Taxonomy of Genus Branchiostoma in Xiamen Waters and Continuous Breeding of Two Lancelets in the Laboratory

文昌鱼是现存无脊椎动物中与脊椎动物直接祖先很相似的一个类群，在动物进化史上占据重要的地位，是进化发育生物学和比较基因组学研究领域的重要模式生物。厦门曾经以盛产文昌鱼并形成世界上唯一的文昌鱼渔场而闻名于世，厦门文昌鱼自1932年被鉴定为白氏文昌鱼BranchiostomabelcheriGray以来一直被认为是同一种，直到2005年应用分子生物学技术进行文昌鱼物种鉴定时，才发现其中包含有2个独立物种，即白氏文昌鱼B.belcheri和日本文昌鱼B.japonicum。本研究详细观察了这2种文昌鱼大量标本，发现了2种文昌鱼的主要形态鉴别特征：白氏文昌鱼喙鳍圆形、前端较钝；臀前鳍鳍室数较多，一般多于...The Cephalochordata, commonly called amphioxus or lancelets, are considered to be basal chordates and have long occupied a key place in studying the origin and evolution of vertebrates. The lancelet is considered to be a promising laboratory model organism. Xiamen has long been famous for having abundant lancelets. Since it was taxonomically identified as Branchiostoma belcheri in 1932 by Boring &...学位：理学博士院系专业：生命科学学院生物学系_动物学学号：B20042600

Programmed cryopreservation of the amphioxus Branchiotoma belcheri embryos

文昌鱼在进化和发育生物学中有重要科学研究价值,然而由于文昌鱼实验室繁殖受产卵季节等因素的限制,以其胚胎为材料的研究工作受到局限,胚胎冷冻保存是解决这一问题的有效途径之一。本文首次对文昌鱼(Branchiostoma japonicum)胚胎的程序化冷冻保存进行了研究,通过筛选合适的程序化抗冻液、适宜的胚胎发育阶段,以及比较不同平衡处理时间、植冰温度、保存温度和洗脱时间对文昌鱼胚胎冷冻保存的影响。结果表明:几种抗冻液中,A2的效果较好,对胚胎的毒性小;原肠中期胚胎较其它时期对抗冻液的耐受力更强,适宜进行冷冻保存;胚胎在抗冻液中的最佳平衡处理时间为40-50min;冷冻降温时,适宜的植冰温度为-7.0℃--10.0℃;植冰后进行程序化冷冻,目前能稳定得到存活胚胎的最低保存温度为-15℃;此外,不同的洗脱时间对胚胎的存活率无显著影响。根据优化后的条件,对文昌鱼胚胎进行程序化冷冻保存(-196℃)实验,获得1粒存活的胚胎,并孵化出膜,发育至两鳃裂时期,幼鱼共存活了8d,但形态畸形。结果提示文昌鱼胚胎冷冻保存的可行性,但需要进一步优化包括抗冻液在内的各种条件,以提高存活率。In the present study, we investigated the possibility of cryopreservation of amphioxus Branchiostoma japonicum embryos. We found that programmed freezing solution A1 was good for the cryopreservation of amphioxus embryos. The test of toxicity tolerance of embryos at various developmental stages towards the solution A1 showed that embryos at the metaphase of gastrulation were more tolerant of the cryoprotectant solution than embryos at other stages. The optimal equilibration time of embryos treated with A1 was 40-50 min. We also compared the effects of seeding temperature, cryopreservation temperature and elution time during the process. The survival rates after seeding at -7℃ and -10℃ were 46% and 42% respectively, but no embryo survived after seeding at -12℃. Some embryos could survive preserved at -15℃, but all embryos perished at lower temperatures. When elution time varied, the survival rate of amphioxus embryos did not vary significantly. Similar to fish, the membrane permeability of amphioxus embryos to water and cryoprotectants is low, which is probably a major obstacle to successful cryopreservation, and causes lethal intracellular ice formation. We used A1 as a programmed freezing solution to cryopreserve different embryonic stages, and stored them in liquid nitrogen (-196℃). Of those embryos, one was successfully recovered and developed to the two-gill-slit stage. Eventually, it died due to morphological abnormality after surviving for eight days. These results indicate the possibility of cryopreservation methods for amphioxus embryos.国家自然科学基金(No.30470938和No.30570208);; 福建省自然科学基金(No.D0510002);; 厦门市科技局科技计划重点项目(No.3502Z20042015)资助~

Chromosome Preparation and Preliminary Observation of Two Amphioxus Species in Xiamen

