ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

Association Study of Germline Variants in CCNB1 and CDK1 with Breast Cancer Susceptibility,Progression, and Survival among Chinese Han Women

The CCNB1 and CDK1 genes encode the proteins of CyclinB1 and CDK1 respectively, which interact with each other and are involved in cell cycle regulation, centrosome duplication and chromosome segregation. This study aimed to investigate whether the genetic variants in these two genes may affect breast cancer (BC) susceptibility, progression, and survival in Chinese Han population using haplotype-based analysis. A total of ten tSNPs spanning from 2kb upstream to 2kb downstream of these genes were genotyped in 1204 cases and 1204 age-matched cancer-free controls. The haplotype blocks were determined according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. For CCNB1, rs2069429 was significantly associated with increased BC susceptibility under recessive model (OR=2.352, 95%CI=1.480-3.737), so was the diplotype TAGT/TAGT (OR=1.947 95%CI=1.154-3.284, P=0.013). In addition, rs164390 was associated with Her2-negative BC. For CDK1, rs2448343 and rs1871446 were significantly associated with decreased BC risk under dominant models, so was the haplotype ATATT. These two SNPs also showed a dose-dependent effect on BC susceptibility. Using stratified association analysis, we found that women with the heterozygotes or minor allele homozygotes of rs2448343 had much less BC susceptibility among women with BMI<23. In CDK1, three closely located SNPs, rs2448343, rs3213048 and rs3213067, were significantly associated with tumor’s PR status: the heterozygotes of rs2448343 were associated with PR-positive tumors, while the minor allele homozygotes of rs3213048 and heterozygotes of rs3213067 were associated with PR-negative BC tumors. In survival analysis, rs1871446 was associated with unfavorable event-free survival under recessive model, so was the CDK1 diplotype ATATG/ATATG, which carried the minor allele homozygote of rs1871446. Our study indicates that genetic polymorphisms of CCNB1 and CDK1 are related to BC susceptibility, progression, and survival in Chinese Han women. Further studies need to be performed in other populations as an independent replication to verify these results.     

血管紧张素Ⅱ在胚胎干细胞向心肌细胞分化中的作用

为检测血管紧张素Ⅱ（angiotensin Ⅱ,AⅡ）对小鼠胚胎干细胞（embryonic stem cells,ESCs）向心肌细胞方向分化的作用,采用10-4 mol/L维生素C诱导小鼠R1胚胎干细胞分化为心肌细胞. Western印记检测胚胎干细胞诱导分化的心肌细胞中表达血管紧张素Ⅱ1 型受体(angiotensin Ⅱ type 1 receptor,AT1R).诱导分化期间用1 μmol/L AⅡ刺激胚胎干细胞,计数搏动拟胚体的比例;诱导分化第14 d用real-time RT-PCR 和Western 印记检测心肌标志物的表达确定其作用. 结果显示,与对照组相比,1 μmol/L AⅡ处理组可显著增加搏动拟胚体的比例,上调心肌标志物mRNA的表达. 预先用1 μmol/L洛沙坦处理1 h后可显著阻碍这种上调作用. 本实验结果表明,AⅡ通过AT1R可促进小鼠R1胚胎干细胞向心肌细胞分化.     

A cytosolic phospholipase A2-initiated lipid mediator pathway induces autophagy in macrophages

Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA(2))-initiated proinflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear. In this study, we report that cPLA(2) and its metabolite lipid mediators induced autophagy in the RAW246.7 macrophage cell line and in primary monocytes. IFN-γ-triggered autophagy involves activation of cPLA(2). Cysteinyl leukotrienes D(4) and E(4) and PGD(2) also induced these effects. The autophagy is independent of changes in mTOR or autophagic flux. cPLA(2) and lipid mediator-induced autophagy is ATG5 dependent. These data suggest that lipid mediators play a role in the regulation of autophagy, demonstrating a connection between the two seemingly separate innate immune responses, induction of autophagy and lipid mediator generation.     

筛选大鼠血管钙化消退差异表达基因及初步分析

目的：探讨大鼠在血管钙化消退过程中基因表达的变化。方法：选取6周龄SPF级雄性SD大鼠24只,随机分成3组（n=8）,分别为对照组、钙化组和消退组。钙化组和消退组制作血管钙化模型（vitamin D3 plus nieotine,VDN）,对照组生理盐水和花生油灌胃。钙化组和对照组于实验第8周处死,消退组继续饲养至16周处死,测定各组大鼠动脉组织钙含量并作病理检查。采用抑制性消减杂交方法将消退组和钙化组大鼠血管cDNA作差减杂交,分离消退组较钙化组高表达或低表达基因的cDNA片段,建立消退组和钙化组的差异表达文库,扩增、鉴定文库并测序阳性克隆,BLAST比对测序序列。随机挑选4个基因进行RT-PCR验证和DNA条带半定量分析。结果：①血管组织钙含量测定显示钙化组（（15．34±2．51）mg／g）较对照组（（5．20±0．75）mg／g）血管组织钙含量明显升高（P〈0．01）;消退组（（12．73±1．89）mg／g）较钙化组降低（P〈0．05）;②构建了血管钙化的差减文库,对所获阳性克隆进行测序和BLAST比对分析,获得28个表达上调基因和22个表达下调基因。RT-PCR验证示Prdx3,Ank2,Ror2,Abcel等基因在消退组和钙化组间差异表达,消退组较钙化组表达增高,平均约为后者的1．7倍。结论：VDN模型诱导的大鼠血管钙化可以发生主动消退。钙化消退过程中焦磷酸合成相关基因、谷氨酸信号相关基因、还原及凋亡调节基因表达上调,同时见较多骨化相关基因及氧化活性等基因表达下调。钙化抑制基因的表达增多而钙化促进基因表达的下降可能是钙化发生主动消退的内源性机制。     

