Effect of silencing LC3 on apoptosis of mouse hepatocellular carcinoma Heap1-6 cells

目的建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系，探讨其对衣霉素诱导的肝癌细胞凋亡的影响。方法设计并合成一段针对小鼠LC3基因的shRNA及不针对任何基因的shRNA作为阴性对照，将它们退火后连接构建重组载体，转化扩增及酶切鉴定之后将重组质粒与病毒包装、包膜质粒共同转染293T，收集病毒上清转染Heap1-6细胞，嘌呤霉素筛选10 d，获得稳定的细胞株。以导入LC3基因shRNA的Heap1-6为实验组（Heap1-6 shLC3），以导入shcoo2的Heap1-6为对照组（Heap1-6 shctrl）。衣霉素处理实验组和对照组的细胞，Western Blot检测LC3Ⅱ、c-Caspase3、Caspase9蛋白的表达，流式细胞术检测细胞凋亡。Western Blot 条带灰度值之间两组比较采用独立样本的t检验。结果成功构建了pLKO.1-shLC3重组慢病毒载体，与野生型的Heap1-6相比，LC3基因沉默之后，LC3Ⅱ蛋白表达水平降低了62.9﹪（P 〈 0.01）；野生型的Heap1-6和LC3基因沉默之后的Heap1-6 shLC3都经Tm处理12 h之后，后者LC3Ⅱ蛋白表达水平降低了58.6﹪（P 〈 0.01）。与对照组Heap1-6 shctrl相比，衣霉素作用12 h后实验组Heap1-6 shLC3 c-Caspase3增加了37.7﹪（P = 0.007），Caspase9增加了37.1﹪（P = 0.023））；衣霉素作用24 h后shLC3组c-Caspase3增加了12.6﹪（P = 0.04）， Caspase9增加了14.3﹪（P = 0.043）。药物干预12 h和24 h后，Heap1-6 shLC3组比对照组Heap1-6 shcoo2凋亡比例分别增加22.8﹪和18.6﹪。结论成功建立小鼠LC3基因沉默的Heap1-6稳定表达细胞系，LC3基因沉默促进衣霉素诱导的小鼠肝癌细胞Heap1-6的凋亡。Objective To establish a stable hepatocellular carcinoma Heap1-6 cell line expressing shRNA against mouse LC3 and to study apoptosis of Heap1-6 cells treated with tunicamycin. Methods shRNA targeting LC3 gene and negative shRNA were designed and synthesized, pLKO.1-TRC-shRNA LC3 vector and negative vector were constructed. After amplification and identification, the recombinant lentivirus vectors were transfected into 293T cells with packaging and envelope plasmids. The supernatant of 293T ceils transfected with recombinant vector was collected, and Heap1-6 cells were transfected with plasmids, and treated with puromycin for ten days to acquire a cell line with stable expression of shRNA against mouse LC3. Western blot analysis was used to detect the expression level of LC3 Ⅱ, cleaved-caspase3, and caspase9 protein respectively. Apoptotic cells were measured by flow cytometry. Results We successfully constructed pLKO. 1-shLC3 lentivirus vector. Before treated by tunicamycin, the level of LC3 Ⅱ in the Heap1-6 shLC3 cells was decreased by 62.9 % compared with that in the WT Heap1-6 cells （P = 0.0001 ）. After being treated by tunicamycin for 12 h, the level of LC3 Ⅱ in Heap1-6 shLC3 cells was decreased by 58.6 % compared with that in the WT Heapl-6 cells P = 0.0003 ）. Compared with the control group （Heap1-6 cells transfected with negative shRNA vector）, the level of cleaved-caspase3 and caspase9 in the shLC3 group was increased by 37.7 % and 37.1% respectively under tunicamycin for 12 h （P = 0.007, 0.023 ）. And the level of the same two proteins in the shRNA group was elevated by 12.6 % and 14.3 % compared with those of Heap1-6 shctrl cells respectively （P = 0.040, 0.043）. The ratio of apoptotic cells of the experiment group was increased by 22.8 % and 18.6 % compared with that of the control treated with ttmicamycin for 12 h and 24 h, respectively. Conclusion LC3 knockdown could promote apoptosis of mouse hepatocellular carcinoma Heap 1-6 cell line induced by tunicamycin.国家自然科学基金面上项目（81270431

Promotion of proliferation of luminal B breast cancer cells by mesenchymal stem cells and its underlying molecular mechanisms

