熊去氧胆酸对阿霉素诱导的H9c2心肌细胞的影响及机制研究

目的:探讨熊去氧胆酸(UDCA)对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响及机制。方法:体外培养H9c2细胞,1μM DOX和不同浓度UDCA处理H9c2,CCK-8法测定细胞活力;实时定量聚合酶链反应检测心肌细胞凋亡分子Bax及炎症因子IL-1β、IL-6的表达;Western blotting检测UDCA对DOX诱导的心肌细胞凋亡相关蛋白Bax、Bcl2、Caspase3表达水平变化。结果:与对照组相比,DOX组心肌细胞活力减弱;炎症因子IL-1β,IL-6表达上调;促凋亡分子Bax和cleaved Caspase3表达增多;抑制凋亡蛋白Bcl2下调(P<0.05)。与DOX组相比,UDCA+DOX组显著恢复心肌细胞活力;炎症因子IL-1β、IL-6表达下调;促凋亡分子Bax、cleaved Caspase3下调;抑制凋亡蛋白Bcl2表达上调(P<0.05)。结论:UDCA能缓解DOX诱导的H9c2心肌细胞损伤,其机制可能与抑制炎症及凋亡有关。本研究为阿霉素心肌毒性的防治提供新的实验基础及理论依据。     

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.     

水稻恢复系M630稻瘟病抗性改良及其代谢组研究

稻瘟病是水稻的主要病害之一,改良水稻稻瘟病抗性对水稻生产、推广具有重要意义。本研究以携带广谱抗稻瘟病基因Pi9材料9311-Pi9为供体,水稻优良恢复系M630为受体,将杂交、回交与分子标记辅助选择和背景筛选相结合,获得改良恢复系M630-Pi9。主要农艺性状和稻米品质分析显示改良后的M630-Pi9及其杂交组合徽两优630-Pi9与M630和徽两优630相比无明显差异。进一步利用安徽省优势混合生理小种进行苗期稻瘟病抗性鉴定,结果表明,改良后的M630-Pi9相对于M630抗性明显增强;稻瘟病抗性自然病圃鉴定结果发现改良后的恢复系及其杂交组合抗性明显增加。为了更好地了解稻瘟病抗性基因对植株发育的代谢机制,对M630-Pi9和M630受稻瘟病病原菌侵染后的植株进行代谢组分析,结果显示与M630相比,M630-Pi9中有212种代谢物合成受到调控,其中155种含量降低,57种含量增加;与细胞壁有关的物质含量明显增加,对生物体具有毒害和免疫作用的生物碱类代谢物含量显著降低,与生物胁迫相关的黄酮类物质受到不同程度的调控。这些数据表明稻瘟病抗性基因Pi9可能通过调控植物体中代谢物质的变化进而抵御病原体的侵害。创制的新恢复系稻瘟病抗性显著提高,对稻瘟病抗性机制研究提供依据,为水稻抗病、高产育种提供新的种质。     

北京市主城区不透水面时空演变及其热环境效应研究

城市不透水面是表征城市化进程的重要指标,开展城市不透水面研究对生态环境监测及城市建设规划具有重要意义。以北京市主城区为研究区,基于Landsat影像采用支持向量机(SVM)和线性光谱混合分解模型(LSMM)相结合的方法提取研究区2002年、2009年和2017年三期不透水面;同时采用辐射传输方程法反演研究区地表温度,分析北京市主城区不透水面时空格局演变及其与城市热环境的相关性。研究结果表明:2002—2017年,北京市主城区近15年不透水面聚集度较高且呈持续增长趋势,东、西城区不透水面面积占比最高,朝阳区和海淀区不透水面增长速率和增长强度相对较大。北京市主城区不透水面呈放射状扩张趋势且增长集中在四环以外的区域,不透水面盖度高值区面积不断增加。研究区近15年地表温度持续升高,热岛效应加剧,地表温度高值区与城市建筑区空间分布存在一致性。不透水面空间格局与热环境高度相关,不透水面的斑块密度和破碎度与地表温度呈负相关,斑块所占景观比例及最大斑块指数与地表温度呈正相关,可通过合理控制相关景观指标、优化不透水面空间格局控制地表温度。在此基础上提出了缓解城市热岛效应的建议:增加树木、草地等用地类型;将不透水面分割成许多小斑块,减少大面积不透水面区域分布;使用透水性材料等。     

生防菌株HG18基因组信息分析与抗菌功能基因挖掘

贝莱斯芽胞杆菌（Bacillus velezensis）HG18是1株低温生防菌株,能够分泌抗菌物质。为挖掘和利用其抗菌功能基因,服务农业生产,采用二、三代相结合测序技术,对其进行全基因组测序,获得菌株完整基因组序列。基因组全长4 461 844 bp,包含一个染色体和一个质粒,GC含量44.06%,编码4 643个基因,编码基因总长度3 893 994 bp,占基因组87.27%。发现6个几丁质降解相关基因,2个葡聚糖酶基因和1个壳聚糖酶基因,2个脂肽类抗菌物质芬芥素与表面活性素合成基因簇,2个细菌素subtilin和bacillolysin合成基因。研究为提高抗菌物质产量的菌株定向遗传改造以及植物抗病育种提供基因资源。     