文昌鱼的进化地位十分重要,对其染色体的研究在进化和比较基因组学方面有重要意义。然而文昌鱼的染色体制备困难,使研究受到了限制。本文介绍了一种改良的文昌鱼胚胎细胞染色体标本制备方法,以及用文昌鱼成体再生细胞制备染色体,首次获得了文昌鱼体细胞中期染色体标本,并观察了厦门2种文昌鱼的染色体,其中白氏文昌鱼(brAnCHIOSTOMA bElCHErI)二倍体2n=40,日本文昌鱼(b.JAPOnICuM)二倍体2n=36。再次从细胞分类学角度证实白氏文昌鱼和日本文昌鱼作为两个独立物种的分类地位。Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates.Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics.The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research.In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells.Chromosomes of two amphioxus species from Xiamen waters in China are also observed.The diploid chromosome number was found to be 40 in Branchiostoma belcheri, while B.japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.SupportedbygrantsfromNSFC(No.30570208);SRFDPofMinistryofEducation;China(20070384041);TheEducationDepartmentofFujianProvince;China(JB07063

Immunohistochemical Study on 5-Hydroxytryptamine Immunoreactive Cells in the Alimentary Tract of Bufo melanostictus and Rana nigromaculata

对黑眶蟾蜍(Bufomelanostictus)和黑斑蛙(Rananigromaculata)消化道5-羟色胺免疫活性细胞的形态、分布进行了免疫组织化学定位。5-羟色胺免疫活性细胞在黑眶蟾蜍和黑斑蛙消化道各段中均有分布,分布密度均呈升-降-升-降的波浪式分布特点,二者在幽门部和回肠都有个分布的高峰值。黑眶蟾蜍回肠最高,空肠、幽门部次之,十二指肠、直肠最低;黑斑蛙幽门部最高,回肠、空肠次之,食道、贲门部、直肠最低。5-羟色胺免疫活性细胞位于胃的胃腺上皮、食道及肠的粘膜上皮,有圆形、椭圆形、梭形、楔形等,有的有胞突。文中讨论了5-羟色胺免疫活性细胞分布型的原因及形态与功能的关系。Using immunohistochemical technique, 5-Hydroxytryptamine immunoreactive (5-HT-IR) cells were observed in the alimentary tract of the toad Bufo melanostictus and the frog Rana nigromaculata . 5-HT-IR cells were distributed throughout the alimentary tracts of both species and located in the gastric glands of the stomach regions and in the intestinal or esophageal epithelium with variable frequencies. The density of the cell in B. melanostictus is the highest in the ileum, moderate in the jejunum and stomachus pyloricus, and the lowest in the duodenum and rectum. In R. nigromaculata the density of 5-HT-IR cells is the highest in the stomachus pyloricus, moderate in the ileum and jejunum, and the lowest in the esophagus, stomachus cardiacus and rectum. 5-HT-IR cells were spherical, elliptoid, coniform or spindle-shaped with long cytoplasmic process reaching to the lumen. The distribution mode of 5-HT-IR cells and the relationship between morphology and function are also discussed.福建省自然科学基金(No.D0320001

Laboratory Culturing and Acquirement of the Second Filial Generation of Amphioxus

文昌鱼特殊的进化地位、简单的器官系统和终生透明的躯体等特征,使其很有希望成为一个新型实验室模式动物。要实现文昌鱼的模式动物化,实验室内全人工条件下繁殖是关键的第一步。为此,我们于2003年9月和2004年4月两次采集产于厦门海域的2种文昌鱼,开展实验室内养殖研究。经过3年多的持续实验室养殖,继2005年夏季于实验室内繁殖出子一代文昌鱼后,又在2006年夏季成功获得了这两种文昌鱼的子二代,初步实现文昌鱼在实验室内的全人工养殖。对子代文昌鱼养殖的初步观察发现,不同水温对生长发育速度有一定影响,提示有可能通过水温控制实现文昌鱼一年多次产卵的目的。目前这两种文昌鱼子二代幼体已完成变态,进入亚成体生长发育阶段,其体长分别已达14.6mm(日本文昌鱼Branchiostomajaponicum)和6.5mm(白氏文昌鱼B.belcheri)。Amphioxus is expected to be a model laboratory animal in the near future because of its important position in evolution, simplified internal organ systems and transparent body. In order to develop the animal into a new laboratory model, we sampled two species of amphioxus (Branchiostoma belcheri and B. japonicum) from Xiamen in September 2003 and April 2004. The captive lancelets were cultured in the laboratory and fed daily with unicellular algae. After three years of laboratory culturing, we successfully obtained a second filial generation of both species, meaning that a full reproductive cycle was achieved in the laboratory. Preliminary observations on the growth of the progeny indicated that warmer water temperature will speed up the growth and development of amphioxus. This hints that amphioxus probably spawns more often in the laboratory, where water temperature is controlled, than in the wild. Most of the second generation lancelets have now metamorphosed into the early sub-adult stage, and their maximum full body lengths were up to 14.6 mm (B. japonicum) and 6.5 mm (B. belcheri), respectively.国家自然科学基金(30470938和30570208);; 福建省自然科学基金(D0510002);; 厦门市科技计划重点项目(3502Z20042015)资

