噬菌体展示TM-TNF-α模拟肽体内杀瘤效应的研究

目的　研究噬菌体展示TM -TNF- α模拟肽的体内杀瘤效应及机制。方法　大量制备所需的噬菌体展示肽,比较不同展示肽对小鼠的肝癌细胞H2 2的体外杀瘤效应,挑取杀瘤效应最好的展示肽并选择其合适的滴度进行体内实验。小鼠接种H2 2细胞3d后,于肿瘤接种部位皮下注射噬菌体展示肽,观察肿瘤的生长情况。结果　噬菌体展示TM- TNF -α模拟肽在体内能明显抑制肿瘤的生长(P <0 .0 1)。且抑瘤率与展示肽之间呈剂量依赖关系。HE染色发现展示肽治疗组肿瘤组织中有大量淋巴细胞浸润,而sTNF- α治疗组除淋巴细胞浸润还可见中性粒细胞、浆细胞浸润。结论　直接注射噬菌体展示TM -TNF -α模拟肽可在体内有效杀瘤,其杀瘤机制可能不同于分泌型TNF -α。     

高三学生时间管理倾向、自尊与成就动机的关系研究

目的探讨时间管理倾向、自尊与成就动机的关系。方法以青少年时间管理倾向量表（ATMD）、成就动机测量表（AMS）和自尊量表（SES）对高三151名文、理和艺术学生进行了研究。结果①高三学生在自尊上有显著性别差异（P〈0.05）;②高三文、理科和艺术学生在时间价值感（0.020）、自尊水平（0.017）上差异显著（P〈0.05）;③时间管理倾向各因子与自尊、自尊与成就动机两因子、时间管理倾向各因子与成就动机因子（时间价值感与避免失败动机除外）均有显著性相关（P〈0.05）。4时间管理总分和自尊对成就动机有显著的预测。结论高三学生时间管理倾向、自尊与成就动机三者密切相关。     

磁共振成像及氧化铁粒子标记在干细胞治疗脑缺血大鼠中的应用

目的 探讨磁共振成像及氧化铁粒子标记在干细胞治疗脑卒中模型中的应用价值.方法 线栓法建立9只大鼠大脑中动脉缺血模型,进行神经行为学评分、磁共振影像学和病理学评价,并对上述3项指标进行相关性分析.用超顺磁性氧化铁体外标记骨髓间充质干细胞,并另筛选18只脑缺血大鼠,分别接受缺血对侧皮层(n.6)、缺血同侧纹状体(n=6)的标记干细胞移植,并设对照组(n=6).分别在脑缺血后1 d、细胞移植后第1天、第7天及第14天进行磁共振扫描,观察各时间点的标记干细胞显示情况,并对各组之间梗死体积的变化进行统计学分析.结果 神经行为学评分、磁共振影像学和病理学评价对大鼠脑缺血模型评价的相关性好.磁共振各序列均可显示局部移植的标记干细胞,梯度回波序列最敏感,T2像空间分辨力高.磁共振成像可显示局部移植标记干细胞随时间推移而产生的分布变化.干细胞治疗各组之间脑梗死体积变化无明显差异.结论 磁共振成像是大鼠脑缺血模型评价的理想工具,配合氧化铁粒子标记干细胞可活体状态下了解干细胞移植后的分布变化.     

低氧和高二氧化碳作用下肺动脉高压形成过程中骨桥蛋白表达与分布的实验研究

目的： 观察慢性低O2高CO2大鼠肺动脉骨桥蛋白（OPN）及其基因的表达与分布情况，探讨OPN在肺动脉高压发病机制中的作用。方法： 48只雄性SD大鼠随机分为4组：即正常对照组（NC组），低O2高CO2 1周、2周和4周组（1HH、2HH和4HH）。采用RT-PCR法测定不同低O2高CO2 组大鼠离体肺动脉和肺组织骨桥蛋白（OPN）mRNA，免疫组织化学染色法测定肺细小动脉骨桥蛋白分布表达情况，ELISA 法定量测定肺组织匀浆OPN水平，Western blotting 法测定离体肺动脉OPN表达水平。结果： ①低O2高CO2各组大鼠mPAP和RV/LV+S均明显高于NC组（P<0.01），4组大鼠间mCAP比较无显著差异（P>0.05）；②低O2高CO2 各组大鼠肺组织和离体肺主动脉OPN mRNA表达水平显著高于对照组（均P<0.01）；③免疫组化法显示NC组大鼠肺组织OPN表达主要见于支气管、肺泡上皮细胞内，1HH组大鼠肺细小动脉内皮细胞、平滑肌细胞以及周围巨噬细胞均见有OPN的表达，其中以中膜平滑肌细胞最为明显，而2HH和4HH组大鼠OPN表达更加明显, 低O2高CO2 各组肺细小动脉OPN相对含量与对照组比较有显著差异（均P<0.01），且随缺氧时间的延长，骨桥蛋白表达也呈增高趋势；④ 低O2高CO2 各组大鼠肺组织匀浆OPN含量分别为正常对照组的1.69倍、2.28倍和2.87倍（均P<0.01）；⑤ Western blotting 分析1HH、2HH和4HH各组大鼠离体肺动脉OPN表达明显增强，与NC组比较有显著差异（均P<0.01）。结论： 慢性低O2高CO2能够刺激大鼠肺组织和肺动脉OPN及其基因表达增强，OPN可能在慢性低O2高CO2性肺动脉高压发病的病理和病理生理过程中起着重要的作用。     