云南特有螺类光肋螺蛳主要生物学特征研究

光肋螺蛳（Margarya mansugi）是仅分布于云南省的特有螺类。受过度捕捞、栖息环境破坏等人为因素影响,光肋螺蛳自然种群急剧萎缩,目前已被世界自然保护联盟列为极危物种。由于光肋螺蛳的研究资料匮乏,其生物学特征尚未被完全掌握,制约了光肋螺蛳保护对策的制定。为此,通过室内人工驯养观察,记述了光肋螺蛳的形态、活动习性、食性、生长和繁殖等主要生物学特征。光肋螺蛳壳质厚且坚硬,呈长圆锥形,褐色,角质厣,螺壳一般有5~6个螺层,体螺层有4条环肋,雌螺体型一般比同龄雄螺大;喜栖息在水体底层,夜晚活动频率明显高于白天;主要以藻类为食,成螺经驯化可摄食人工配合饲料;仔螺、幼螺和成螺的生长速度顺次降低;性成熟时间需要两年以上,卵胎生,终年生产,一般在白天交配,交配时间7~10 h,分批产仔,每次产仔1~2只,观察到的一只雌螺分9批产仔,共产仔螺14只。从受精到仔螺产出需85 d以上。本研究可为光肋螺蛳基础生物学研究积累资料,同时也可为高原湖泊特有螺类的人工驯养繁育提供参考。     

基于H原子和O原子代谢的葡萄糖有氧氧化解析

通过解析葡萄糖有氧氧化偶联电子传递链合成ATP过程中的物质代谢与能量代谢,特别是基于H原子和O原子的来源与去路的全面追踪,明晰了葡萄糖有氧氧化途径不仅仅只是葡萄糖分子彻底分解代谢为CO2和H2O并合成ATP,还有葡萄糖和O2以外的物质(如Pi、Pi+GDP和H2O等)提供H原子和O原子参与合成ATP和脱羧生成CO2。     

分子生物学教材中的真核生物基因组重复序列

现行的高校分子生物学教材中主要以重复频率为依据对重复序列进行分类,对于小卫星DNA及微卫星DNA是属于高度或是中度重复序列存在不同见解。提出依据重复频率及空间结构分布两个方面对重复序列进行分类,并建议按照重复频率将小卫星DNA及微卫星DNA归属于中度重复序列。     

Endothelial Dysfunction Exacerbates Renal Interstitial Fibrosis through Enhancing Fibroblast Smad3 Linker Phosphorylation in the Mouse Obstructed Kidney

Endothelial dysfunction and enhanced transforming growth factor-β (TGF-β)/Smad3 signalling are common features of progressive renal fibrosis. This study investigated a potential link between these mechanisms. In unilateral ureteric obstruction (UUO) we observed an acute (6 hr) down-regulation of nitric oxide synthase 3 (NOS3/eNOS) levels and increased phosphorylation of the linker region of Smad3 at T179 and S208 in Smad3/JNK complexes. These events preceded Smad3 C-terminal domain phosphorylation and the induction of myofibroblast proliferation at 48 hrs. Mice deficient in NOS3 showed enhanced myofibroblast proliferation and collagen accumulation compared to wild type mice in a 7 day UUO model. This was associated with enhanced phosphorylation of Smad3 T179 and S208 by 92% and 88%, respectively, whereas Smad3-C-terminal phosphorylation was not affected. Resolvin D1 (RvD1) can suppress renal fibrosis in the UUO model, and further analysis herein showed that RvD1 protected against endothelial dysfunction and suppressed Smad3/JNK complex formation with a consequent reduction in phosphorylation of Smad3 T179 and S208 by 78% and 65%, respectively, while Smad3 C-terminal phosphorylation was unaltered. In vitro, conditioned media from mouse microvascular endothelial cells (MMEC) treated with a general inhibitor of nitric oxide synthase (L-NAME) augmented the proliferation and collagen production of renal fibroblasts (NRK49F cells) compared to control MMEC media and this was associated with increased phosphorylation of JNK and Smad3 T179 and S208, whereas Smad3-C-terminal domain phosphorylation was unaffected. The addition of RvD1 to L-NAME treated MMEC abrogated these effects of the conditioned media on renal fibroblasts. Finally, Smad3 T179/V and S208/A mutations significantly inhibit TGF-β1 induced up-regulation collagen I promoter. In conclusion, these data suggest that endothelial dysfunction can exacerbate renal interstitial fibrosis through increased fibroblast proliferation and collagen production via enhanced Smad3 linker phosphorylation.     

“微生物学实验”混合式教学实践与评价

生命科学的本科教学中,实验教学是举足轻重的一个部分。本文以"微生物学实验"课程为研究对象,利用互动网站进行线上线下混合式教学,评价学生的学习效果和规律,探索生命科学实验类课程采用混合式教学的效果,并探讨推广可能性。在连续三年的教学实践中,统计分析各维度数据,发现在混合式教学模式下,绝大部分学生都具有主动自学的能力。同时,实验课程中的深度和广度都有所提升。学习规律的监控和综合评价显示,深度学习的学生在各种评价指标中都显著表现优秀。因此,在混合式教学模式下,如何引导学生进行真实有效的深度学习是教师在新教学模式下的重任。