目的分析人脐带间充质干细胞(hUC-MSCs)对Luminal B型乳腺癌细胞生长增殖的影响,并初步探讨其可能的分子机理。方法绿色荧光蛋白(GFP)和荧光素酶共表达慢病毒感染人Luminal B型乳腺癌细胞BT474,并经嘌呤霉素筛选两周后,于荧光显微镜下观察GFP的表达情况,IVIS Kinetic成像系统拍照以观察和记录慢病毒感染后BT474细胞荧光素酶的表达情况;荧光显微镜下直接观察,结合MTS实验分析hUC-MSCs共培养或其浓缩上清处理对GFP和荧光素酶共表达BT474细胞生长增殖的影响;Western blot法检测hUC-MSCs浓缩上清处理对BT474细胞Akt和MAPK信号通路激活情况以及下游细胞周期调控蛋白Cyclin D1表达的影响;常规RT-PCR法检测hUC-MSCs中NRG-1、NRG-2、IGF-Ⅰ、IGF-Ⅱ和EGF等配体的表达。荧光素酶表达强度与细胞数量的相关性经由Excel软件行统计学分析,MTS实验数据则经由SPSS13.0统计软件行统计学分析。结果荧光显微镜和IVIS Kinetic成像系统的观察结果分别证实,GFP和荧光素酶经慢病毒载体系统的介导可在BT474细胞中成功地共表达,且荧光素酶的表达强度与细胞数量呈直线相关。MSCs共培养或其浓缩上清处理均可显著促进Luminal B型乳腺癌细胞BT474的生长增殖,其细胞存活比例分别为各自对照组的148.06%(P<0.005)和147.99%(P<0.001);MSCs浓缩上清处理同时激活BT474细胞内Akt和MAPK信号通路,并上调细胞周期调控蛋白Cyclin D1表达。此外,RT-PCR结果显示,hUC-MSCs中NRG-1和EGF的mRNAs水平呈高表达,而NRG-2、IGF-Ⅰ和IGF-Ⅱ等配体的mRNAs表达也可见。结论 MSCs可通过表达并分泌NRG-1等配体,从而激活Luminal B型乳腺癌细胞BT474的下游Akt和MAPK信号转导通路以上调细胞周期调控蛋白Cyclin D1的表达,进而促进其生长增殖。Objective To investigate the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs) on proliferation of luminal B breast cancer cells and its underlying molecular mechanisms. Methods Human luminal B breast cancer cells BT474 were infected with GFP and luciferase co-expressing lentiviruses and then subjected for selection with Puromycin for 2 weeks. The expression of GFP and luciferase was detected by fluorescent microscopy and IVIS Kinetic image system, respectively. The effect of coculture or treatment with conditioned medium of hUCMSCs on proliferation of BT474 was analyzed with MTS assay. Western blot was carried out to detect the effect of treatment with conditioned medium of hUC-MSCs on the activation of both Akt and MAPK signalings in BT474, as well as the expression of downstream cell cycle regulator Cyclin D1. Regular RT-PCR was applied to analyze the mRNAs expression of ligands such as NRG-1, NRG-2, IGF-Ⅰ, IGF-Ⅱ, and EGF in hUC-MSCs. The correlation between relative luciferase activity and cell number was analyzed with Excel software, while MTS assay data was statistically analyzed with SPSS 13.0 software. Results The co-expression of GFP and luciferase in BT474 via lentiviral expression system was visualized by fluorescent microscopy and IVIS Kinetic image system. The linear correlation between relative luciferase activity and cell number was determined by curve fitting analysis. Coculture or treatment with conditioned medium of hUC-MSC significantly promoted the proliferation of BT474, with survival rates being 148.06 %(P < 0.005)and 147.99 %(P < 0.001)of control, respectively. In addition, treatment with conditioned medium of hUC-MSC was shown to induce activation of both Akt and MAPK signalings, which further upregulated the expression of Cyclin D1. Moreover, high mRNAs expression levels of both NRG-1 and EGF, as well as moderate mRNAs expression levels NRG-2, IGF-Ⅰ, and IGF-Ⅱ were showed by RT-PCR. Conclusion Our results here demonstrated that MSCs may promote the proliferation of luminal B breast cancer cells through paracrine of ligands such as NRG-1, which in turn results in the activation of both Akt and MAPK signalings and upregulation of the expression of Cyclin D1.国家自然科学基金面上项目(81272922);; 福建省自然科学基金面上项目(2016J01577);; 福州总医院院内课题国际合作研究专项(2015G01

基于变速器振动特性的齿轮异常磨损分析

变速器由于各种激振产生复杂的振动，不仅关系到乘坐舒适性，对传动系统各零件寿命和可靠性都有重要的影响。在设计开发阶段考虑变速器的激振来源以及内部零件与外部换挡操纵的模态，可有效改善变速器相关振动问题。通过某机械式变速器整车耐久试验中挡位齿轮结合齿早期异常磨损引起的换挡手柄抖动分析，从振动特性为出发点，推论出主减齿轮为激振源，一轴总成、平衡块等产生叠加共振。提出改善对策并通过了试验验证