太湖夏季浮游细菌群落多样性的空间格局简

为了研究太湖夏季浮游细菌群落多样性与水体营养盐的关系,在太湖全湖范围内开展了一次大规模浮游细菌采样调查,分析了太湖不同湖区浮游细菌丰度和多样性组成。研究发现,浮游细菌丰度在不同湖区中存在明显的空间差异,从北部和西部湖区沿湖流向东南方向至湖心和南部沿岸再到东部湖区呈下降趋势,这与太湖水体营养水平从高到低变化趋势一致。浮游细菌丰度与营养盐浓度回归分析结果显示,总磷(TP)与细菌丰度存在较好的正相关(R2=0.6392,n=29,P0.01),而总氮(TN)与细菌丰度无显著相关(R2=0.0663,n=29,P0.05)。因此,磷是太湖夏季浮游细菌生长的限制因子。不同湖区营养盐与浮游细菌群落多样性也具有显著的正相关,随着营养水平的升高,浮游细菌多样性增加。此外,细菌群落的组成在不同湖区间亦具有明显的空间异质性,与不同湖区营养水平空间变化一致。研究结果将有助于人们更好地理解淡水湖泊中微生物循环和生态系统功能。     

微酸性电解水和紫外光结合对采后六妹羊肚菌的保鲜作用

联合使用微酸性电解水和紫外光（SAEW+UV）处理食品是一种延长食品保质期的有效方法。本研究在20℃条件下,观察经SAEW+UV处理六妹羊肚菌Morchella sextelata采后9d内品质的变化。SAEW+UV处理能减少六妹羊肚菌表面附着的细菌和真菌数,提高六妹羊肚菌中超氧化物歧化酶和维生素C含量,降低多酚氧化酶、过氧化物酶含量,并在此基础上改善六妹羊肚菌储存过程中褐变及质地软化,而对六妹羊肚菌失重率、风味和营养成分无明显作用。转录组分析发现,SAEW+UV处理能上调六妹羊肚菌涉及抗氧化酶的基因表达,下调细胞壁降解、细胞凋亡和黑色素合成的基因表达,该结论与上述品质分析结果一致。综上,SAEW+UV处理能够延长六妹羊肚菌的保鲜期。     

湖南沅水下游?繁殖期内繁殖力和卵径的变化研究

为分析鱼类繁殖期内繁殖力和卵径变化趋势, 研究于4—10月在沅水下游逐月采集180尾?(Hemiculter leucisculus), 分析81尾雌性成熟个体的繁殖力。研究结果表明: ?的性体指数在5、6月最高, 其次在7、8和9月, 在4和10月最低。绝对繁殖力平均值为(30116±19390)粒, 以10000—30000粒为主, 相对繁殖力的平均值为(650±324)粒/g, 以400—800粒/g为主。绝对繁殖力与体长、空壳重呈幂函数关系(P<0.05)。绝对繁殖力和相对繁殖力在5和6月最大, 7、8和9月次之, 在4和10月最小。不同月份成熟卵子的卵径无显著差异, 补充卵子的卵径在5和6月最大, 暗示5和6月分批繁殖次数较多。不同繁殖时期的分批繁殖力、繁殖次数不一样, 对评估种群的繁殖潜能具有重要的指导意义。比较不同水系繁殖力发现, 湖泊种群的个体繁殖力普遍大于河流种群, 沅水种群的个体繁殖力跟湖泊种群的相似, 可能与其具有类似湖泊的缓流水或静水生境有关。研究揭示了?的基本繁殖特征, 为渔业资源管理提供了基础数据资料。     

凝血因子FXIII A链基因第1内含子内顺式调节元件的鉴定

探讨FⅩⅢ A基因表达的分子机制.凝胶阻滞实验（EMSA）结果证明,FⅩⅢ A基因第1内含子5′区的前12碱基与转录因子结合并由此调控基因表达.FⅩⅢ A基因第1内含子第12位碱基由C突变为A(内含子1 (+12)C→A)导致与转录因子结合能力降低.构建不同的荧光素酶表达质粒Luc 1、Luc 2、Luc 3、Luc 4、Luc 5、Luc 6,并转染U937细胞和HepG2细胞.结果显示,如果Luc 5（具有最高表达活性）的内含子1(+12)由C突变为A,启动子活性显著降低.与Luc 5相比,突变后的Luc 5的活性分别下降了52.9％（U937, P＜0.01）和47.6％（HepG2, P＜0.01）.将Luc 5与PN3 Sp1共同转染U937细胞和HepG2细胞后,Luc 5的荧光素酶活性分别提高了42.4％（U937,与Luc 5单独转染比较P＜0.01）和54.9％（HepG2, 与Luc 5单独转染比较P＜0.01）,而突变后的Luc 5（Mut）与PN3 Sp1共同转染则没有明显的改变.表明转录因子Sp1在FⅩⅢ A基因表达的重要性.这些结果也表明,内含子在FⅩⅢ A基因表达过程中起重要作用,为遗传性凝血因子发病机理的研究提供了新的证据.