组成型一氧化氮合酶的表达与胃癌的侵袭转移和预后的关系

目的　观察胃癌组织中cNOSmRNA表达分布情况 ,探讨其与胃癌侵袭转移和预后的关系。　方法　运用原位杂交技术 ,检测 119例原发性胃癌标本中cNOSmRNA的表达 ,并对所获得的 82份病例随访资料进行生存分析。　结果　胃癌组织中胃腺癌细胞、血管 淋巴管内皮细胞及巨噬细胞中均可见有cNOSmRNA表达 ;肿瘤细胞分化程度越低、恶性度越高 ,胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率就越高 ;胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA阳性表达率与胃癌侵袭深度、淋巴结转移以及TNM分期呈正相关 ;胃癌血管 淋巴管内皮细胞cNOSmRNA阳性表达者术后生存率较低 ;巨噬细胞中cNOSmRNA表达与胃癌组织学分型、侵袭深度、淋巴结转移、TNM分期及患者术后生存期无关。　结论　胃腺癌细胞和血管 淋巴管内皮细胞cNOSmRNA表达情况与肿瘤恶性程度、侵袭和转移潜能有关 ,血管 淋巴管内皮细胞上cNOSmRNA阳性表达提示患者预后不良     

A HIT-trapping strategy for rapid generation of reversible and conditional alleles using a universal donor

Targeted mutagenesis in model organisms is key for gene functional annotation and biomedical research. Despite technological advances in gene editing by the CRISPR-Cas9 systems, rapid and efficient introduction of site-directed mutations remains a challenge in large animal models. Here, we developed a robust and flexible insertional mutagenesis strategy, homology-independent targeted trapping (HIT-trapping), which is generic and can efficiently target-trap an endogenous gene of interest independent of homology arm and embryonic stem cells. Further optimization and equipping the HIT-trap donor with a site-specific DNA inversion mechanism enabled one-step generation of reversible and conditional alleles in a single experiment. As a proof of concept, we successfully created mutant alleles for 21 disease-related genes in primary porcine fibroblasts with an average knock-in frequency of 53.2%, a great improvement over previous approaches. The versatile HIT-trapping strategy presented here is expected to simplify the targeted generation of mutant alleles and facilitate large-scale mutagenesis in large mammals such as pigs.

