基于CRISPR/Cas9基因编辑技术的本科教学实验体系的创建与实践

CRISPR/Cas9是新兴的基因编辑技术,在生命科学研究中发挥着重要的作用。将它引入本科生的实验教学,使本科生了解这项前沿科研技术很有意义。我们创建了一个基于CRISPR/ Cas9技术的本科教学实验体系。该实验体系侧重CRISPR/Cas9技术在哺乳动物细胞中的应用,选用一株基因组上被插入mCherry基因的小鼠胚胎成纤维细胞为实验材料,命名为STO-82。首先设计靶向mCherry的sgRNA,构建CRISPR-Cas9/sgRNA共表达质粒。经测序验证无误后,转染到STO-82细胞。采用流式细胞仪分析检测mCherry阴性和阳性两群细胞,分选出阴性单细胞并扩大培养。最后用测序检验单克隆细胞中靶标DNA序列的编辑情况。结果显示,靶位点有插入或缺失突变,说明体系创建成功。该实验体系将sgRNA设计、CRISPR-Cas9/sgRNA共表达质粒的构建、细胞转染、单细胞分选、单克隆细胞培养、测序序列分析等内容融合为一个综合实验,用于高年级本科生的实验教学。根据实际情况,将教学实践内容分解分块教学,也可以做完整性项目教学。本教学实践采用10人左右的小班分块教学,2人一组,经过3个班（共13组）的实践,绝大部分学生都能完成实验,得到预期结果。通过这个实验,学生加深了对CRISPR/Cas9技术的原理和实验流程的理解,锻炼了实验操作能力和严谨的科研思维,也使学生对该技术的医疗应用风险有了一些认识。     

红角鸮的食性及育雏行为初报

红角鸮(Otus sunia)广泛分布于东亚和南亚。2012和2013年的两个繁殖季节,在江苏省南京大学浦口校区通过红外摄像观察记录了红角鸮的摄食及育雏行为,统计了亲代红角鸮育雏食物的种类、育雏的时间和次数。以1 h为一个时段,利用单因素方差分析(ANOVA)及Student-Newman-Keulsa多重比较法分析各时段之间育雏频次的差异。亲鸟育雏的食物大部分为直翅目(62%)和鳞翅目(18%)昆虫,以及非昆虫类的无脊椎动物,如蜘蛛(8%)和唇足动物(7%),红角鸮也捕食小型陆栖脊椎动物例如田鼠、壁虎等育雏,但比例甚低(3%)。红角鸮育雏节律较为明显,亲鸟主要集中在夜间外出觅食,但在白天也有觅食行为。夜间共有三个育雏高峰,分别为日落之后的19:00~20:00时和21:00~22:00时以及日出之前的04:00~05:00时。     

microRNA-329 suppresses epithelial-to-mesenchymal transition and lymph node metastasis in bile duct cancer by inhibiting laminin subunit beta 3

Bile duct cancer (BDC), also known as cholangiocarcinoma, is a highly desmoplastic cancer with a growth pattern characterized by periductal extension and infiltration. Studies have suggested that microRNAs (miRNAs) play an important role in BDC progression. Here we aim at investigating the effects of miR-329 on BDC development, focusing especially on epithelial-to-mesenchymal transition (EMT) in vitro and lymph node metastasis in vivo. Expression microarrays associated with BDC tissues were collected and differentially expressed genes were analyzed, followed by miRNA target prediction and verification. The role miR-329 played in BDC was examined using gain-of-function and loss-of-function methods. The expressions of miR-329, laminin subunit beta 3 (LAMB3), and EMT markers, in addition to cell proliferation, migration, and invasion were evaluated. Furthermore, nude mice models of BDC were established to observe tumor growth and metastatic lymph nodes. The LAMB3 was identified as an upregulated gene based on the GSE77984 and GSE45001 microarray analysis. LAMB3 was also predicted and confirmed to be a target gene of miR-329 by dual-luciferase reporter assay. Through further cell experiments, the EMT process was reversed, cell proliferation, invasion, and migration were suppressed, when miR-329 was upregulated. Furthermore, in vivo experiments exhibited that the overexpression of miR-329 inhibited tumor growth and the number of metastatic lymph nodes. This study provides in vivo and in vitro evidence that miR-329 inhibits BDC progression through translational repression of LAMB3. Therefore, the obtained results may aid as an experimental basis for improving prognosis of BDC.     

心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach

Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4-70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.