双管基因枪介导的基因瞬时表达技术在拟南芥中的应用

基因瞬时表达是植物中研究目标基因功能的常用手段。在模式植物拟南芥(Arabidopsis thaliana)中, 相比原生质体和农杆菌介导的基因异源表达技术, 利用粒子轰击进行基因瞬时表达一直鲜有报道。其主要原因是拟南芥叶型相对较小、基因枪操作相对烦琐以及基因表达效率差异较大。该研究通过优化双管基因枪系统, 在营养生长旺盛的拟南芥莲座叶中实现GFP和GUS基因高效表达。同时, 通过GUS报告基因明确了坏死诱导因子BAX、Avh238和ATR13/Rpp13激发拟南芥细胞坏死的表型。但在本氏烟(Nicotiana benthamiana)中明显诱导细胞坏死的Avrblb1/RB基因对, 在拟南芥中却丧失了诱导细胞坏死的活性。由于双管基因枪系统每次轰击时设置平行对照, 可有效降低转化实验中的样本变异度, 为拟南芥及其突变体研究中准确评价基因功能和高通量筛选目标基因提供新的技术参考。     

液血分流吸引器在剖宫产术中失血量估算的临床应用

目的为了及时诊断产后出血和正确测量剖宫产术中的失血量,及时补充血容量,降低孕产妇死亡率。方法同一剖宫术孕妇分别采用精测法（称重法和液血分流吸引器相结合）和目测法两种方法进行失血量的比较。结果目测法估算失血量明显低于精测法,结果显示用精测法对剖宫产术中进行失血量的估算是目测法的1．5倍（1．5：1）。结论在剖宫产术中精确测量失血量,及时诊断产后出血,有利于及时补充血容量,提高孕产妇身心健康。     

H9N2禽流感病毒反向遗传系统转录／表达载体的构建和验证

设计带有BsmBI、BsaI或AarI酶切位点的引物,用RT-PCR扩增H9N2亚型禽流感病毒(AIV)的8个基因全长片段,克隆入双向转录/表达载体pHW2000,并在PB2、PB1和NA基因中共引入了3个沉默突变标签。将其2个表面基因(HA和NA基因)加上任意1个内部基因,而其它5个内部基因来自A/WSN/33,进行了6种3 5组合形式的基因重排,把相应组合的转录/表达质粒共转染COS-1细胞,均产生了预期组合、有感染性的H9N2亚型流感病毒,表明亲缘关系遥远的流感病毒可以互相获取基因片段产生重组病毒,提示表面结构基因和单个内部基因不足以限制H9N2AIV在哺乳动物细胞上的宿主范围,同时也验证了构建的8个转录/表达载体均能有效工作,为进一步研究H9N2亚型AIV基因结构与功能、AIV与宿主之间的关系打下了基础。     

干旱胁迫下硫营养对小麦光合色素及MDA含量的影响

选用郑引1号（高水肥型）和陕合6号（抗旱型）2个小麦品种,通过室内盆栽试验比较了干旱及硫胁迫情况下小麦苗期功能叶片中叶绿素（Chla和Chlb）及丙二醛（MDA）含量的变化。采用自然渐进干旱法控制水分,每天称重。结果表明：无论在正常供水还是干旱胁迫时,供硫处理的叶绿素含量始终高于无硫处理,郑引1号的这种优势明显优于陕合6号,硫营养显示出对叶绿素含量的调节能力。干旱胁迫下,供硫处理的小麦叶片MDA含量始终低于无硫处理。两个试验结果均证实了硫营养能通过调节光合色素及MDA的含量增强作物的抗旱能力。     

一种利用STO饲养层细胞制备拟胚体的新方法

建立了一种利用STO饲养层细胞制备拟胚体的新方法。该方法选用生长至80%饱和密度的STO细胞,经丝裂霉素C(10 mg/ml)处理4 h后以8×104 cm-2的密度接种培养12 h,制备饲养层,再将ES-D3细胞以1×104 cm-2的密度接种其上,首先用含mLIF的DMEM培养液培养24 h,再更换拟胚体诱导培养液,5～9天后获得了各成熟阶段的拟胚体。形态结构和分化潜能等研究表明,该方法制备的拟胚体结构典型,具有产生3个胚层谱系来源的功能细胞的潜能。与传统拟胚体制作方法如悬滴培养法相比,具有操作简便,拟胚体形成率高,重复性好等优点,是开展哺乳动物早期胚胎发育和干细胞分化研究的理想工具。     

