine (150 mg/kg body weight ip) at various times relative to hCG (-2, 0, and +2h); in an in vitro study, 1, 5 or 10 mM caffeine was added to the maturation culture. Caffeine had no effect on the quality of oocytes in vivo maturation, but caffeine was detrimental to the quality of oocytes matured in vitro. Further studies are needed to determine caffeine concentration in follicles relative to that in culture medium.     

Comparison of histone modifications in in vivo and in vitro fertilization mouse embryos

Histone modifications are thought to play important roles in various cellular functions. In this article, the distribution patterns of acetylation on histone H4, methylation on histone H3 lysine 9, and phosphorylation on histone H3 serine 10 were examined in in vivo and in vitro fertilization (IVF) preimplantation mouse embryos by using indirect immunofluorescence and scanning confocal microscopy. We desired to know whether the IVF, which has been widely used as a routine assisted reproductive technology in animal and human, was safe at the epigenetic level. As results, we found that there was no difference in these histone modification patterns in in vivo and IVF mouse embryos from zygote to blastocyst stage. Moreover, these histone modifications had different distributions at all examined stages, but they were consistent with the mouse embryo developmental stages.     

Cytoskeletal and nuclear organization in mouse embryos derived from nuclear transfer and ICSI: a comparison of agamogony and syngamy before and during the first cell cycle

In this study, somatic cell nuclear transfer (SCNT) and intracytoplasmic sperm injection (ICSI) are used as models of agamogony and syngamy, respectively. In order to elucidate the reasons of low efficiency of somatic cell cloning, cytoskeletal and nuclear organization in cloned mouse embryos was monitored before and during the first cell cycle, and compared with the pattern of ICSI zygote. A metaphase-like spindle with alignment of condensed donor chromosomes was assembled within 3 hr after NT, followed by formation of pronuclear-like structures at 3-6 hr after activation, indicating that somatic nuclear remodeling depends on microtubular network organization. The percentage of two (pseudo-) pronuclei in cloned embryos derived from delayed activation was greater than that in immediate activation group (68.5% vs. 30.8%, P<0.01), but similar to that of ICSI group (68.5% vs. 65.5%, P>0.05). The 2-cell rate in NT embryos was significantly lower than that in zygotes produced by ICSI (64.8% vs. 82.5%, P<0.01). Further studies testified that the cloned embryos reached the metaphase of the first mitosis 10 hr after activation, whereas this occurred at 18 hr in the ICSI zygotes. Comparision of the pattern of microfilament assembly in early NT embryos with that in syngamic zygotes suggested that abnormal microfilamental pattern in cloned embryos may threaten subsequent embryonic development. In conclusion, agamogony, in contrast to syngamy, displays some unique features in respect of cytoskeletal organization, the most remarkable of which is that the first cell cycle is initiated ahead distinctly, which probably leads to incomplete organization of the first mitotic spindle, and contributes to low efficiency of cloning.     

Time course of meiotic progression after transferring primary spermatocyte into ooplasm at different stages

This study attempted to investigate the time course of meiotic progression after transferring primary spermatocyte (PS) into ooplasm at different maturing stages. In present experiments, PSs were introduced into maturing ooplasts or oocytes by electrofusion. Higher fusion rate was obtained by phytohemagglutinin (PHA) agglutination than by perivitelline space (PVS) insertion. When the ooplasms prepared at 0, 2, 5, and 8.5 hr of in vitro maturation (IVM) were used as recipients and PSs were used as donors, the reconstructed cells extruded the first polar body (PB1) approximately 8.5, 7, 5.5, and 3 hr after electrofusion, respectively. Especially, when ooplasm cultured for 8.5 hr in vitro after GV removal was fused with PS, the PB1 was emitted 7-11 hr after electrofusion. Additionally, the PB1 extrusions of GV and pro-MI oocytes fertilized with PSs were 2.5 hr earlier than control oocytes. The results suggest that (1) PSs undergo the first meiosis in different time courses when introduced into ooplasm at different maturing stages; (2) GV material plays an important role in determining the timing of PB1 extrusion; and (3) first meiotic division of GV and pro-MI oocytes can be accelerated by introducing PS.