THE LABORATORY CULTURE AND REPRODUCTION of TWO IANCELETS IN XIAMEN

国家自然科学基金(30570208);教育部高等学校博士学科点专项科研基金(20070384041)资

中国国际科学合作中的”华人现象“

将8 个国家的合作论文分成五种类型，重点考察了中国在与8 个国家的合作中所呈现的“华人现 象”。分析表明，海外华人（包括短期在外的科研人员）在现阶段正在发挥桥梁作用，在中国国际合作中表 现得非常活跃。分析了导致“华人现象”的社会基础和政策效应

蒿芩清胆汤及其拆方对湿热型流感病毒性肺炎及NF-κB水平的作用研究

目的:通过观察蒿芩清胆汤及其拆方对湿热型流感病毒性肺炎及NF-κB的干预作用,以期探讨其治疗机制。方法:通过气候仓、肥甘饮食加流感病毒滴鼻感染造模成功后,蒿芩清胆汤及其拆方组以相应中药干预,比较各组肺指数、光镜下病变程度半定量、肺组织NF-κB mRNA表达水平。结果:蒿芩清胆汤及其拆方均能不同程度降低肺指数、肺部病理改变及NF-κB mRNA表达水平,其中全方效果最佳。结论:蒿芩清胆汤具有抗湿热型流感病毒性肺炎的作用,其机制可能与抑制NF-κB活化从而减少其下游炎性细胞因子分泌,截断"炎症瀑布"效应有关

Detection of point mutations of Axin gene and its expression in gliomas

目的　检测胶质瘤中Axin基因的点突变及其表达情况 ,初步探讨Axin与胶质瘤发生的关系。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序方法检测Axin基因外显子 8,9及 10在 2 8例胶质瘤中的突变情况 ;同时对上述胶质瘤及正常脑组织进行免疫组化染色。结果　在 2 8例胶质瘤组织中Axin的第 10个外显子共有 6例样本 (2 1.4 % ) 3处发生了错义突变 ;3例 3处发生了同义突变 ;2 8例胶质瘤中 8例 (2 8.6 % )Axin表达阳性 ,正常脑组织中神经元表达阳性 ,神经胶质细胞表达阴性 ,检测到突变的样本中 1例表达阳性。结论　Axin基因的点突变可能参与胶质瘤的发生 【英文摘要】 Objective To detect the point mutations of Axin gene and its expression in glioma and explore the relationship between Axin gene and the occurrence of human glioma.Methods The point mutations of exon 8,9,10 of Axin gene were analyzed in 28 cases of glioma by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) analysis, silver staining and DNA sequencing. Immunohistochemistry was used to detect the Axin expression in these cases and normal brain tissues.Results Three missense poi...高等学校骨干教师计划资助项目;; 归国留学人员科研启动基金资助项目 (1 999747

Detection of point mutations of Axin gene and its expression in gliomas

目的　检测胶质瘤中Axin基因的点突变及其表达情况 ,初步探讨Axin与胶质瘤发生的关系。方法　采用聚合酶链反应 单链构象多态性 (PCR SSCP)技术及DNA测序方法检测Axin基因外显子 8,9及 10在 2 8例胶质瘤中的突变情况 ;同时对上述胶质瘤及正常脑组织进行免疫组化染色。结果　在 2 8例胶质瘤组织中Axin的第 10个外显子共有 6例样本 (2 1.4 % ) 3处发生了错义突变 ;3例 3处发生了同义突变 ;2 8例胶质瘤中 8例 (2 8.6 % )Axin表达阳性 ,正常脑组织中神经元表达阳性 ,神经胶质细胞表达阴性 ,检测到突变的样本中 1例表达阳性。结论　Axin基因的点突变可能参与胶质瘤的发生高等学校骨干教师计划资助项目;; 归国留学人员科研启动基金资助项目 (1 99974