过表达Lin28a/Lin28b对let-7家族活性的影响

研究在HeLaS3细胞中过表达Lin28a/Lin28b对let-7家族miRNA表达水平和活性的影响。首先,构建Lin28a和Lin28b的表达载体pAAV2neo-Lin28a和pAAV2neo-Lin28b,分别转染HeLaS3细胞并筛选获得Lin28a和Lin28b的稳定表达细胞株HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b。然后,以pAAV2neo-Gluc-(Fluc)为基本骨架,构建并获得检测let-7家族miRNA活性的8种质粒型载体,并包装为相应的重组腺相关病毒(Recombinant adeno-associated virus,rAAV),作为检测miRNA靶序列介导的转录后抑制活性的传感器,命名为Asensor。在此基础上,以HeLaS3细胞为对照,用Western blot检测HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b细胞中Lin28a和Lin28b表达水平,用QRT-PCR测定let-7家族各成员表达水平,用Asensor检测let-7家族各成员活性。Western blot结果显示,HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b均能有效地表达Lin28a和Lin28b蛋白;QRT-PCR检测结果显示,相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28a细胞中let-7家族各成员表达水平都下降(除let-7e外),但不同成员下降幅度存在差异;Asensor检测结果显示,let-7家族所有成员活性水平都下降,但不同成员下降幅度也存在差异,且同一成员活性水平与表达水平及其下降趋势也不一致。相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28b细胞中let-7家族成员的表达和活性水平均明显下降,但表达水平的下降幅度比HeLaS3/pAAV2neo-Lin28a细胞大,而活性的下降幅度却与之相近。本研究建立了一种检测和比较miRNA靶序列介导的转录后抑制活性的方法,首次研究了过表达Lin28a和Lin28b对细胞中的let-7家族miRNA活性影响,并发现Lin28a和Lin28b对let-7家族miRNA表达水平的影响和对其相应活性的影响不一致性,说明在检测miRNA表达水平的同时检测miRNA活性能更全面揭示miRNA的调节功能,为进一步研究let-7家族的调控机制奠定了基础。     

Genome-Wide Gene Expression Profiles in Lung Tissues of Pig Breeds Differing in Resistance to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, typical respiratory symptoms in piglets, and high mortality rate of piglets. In this study, we employed an Affymetrix microarray chip to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited a range of clinical features that typify the disease, whereas the DPL pigs showed only mild signs of the disease. Overall, the DPL group had a lower percentage of CD4⁺ cells and lower CD4⁺/CD8⁺ratios than the DLY group (p<0.05). For both IL-10 and TNF-α, the DLY pigs had significantly higher levels than the DPL pigs (p<0.01). The DLY pigs have lower serum IFN-γ levels than the DPL pigs (p<0.01). The serum IgG levels increased slightly from 0 dpi to 7 dpi, and peaked at 14 dpi (p<0.0001). Microarray data analysis revealed 16 differentially expressed (DE) genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The mRNA expression levels of 10 out of the 16 DE genes were validated by real-time quantitative RT-PCR and their fold change was consistent with the result of microarray data analysis. We further analyzed the mRNA expression level of 8 differentially expressed genes between the DPL and DLY pigs for both uninfected and infected groups, and found that TF and USP18 genes were important in underlying porcine resistance or susceptibility to PRRSV.     

P-glycoprotein expression in extracellular matrix formation of chondrogenic differentiation of human adult stem cells

Mesenchymal stem cell (MSC) has been known as a good source of progenitor for multiple connective tissue including cartilage, muscle, adipocyte, and bone. P-glycoproteins (P-gps) also known as ABCB1 that exports diverse substrates are the product of the multidrug resistance-1 (MDR-1) gene. P-gp expression has been reported in chondrosarcoma and hypertrophic chondrocyte in the human growth plate. This study was designed to investigate the expression of P-gp during chondrogenic differentiation of adult human stem cells. Bone marrow samples were obtained from nine human donors after informed consent. The isolated mononuclear cells (MNCs) were incubated as one pellet/tube and 0.5ml chondrogenic medium in the presence of 10ng/ml of TGF-beta 1 and TGF-beta 3 for 28 days. The expression of surface P-gps was analyzed by flow cytometry and quantitative RT-PCR was performed for the detection of mRNA expression of MDR-1 and type II collagen gene. Total collagen and glycosaminoglycan (GAG) contents of the pellets were measured. Surface P-gp expression of the MSCs was decreased during chondrogenic differentiation. MDR-1 gene was decreased 10-fold after the 2-week incubation whereas type II collagen gene was increased 491-fold after the 4-week incubation in chondrogenic medium. The total amount of collagen and GAG were increased during pellet culture. This study has demonstrated a decrease in expression of P-gp and down regulation of MDR-1 gene consistently by flow cytometry and quantitative RT-PCR, but an increased expression of type II collagen on MSC during chondrogenesis.     

Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis

Spermatogenesis is a complex process of terminal differentiation wherein mature sperm are produced. In the first wave of mouse spermatogenesis, different spermatogenic cells appear at specific time points, and their appearance is expected to be accompanied by changes in specific protein expression patterns. In this study, we used 2D-PAGE and MALDI-TOF/TOF technology to construct a comparative proteome profile for mouse testis at specific time points (days 0, 7, 14, 21, 28, and 60 postpartum). We identified 362 differential protein spots corresponding to 257 different proteins. Further cluster analysis revealed 6 expression patterns, and bioinformatics analysis revealed that each pattern was related to many specific cell processes. Among them, 28 novel proteins with unknown functions neither in somatic cells nor germ cells were identified, 8 of which were found to be uniquely or highly expressed in mouse testes via comparison with the GNF SymAtlas database. Further, we randomly selected 7 protein spots and the above 8 novel proteins to verify the expression pattern via Western blotting and RT-PCR, and 6 proteins with little information in testis were further investigated to explore their cellular localization during spermatogenesis by performing immunohistochemistry for the mouse testis tissue. Taken together, the above results reveal an important proteome profile that is functional during the first wave of mouse spermatogenesis, and they provide a strong basis for further research.     

Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury

Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability.     

Effect of Vitamin B Deprivation during Pregnancy and Lactation on Homocysteine Metabolism and Related Metabolites in Brain and Plasma of Mice Offspring

Epidemiological and experimental studies indicate that the altered fetal and neonatal environment influences physiological functions and may increase the risk of developing chronic diseases in adulthood. Because homocysteine (Hcy) metabolic imbalance is considered a risk factor for neurodegenerative diseases, we investigated whether maternal Vitamin B deficiency during early development alters the offspring''s methionine-homocysteine metabolism in their brain. To this end, the dams were submitted to experimental diet one month before and during pregnancy or pregnancy/lactation. After birth, the offspring were organized into the following groups: control (CT), deficient diet during pregnancy and lactation (DPL) and deficient diet during pregnancy (DP). The mice were euthanized at various stages of development. Hcy, cysteine, glutathione (GSH), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), folate and cobalamin concentrations were measured in the plasma and/or brain. At postnatal day (PND) 0, total brain of female and male offspring exhibited decreased SAM/SAH ratios. Moreover, at PND 28, we observed decreased GSH/GSSG ratios in both females and males in the DPL group. Exposure to a Vitamin B-deficient diet during the ontogenic plasticity period had a negative impact on plasma folate and brain cortex SAM concentrations in aged DPL males. We also observed decreased plasma GSH concentrations in both DP and DPL males (PND 210). Additionally, this manipulation seemed to affect the female and male offspring differently. The decreased plasma GSH concentration may reflect redox changes in tissues and the decreased brain cortex SAM may be involved in changes of gene expression, which could contribute to neurodegenerative diseases over the long term.     

Effect of early decrease in the lesion size on late brain tissue loss, synaptophysin expression and functionality after a focal brain lesion in rats

The purpose of the present study is to determine the effects of early decrease in the lesion size on late brain tissue loss, synaptogenesis and functionality after a focal brain lesion in rats. The lesion was induced either to the cortex using the photothrombotic ischemic stroke or to the striatum using the malonate poisoning model. The cortical and striatal lesions amounted to 66-80 mm(3) at day 1 post-lesion and were reduced by 50% after the acute administration of dipyridyl (a liposoluble iron chelator) and aminoguanidine (an inhibitor of the inducible nitric oxide synthase), respectively. Loss of histologically intact tissue and synaptophysin expression as an indicator of synaptogenesis were examined at day 35 post-lesion. Both types of lesion resulted in synaptophysin upregulation in contralateral and ipsilateral cortical areas. On the contrary, brain tissue loss was greater after the striatal (-17%) than the cortical lesion (-5%). Synaptophysin expression and tissue loss were not different between drug- and vehicle-treated rats. Moreover, a set of standard neurological tests revealed a difference in deficit between the both types of lesion, yet only in the acute post-lesion stage. However, it did not distinguish between vehicle- and drug-treated rats whatever the lesion location. Our results indicate that late histological endpoints measurements are not recommended to probe the potential neuroprotective properties of a drug administered within the acute post-lesion stage. They also suggest that inhibition of cytotoxic mechanisms involved in lesion growth is of no clinical interest when it cannot lead to a long-term histological protection and/or increased synaptogenesis.     

Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.     

RNA干扰沉默缺氧诱导因子1α逆转肺癌细胞耐药性

目的：观察RNA干扰沉默缺氧诱导因子1α（HIF-1α）对肺癌细胞耐药性的影响。方法：构建靶向HIF-1α小干扰RNA基因,并转染到人肺腺癌耐顺铂细胞株A549／DDP细胞中。逆转录聚合酶链反应RT—PCR）检测细胞的HIF-1α、多药耐药基因-（MDR-1）以多药耐药相关蛋白基因（MRP）mRNA变化,免疫细胞化学法观察干扰后HIF-1α、P-糖蛋白以及MRP蛋白的变化。MTT法检测不同浓度的顺铂作用下细胞死亡率。结果：HIF-1αsiRNA组中H1F-1α、MDR—1、MRPmRNA水平显著降低（P〈0．05）。且蛋白水平也显著下降（P〈0．05）。HIF-1αsiRNA组细胞死亡率较未转染组均明显增高（P〈0．05）,转染siRNA阴性组不影响肿瘤细胞的耐药性。结论：HIF-1αsiRNA可显著降低A549／DDP细胞中H1F-1α、MDR-1、MRP表达,从而起到逆转肺腺癌A549／DDP细胞的耐药作用。     

Fluorescent probes of the isoxazole–dihydropyridine scaffold: MDR-1 binding and homology model

Isoxazole-1,4-dihydropyridines (IDHPs) were tethered to fluorescent moieties using double activation via a lanthanide assisted Weinreb amidation. IDHP–fluorophore conjugate 3c exhibits the highest binding to date for IDHPs at the multidrug-resistance transporter (MDR-1), and IDHP–fluorophore conjugates 3c and 7 distribute selectively in SH-SY5Y cells. A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis.     

Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.