Effects of ocean acidification and copper ions on the ephyrae of moon jellyfish Aurelia coerulea

Ocean acidification and heavy metal pollution are two global marine environmental problems,both of which have influences on the survival of marine organisms and the health of ecosystems. As a widely distributed glial zooplankton in the ocean,Aurelia coerulea plays an important role in biogeochemical cycle of important elements,such as carbon,nitrogen, and phosphorus. Many previous studies have reported the growth and population abundance of A. coerulea are susceptible to changes of the marine environment. In this study,the physiological response of A. coerulea ephyrae to ocean acidification (pH 8.1 and pH 7.6) and Cu~(2+)(0,10 mug/L and 25 mug/L) stress was analyzed by measuring the activities of physiological metabolic enzymes including catalase (CAT),superoxide dismutase (SOD),Ca~(2+)-ATPase,as well as respiration rates, pulsation rates and bell diameter of A. coerulea. The results showed that ocean acidification and Cu~(2+) stress had different effects on physiological indices of A. coerulea ephyrae. Cu~(2+) exposure can significantly inhibit the activities of CAT and Ca~(2+)- ATPase,as well as the pulsation rates and growth rate of A. coerulea ephyrae. Besides,Cu~(2+) exposure can also lead to a significant increase in SOD activity and respiratory rate. While copper pollution might affect the swimming behavior of A. coerulea,leading to the significant decline in their predation ability and ontogeny. Furthermore,a negative correlation was observed between the bell diameter and Cu~(2+) concentration. Seawater acidification had an inhibitory effect on activity of CAT,SOD,and Ca~(2+)-ATPase,as well as growth of A. coerulea ephyrae,and promoted its respiration. However,when A. coerulea ephyrae were exposed to co-exposure of seawater acidification and Cu~(2+),there were significant antagonistic effect between seawater acidification and Cu~(2+) on the growth and respiration of A. coerulea ephyrae. Our study indicates that the adaptability of A. coerulea ephyrae to copper pollution is enhanced with the background of ocean acidification. The different physiological effects of ocean acidification and Cu~(2+)on A. coerulea identified in current study may lead to changes in marine biodiversity and ecosystems in the future

水母暴发对海洋水产养殖业的影响研究进展

As a marine biological disaster, the jellyfish blooms has been a hot issue in the field of marine ecology. Hydromedu sae, siphonophore, scyphomedusae, cubomedusae and ctenophore are found in aquaculture ponds or cages, which has a serious impact on aquaculture production. This study integrally describes the harmful jellyfish types and main related hazards to aquaculture, analyzes the possible harms of aquaculture activities on jellyfish blooms. And based on the previous study, we put forward several proposals in prevention of jellyfish disasters in aquaculture.</p

生物质基金属吸附材料的研究进展

通过对生物质吸附材料的原料、制备方法及在金属离子吸附方面的应用进行分析,对用于金属离子吸附的生物质基吸附材料进行了分类.总结了细菌、真菌和藻类,生物质废弃物和天然生物高分子三类生物质基吸附材料吸附金属离子的特点和重要发展方向.根据目前生物质基金属吸附材料研究中存在的问题和不足,提出提高生物质吸附剂吸附机理认识和加快开展实际应用是今后的研究重点.</p

太阳盐/钢渣定型复合相变储热材料的制备与性能研究

全球范围内的能源短缺和环境污染问题迫使人们积极开发可再生新能源。储热技术是解决新能源不稳定性问题的关键技术。相变材料是重要的储热介质之一。熔盐相变材料因其储热密度高,可操作温度范围广的优势,成为储热材料领域研究的热点。为解决熔盐液相易泄漏、低导热和高成本的问题,选择钢渣为基体材料,制备了太阳盐/钢渣定型复合相变储热材料,并通过扫描电子显微镜(SEM),热重–差示扫描量热法(TG–DSC),闪射法导热仪(LFA)和X射线衍射仪(XRD)对复合材料的微观结构、热性能和化学相容性进行了测试与表征。结果表明,钢渣与熔盐质量比5:5的复合材料定型效果最优。复合材料结构紧密;钢渣与熔盐化学相容性良好;复合材料潜热为64.0 kJ/kg,100～500℃内储热密度为945 kJ/kg,热导率高达2.23 W/(m·K)。太阳盐/钢渣复合相变储热材料不仅有利于储热技术的大规模应用,而且为钢铁工业废弃物回收利用提供了良好的参考,对节约资源、保护环境以及提高经济效益具有重要的意义