基于高通量测序技术的三叉苦幼苗转录组数据分析

目的获得三叉苦Melicope pteleifolia转录组信息特征。方法以三叉苦幼苗根、茎、叶混合样品为对象,采用二代高通量测序平台Illumina HiSeq~(TM) 2000进行转录组测序并进行系统的生物信息学分析。结果转录组测序分析共获得47 045 040条高质量序列(clean reads),Trinity de novo组装获得67 956条unigenes,平均长度787 nt。BLAST分析显示分别有42 749(61.92%)、31 152(45.84%)、26 563(39.0 9%)、17 481(25.72%)条unigenes在NR、Swiss-port、KOG、KEGG数据库得到注释信息,参与生物过程、细胞组分和分子功能3个GO类别的47个小组,共9807条unigenes注释到130个KEGG代谢通路中,筛选到19条次生代谢通路,KOG功能分类分析获得25个不同的KOG功能类群。预测共有高等植物转录因子56个家族;借助MISA软件发现7 748个SSRs,三碱基重复SSRs数量最丰富,有4 117个,出现频率为53.1%,五碱基重复SSRs相对较少,占2.2%。结论利用高通量测序技术和生物信息分析获得三叉苦转录组信息特征,为后续三叉苦功能基因的挖掘、次生代谢途径解析及其调控机制研究奠定基础。     

LncRNA MEG3靶向miR-181a-5p调控宫颈癌细胞放射敏感性

目的 研究LncRNA MEG3对宫颈癌细胞放射敏感性的影响,并探讨其作用机制。方法 运用qRT-PCR法检测放射抗性和放射敏感性宫颈癌细胞中LncRNA MEG3的表达;将过表达对照组(转染pcDNA 3.1)、过表达LncRNA MEG3组(转染pcDNA 3.1-LncRNA MEG3)、抑制miR-NC组(转染anti-miR-NC)、抑制miR-181a-5p组(转染anti-miR-181a-5p)、过表达LncRNA MEG3+过表达miR-NC组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-NC)、过表达LncRNA MEG3+过表达miR-181a-5p组(共转染pcDNA 3.1-LncRNA MEG3和anti-miR-181a-5p),均用脂质体法转染至SiHa细胞;克隆形成实验检测细胞的存活分数;流式细胞术检测细胞的凋亡率;双荧光素酶报告基因检测实验检测细胞的荧光活性;Western blot检测细胞中PTEN、p-Akt、Akt的蛋白表达。结果 与放射敏感组相比,放射抗性宫颈癌组织中LncRNA MEG3的表达明显降低(P<0.05),其表达量与宫颈癌细胞的放射敏感性呈正相关;过表达LncRNA MEG3、抑制miR-181a-5p均可显著增强宫颈癌细胞SiHa放射敏感性,促进凋亡(P<0.05);野生型LncRNA MEG3细胞的荧光活性受miR-181a-5p的抑制。过表达miR-181a-5p逆转了LncRNA MEG3对宫颈癌细胞放射增敏和促凋亡作用及对PTEN/Akt信号通路的调控。结论 长链非编码RNA LncRNA MEG3可增强宫颈癌细胞放射敏感性,其机制可能与靶向miR-181a-5p调控PTEN/Akt 信号通路有关,可为提高宫颈癌的预后提供新方向。     

PI3K/AKT/mTOR通路及其抑制剂在乳腺癌中的应用现状

磷脂酰肌醇3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）信号转导通路在多种肿瘤中异常激活，参与肿瘤细胞增殖、分化和凋亡等生命过程的调控，是抗肿瘤药物研发的重要靶点。对目前已应用于乳腺癌临床或处于临床试验阶段的PI3K/AKT/mTOR信号通路抑制剂进行归纳，并综述该通路抑制剂的联合用药策略，以期为不同亚型乳腺癌提供个体化靶向治疗方案。     

Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

癫痫持续状态后小鼠海马组织神经元中微管蛋白和内体-溶酶体系统表达的变化及其意义

目的：探讨癫痫持续状态（SE）后小鼠海马组织神经元中微管蛋白β-tubulin和内体-溶酶体系统表达的变化，阐明神经元迟发性死亡过程中微管和内体-溶酶体系统的变化规律。方法：40只雄性ICR小鼠分为对照组（n=7，给予生理盐水）和实验组（n=33，给予匹鲁卡品），实验组中达到SE标准的SE小鼠根据SE后时间分为SE1d、SE2d、SE3d和SE7d组（n=5）。Nissl和Fluoro-Jade B （F-JB）染色检测各组小鼠海马组织神经元损伤情况，免疫荧光法检测各组小鼠海马组织神经元中微管蛋白β-tubulin、内体蛋白Rab5和溶酶体结构蛋白LAMP1表达强度及β-tubulin、Rab5和LAMP1阳性面积百分比；双重荧光法检测各组小鼠海马组织CA1区β-tubulin与Rab5和LAMP1表达的关系。结果：与对照组比较，SE1d组小鼠海马CA1和CA3区神经元数减少（P<0.01），F-JB阳性细胞数增加（P<0.01）；SE2d、SE3d和SE7d组小鼠海马组织中Nissl阳性神经元数明显减少（P<0.01）。与对照组比较，SE2d、SE3d和SE7d组小鼠海马组织中F-JB阳性神经元数增加（P<0.01）。与对照组比较，SE2d、SE3d和SE7d组小鼠海马CA1和CA3区神经元树突中β-tubulin阳性面积百分比明显降低（P<0.05），其变化趋势与神经元损伤相似。与对照组比较，SE1d、SE 2d、SE 3d和SE 7d组小鼠海马组织神经元中Rab5和LAMP1表达强度随着时间延长呈下降趋势；Rab5阳性面积百分比明显降低（P<0.05或P<0.01）；LAMP1阳性面积百分比在SE后第1天出现一过性增多，之后逐渐减少（P<0.05或P<0.01）。双重荧光染色检测，SE后1 d是早期内体减少和溶酶体一过性增多的关键点，并与神经元微管损伤的出现时间极为一致。结论：SE引起神经元迟发性死亡的同时神经元微管骨架受损，内体-溶酶体系统的定位和功能也发生异常改变。     

穴位埋线对肾虚血瘀型子宫内膜异位症患者IL-1β、TNF-α、VEGF和MMP-2水平的影响

目的:基于PI3K/Akt/mTOR信号通路,探讨穴位埋线疗法治疗肾虚血瘀型子宫内膜异位症的临床疗效及对IL-1β、TNF-α、VEGF和MMP-2等相关信号通路下游细胞因子的水平的影响。方法:选取肾虚血瘀型子宫内膜异位症的门诊和住院患者86例,按照随机数字表法随机分为穴位埋线组和西药组,西药组口服孕三烯酮胶囊(内美通),穴位埋线组在西药组的基础上采用穴位埋线进行治疗,治疗3个月后观察两组患者的临床疗效和IL-1β、TNF-α、VEGF、MMP-2等相关信号通路下游细胞因子的水平。结果:治疗后穴位埋线组总有效率93.02%(40/43),西药组总有效率83.72%(36/43),两组比较差异无统计学意义(P>0.05)。治疗3个月后,穴位埋线组和西药组的VAS评分、血清IL-1β、TNF-α、VEGF和MMP-2水平均较治疗前改善(P<0.01),但穴位埋线组较西药组改善更为明显(P<0.05或P<0.01)。治疗过程中,西药组不良反应发生率为18.60%(8/43),穴位埋线组不良反应发生率为4.65%(2/43),两组不良反应发生率比较,差异有统计学意义(P<0.05)。结论:穴位埋线可以显著改善子宫内膜异位症患者的临床症状和体征,并能显著降低孕三烯引起的不良反应,可见穴位埋线疗法可以作用PI3K/Akt/mTOR信号通路,通过改善通路下游的IL-1β、TNF-α、VEGF和MMP-2水平,发挥治疗子宫内膜异位症的作用。     

Effects of mild intrauterine hypoperfusion in the second trimester on memory and learning function in rat offspring