Following the completion of animal genome sequencing projects, rapid and efficient mutagenesis strategies are needed for analyzing gene function and for creating human disease models. Gene trapping is a high-throughput mutagenesis strategy whereby random vector insertion can be achieved across the mouse genome. A typical gene-trap vector contains a promoter-less reporter/selection gene flanked by an upstream splice acceptor (SA) and a downstream poly(A) signal. Upon insertion into an intron of a gene, the vector both inactivates the trapped gene and enables the gene-specific expression of a reporter gene (Gossler et al. 1989; Stanford et al. 2001). To date, gene-trapping approaches have been successfully applied toward large-scale mutagenesis in mouse embryonic stem cells (mESCs) and generation of gene knockout mice (Skarnes et al. 2004). The main drawback of random gene trapping is that gene-trap alleles are not specifically engineered to target genes of interest in advance. Therefore, methods to streamline the introduction of predesigned, site-specific modifications into the genome by homologous recombination would represent a significant technological advance. Previously, a hybrid approach combining gene targeting and gene trapping (targeted trapping) enabled mutation of expressed genes in mESCs with high efficiency, using a gene-trap construct flanked by homologous sequences of the target locus (Friedel et al. 2005). Also, homologous recombination is commonly used for creating conditional alleles, which is essential to avoid embryonic lethality and to study the stage- and tissue-specific functions of genes (Branda and Dymecki 2004). However, both standard gene trapping and targeted trapping are only suitable for genes expressed in embryonic stem (ES) cells. Furthermore, construction of targeting donor vectors with homology arms is labor intensive and costly, and the low efficiency of homologous recombination is also a rate-limiting step for gene targeting in mammalian genomes.Recently, by taking advantage of precise genomic double-strand breaks (DSBs) created by the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system (Ran et al. 2013; Doudna and Charpentier 2014; Hsu et al. 2014), homology-directed repair (HDR) efficiency was substantially enhanced (Porteus and Carroll 2005), and even donors with short homology arms (Orlando et al. 2010) or single-stranded DNA oligonucleotides (Chen et al. 2011; Quadros et al. 2017) were found to be compatible with site-specific integration. However, each targeting donor for HDR still needs to be customized with gene-specific homology sequences. Because of the lack of ES cells for certain animals such as pigs, sheep, and cattle, the genome must be edited either in a zygote embryo or in a somatic cell for somatic cell nuclear transfer (SCNT) (Reddy et al. 2020). It is still not feasible to achieve large-scale insertional mutagenesis including conditional knockouts in these important species with random gene trapping or HDR-based methods. Also, the problem of genetic mosaicism in embryo editing remains unresolved (Mehravar et al. 2019), prompting a need for technological advances to accelerate genetic modification in somatic cells.Alternatively, the generally more efficient nonhomologous end joining (NHEJ) pathway has been exploited for site-specific insertion of exogenous DNA by simultaneous cleavage of both donor plasmid and genome using programmable nucleases (Cristea et al. 2013; Maresca et al. 2013; Brown et al. 2016; Suzuki et al. 2016; Sawatsubashi et al. 2018). In contrast to HDR-based strategies, NHEJ-mediated insertions do not require gene-specific homology arms, enabling diverse sites to be targeted with a universal donor vector. Therefore, we speculated that a gene-trap cassette could be inserted into a specific locus easily through this mechanism in any cell type.Here, by combining NHEJ-mediated knock-in and gene trapping, we developed a strategy for targeted mutagenesis, especially in somatic cells with low HDR activity, referred to as HIT-trapping. By using a universal donor, this strategy allows us to (1) create null alleles, (2) produce a fluorescent reporter signal that could potentially allow cells with null alleles to be identified very quickly, and (3) produce reversible and conditional alleles that would be very helpful to have in most animal models but are often cumbersome to create.     

碳纳米管及其复合材料在生物医学领域的研究进展

碳纳米管是一种新型碳材料，具有特殊的纳米结构和优异的力学、电学和磁学性能，在生物医学领域显示出诱人的潜在应用价值和前景，吸引了越来越多的研究者的关注。本文简要介绍了碳纳米管的基本结构与性能，并对近年来碳纳米管在生物大分子的修饰与特异性识别、细胞体外生长支架、植入性生物医学材料、生物医学传感器以及生物医学微电子器件等方面的研究进展和发展趋势进行了综合评述。     

Gene-lifecourse interaction for alcohol consumption in adolescence and young adulthood: five monoamine genes.

Association analysis has suggested that common sequence variants of genes that affect monoamine function can affect substance use and abuse. Demonstration of these associations has been inconsistent because of limited sample sizes and phenotype definition. Drawing on the life course perspective, we predicted a stronger association between the polymorphisms in 5HTT, DAT1, DRD4, DRD2, and MAOA and alcohol consumption in young adulthood than adolescence. This analysis tested for the gene-lifecourse interaction for the frequency of alcohol consumption in a nationally representative non-alcohol-dependent sample of 2,466 individuals that were visited during adolescence and young adulthood for four times between 1994 and 2002. All five genes are significantly associated with the frequency of alcohol consumption, with the genotype effects ranging 7%-20% of the mean score of alcohol consumption and their P values being 0.014, 0.0003, 0.003, 0.007, 0.005, and 0.003, respectively. The association is only observed in the life stage of young adulthood and not in adolescence. This analysis has demonstrated the potential usefulness of the life course perspective in genetic studies of human behaviors such as alcohol consumption.     

具有三层管壁结构组织工程血管支架的生物力学性能

目的针对组织工程血管的体内培养技术路线，对所制备的具有三层管壁结构的组织工程血管支架的生物力学性能进行测试，并研究了壁厚对支架力学性能的影响，以保证后续的动物体内移植实验能顺利进行。方法采用涂敷，喷涂．滤沥的方法制备了具有三层管壁结构（多孔PLGA层．致密PU层．多孔PLGA层）的可降解组织工程血管支架，用自制的设备测试了其爆破强度和径向顺应性，并对血管支架进行了缝合强度的测试。结果所制备的厚度为0．295mm-0．432mm的三层结构血管支架的径向顺应性为3．80％／100mmHg-0．57％／100mmHg，爆破强度为160kPa～183kPa，缝合强度为0．63N／针～1．52N／针。结论支架的管壁厚度，尤其是中间层厚度，对支架的力学性能有重要影响。增大壁厚可导致径向顺应性急剧下降，爆破强度和缝合强度线性提高。在所制备的样品中，管壁厚度为0．295mm的支架其综合力学性能最优，可满足血管组织工程体内植入的力学性能要求。