利用SSR分子标记解析遗传学三大规律

分离规律、自由组合规律和连锁规律被称为遗传学三大规律,是认识生物遗传和变异的基础。一般教学中,主要通过对分离世代性状的表型分离特性进行观察和统计分析,是一种基于表型的分析,而利用位于同一染色体和不同染色体上的微卫星(Simple sequence repeats,SSR)分子标记,在分离世代的基因型数据及PCR扩增产物带型的分析,可以从核酸水平上比较直观地解释遗传规律,提高教学效果,加深学生对遗传规律的认识。     

全反式维甲酸协同DAPT抑制胶质瘤干细胞的自我更新

前期研究脑表明,脑胶质瘤干细胞（glioma stem cells,GSCs）是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和Y-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGCl和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGCl生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA（80μmol/L）能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5％～8％,而正常对照组为32％～35％;同样,DAPT（40μmol／L）也能降低PGCs成球大小,且成球效率降至2％～3％;低浓度ATRA（20μmol／L）和DAPT（5gmol／L）对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3％～5％,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CDl33、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。     

Genetic variants associated with breast cancer risk for Ashkenazi Jewish women with strong family histories but no identifiable BRCA1/2 mutation

The ability to establish genetic risk models is critical for early identification and optimal treatment of breast cancer. For such a model to gain clinical utility, more variants must be identified beyond those discovered in previous genome-wide association studies (GWAS). This is especially true for women at high risk because of family history, but without BRCA1/2 mutations. This study incorporates three datasets in a GWAS analysis of women with Ashkenazi Jewish (AJ) homogeneous ancestry. Two independent discovery cohorts comprised 239 and 238 AJ women with invasive breast cancer or preinvasive ductal carcinoma in situ and strong family histories of breast cancer, but lacking the three BRCA1/2 founder mutations, along with 294 and 230 AJ controls, respectively. An independent, third cohort of 203 AJ cases with familial breast cancer history and 263 healthy controls of AJ women was used for validation. A total of 19 SNPs were identified as associated with familial breast cancer risk in AJ women. Among these SNPs, 13 were identified from a panel of 109 discovery SNPs, including an FGFR2 haplotype. In addition, six previously identified breast cancer GWAS SNPs were confirmed in this population. Seven of the 19 markers were significant in a multivariate predictive model of familial breast cancer in AJ women, three novel SNPs [rs17663555(5q13.2), rs566164(6q21), and rs11075884(16q22.2)], the FGFR2 haplotype, and three previously published SNPs [rs13387042(2q35), rs2046210(ESR1), and rs3112612(TOX3)], yielding moderate predictive power with an area under the curve (AUC) of the ROC (receiver-operator characteristic curve) of 0.74. Population-specific genetic variants in addition to variants shared with populations of European ancestry may improve breast cancer risk prediction among AJ women from high-risk families without founder BRCA1/2 mutations.     

Capsaicin induces apoptosis in human osteosarcoma cells through AMPK-dependent and AMPK-independent signaling pathways

Recent studies have focused on the anti-tumor activity of capsaicin. However, the potential effects of capsaicin in osteosarcoma cells and the underlying mechanisms are not fully understood. In the current study, we observed that capsaicin-induced growth inhibition and apoptosis in cultured osteosarcoma cells (U2OS and MG63), which were associated with a significant AMP-activated protein kinase (AMPK) activation. AMPK inhibition by compound C or RNA interference suppressed capsaicin-induced cytotoxicity, while AMPK activators (AICAR and A769662) promoted osteosarcoma cell death. For the mechanism study, we found that AMPK activation was required for capsaicin-induced mTORC1 (mTOR complex 1) inhibition, B cell lymphoma 2 (Bcl-2) downregulation and Bax upregulation in MG63 cells. Capsaicin administration induced p53 activation, mitochondrial translocation and Bcl-2 killer association, such effects were dependent on AMPK activation. Interestingly, we observed a significant pro-apoptotic c-Jun NH2-terminal kinases activation by capsaicin in MG63 cells, which appeared to be AMPK independent. In conclusion, capsaicin possessed strong efficacy against human osteosarcoma cells. Molecular studies revealed that capsaicin activated AMPK-dependent and AMPK-independent signalings to mediate cell apoptosis. The results of this study should have significant translational relevance in managing this deadly malignancy.