Mild intrauterine hypoperfusion(MIUH) is a serious pathological event that affects the growth and development of fetuses and offspring. MIUH can lead to growth restriction, low birth weight, neurodevelopmental disorders, and other adverse clinical outcomes. To study the effects of MIUH on learning and memory function in offspring, a model of MIUH was established by placing a coil(length 2.5 mm, diameter 0.24 mm) on the uterine artery and ovarian uterine artery of Sprague-Dawley rats in the second trimester of pregnancy(day 17). Next, 120 mg/kg lithium chloride(the MIUH + Li group) or normal saline(the MIUH group) was injected intraperitoneally into these rats. In addition, 120 mg/kg lithium chloride(the Li group) or normal saline(the SHAM group) was injected intraperitoneally into pregnant rats without coil placement. The Morris water maze was used to detect changes in learning and memory ability in the offspring at 4 weeks after birth. In the MIUH group, the escape latency and journey length before reaching the platform were both increased, and the number of times that the platform was crossed and the activity time in the target quadrant within 90 seconds were both decreased compared with the SHAM group. Immunofluorescence double staining and western blot assays demonstrated that hippocampal nestin and Ki67(both cell-proliferation-related proteins) expression was significantly downregulated in the MIUH group compared with the SHAM group. Furthermore, western blot assays were conducted to investigate changes in related signaling pathway proteins in the brains of offspring rats, and revealed that glycogen synthase kinase 3β(GSK3β) expression was upregulated and β-catenin expression was downregulated in the MIUH group compared with the SHAM group. In addition, compared with the MIUH group, the expression levels of p-GSK3β and β-catenin were upregulated in the MIUH + Li group. These results suggest that MIUH may affect learning and memory function in rat offspring by regulating the GSK3β signaling pathway. The experimental procedures were approved by Animal Ethics Committee of Shengjing Hospital of China Medical University(approval No. 2018 PS07 K) in June 2018.     

丹蛭降糖胶囊通过mTOR/S6K1信号通路对糖尿病肾病大鼠的干预作用研究

目的研究丹蛭降糖胶囊对糖尿病肾病大鼠的肾脏病理改变和足细胞自噬水平的影响,初步探讨其相应的作用机制。方法选取GK大鼠40只,采用醋酸脱氧皮质酮-盐皮下注射联合高脂饲料喂养诱导糖尿病肾病模型。造模成功后随机分为模型组、丹蛭降糖胶囊低剂量组[0.54 g/(kg·d)]、高剂量组[1.08 g/(kg·d)]、缬沙坦组[10 mg/(kg·d)],每组10只。另选取10只同周龄正常Wistar大鼠作为正常组。模型组和正常组给予等容积生理盐水。连续灌胃10周后检测空腹血糖(FBG)、血肌酐(SCr)、尿素氮(BUN)和尿微量蛋白(U-mAlb)。Western Blot检测肾小球p-mTOR、p-S6K1、Beclin-1、LC3和Nephrin蛋白的表达,HE染色和PAS染色后于光镜下观察肾脏病理变化,电镜下观察各组亚细胞形态结构变化。结果与正常组比较,模型组FBG、SCr、BUN、U-mAlb水平升高(P<0.01);与模型组比较,丹蛭降糖胶囊低、高剂量组和缬沙坦组U-mAlb水平降低(P<0.01)。模型组可见肾小球基底膜增厚,系膜基质沉积,足细胞损伤,各用药组均有不同程度改善,丹蛭降糖胶囊高剂量组足细胞内有较多自噬体形成。与正常组比较,模型组p-mTOR、p-S6K1的表达增高,Nephrin、Beclin-1和LC3的表达水平降低(P<0.01);与模型组比较,各用药组p-mTOR、p-S6K1的表达下降,Nephrin、Beclin-1和LC3的表达增高(P<0.01)。丹蛭降糖胶囊高剂量组和缬沙坦组Beclin1、LC3Ⅱ/LC3Ⅰ水平较丹蛭降糖胶囊低剂量组升高(P<0.05)。结论丹蛭降糖胶囊具有减轻糖尿病肾病大鼠U-mAIb、足细胞损伤及相关肾脏病理改变的作用,其机制可能与抑制mTOR/S6K1信号通路进而提高足细胞的自噬活性有关。