以纤连蛋白(FN)细胞结合片段研究大鼠肝星状细胞FN受体表达及调控

应用亲和层析、凝胶过滤法以及X型蛋白酶消化结合制备电泳分别提取纯化大鼠FN及FN细胞结合片段（120 KDa FN-f）,后经生物素标记后,用亲和细胞化学方法研究大鼠肝星状细胞（HSC）FN受体（FNR）表达及调控。结果显示,生物素标记的120KDa FN－f与FNR的结合具有特异性。原代培养5d、7d的正常大鼠HSC表达FNR较培养1d、3d的明显增强,血小板源性生长因子（PDGF）和转化生长因子－β、（TGF－β1）可上调大鼠HSC表达FNR,全反式维甲酸（atRA）则下调细胞因子再激活的HSC表达FNR,并呈剂量依赖性。本建立了检测FNR的一种新方法,即配体（120KDa FN-f）－受体（FNR）亲和细胞化学方法,该方法能反映FNR的总体水平及活性状态。同时初步探讨了影响大鼠HSC表达FNR的因素,确切机制有待进一步研究。     

不同打靶位点影响人诱导多能干细胞MYO7A杂合突变位点的基因校正中CRISPR/Cas9介导的同源重组效率

应用CRISPR-Cas9系统对人诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）进行基因编辑,为疾病模型的建立、致病机制研究、药物筛选及基因校正治疗疾病提供了更广阔的平台。相对于CRISPR-Cas9介导的基因敲除,应用该系统介导的同源重组实现基因点突变或突变校正效率要低、且难度偏大。为了实现对MYO7A杂合点突变（c.4118C>T）的人iPSCs的点突变校正,本文构建了表达maxGFP的pX330质粒。针对需校正的突变位点,设计5组识别序列并连接到maxGFP-pX330中构建靶向质粒。将5组打靶质粒分别转染HEK 293FT细胞48 h,细胞表达GFP;测序结果显示,MYO7A基因相应位点出现杂峰,表明打靶质粒具有打断活性。将同源模版单链寡核苷酸链（single-stranded DNA oligonucleotides, ssODN）与打靶质粒共同电转入人iPSCs后48 h,经流式分选出(5.8±2.2)%的细胞表达GFP。分选后细胞行单克隆扩增并测序。结果显示,打靶质粒1和ssODN组合对点突变校正未成功;打靶质粒2、3、4、5与ssODN组合均获得了校正后的细胞株。本研究表明,打断位点是影响同源重组校正效率的关键因素。当应用CRISPR/Cas9（或其它核酸酶）介导的同源重组进行基因编辑操作时,可以同时选择多个打靶位点造成基因组不同位置上的双链打断（double-stranded break, DSB）位点,以获得目的单克隆细胞株。本研究为应用CRISPR-Cas9系统对人诱导多能干细胞进行基因编辑提供了有力参考。     

反义及正义表达癌基因c-Ha-ras重组质粒的构建

c-Ha-ras癌基因通过其产物p21蛋白的点突变或过量表达使细胞恶变。国外一些实验室的初步研究表明,导入一段与c-Ha-ras基因转录出的mRNA(sense RNA,正义RNA)顺序互补的RNA(anti-sense RNA,反义RNA),有可能通过阻断DNA复制,RNA转录或蛋白质翻译等过程,减少p21蛋白的合成,从而使转化细胞逆转。为了进一步系统研究反义c-Ha-ras RNA在转化细胞逆转中的     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还     

可随HeLa细胞传代而传递的反义RNA真核表达系统

为研究反义ＲＮＡ表达载体在细胞内的稳定性,构建了一个特异性针对β地中海贫血基因ＩＶＳ－２－６５４Ｃ→Ｔ（β６５４）突变ｍＲＮＡ前体异常剪接位点的反义ＲＮＡ表达载体ｐＣＭＶＡ．ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,通过ＲＮＡ定量检测反义片段对β６５４ｍＲＮＡ异常剪接的纠正作用;再从转染后传代５次并冰冻保存１年的ＨｅＬａ细胞中回收反义表达载体,转染另外的β６５４ＨｅＬａ细胞,同样检测它对β６５４ｍＲＮＡ异常剪接的纠正作用．结果显示该载体在细胞传代前后均能阻断β６５４异常剪接,部分恢复其正常剪接途径：用回收的ｐＣＭＶＡ转染β６５４ＨｅＬａ细胞后,正常剪接的βｍＲＮＡ水平［β／（β＋β＊）］由０．０５上升到处理后１５ｄ的０．４８,而２种对照质粒处理后对这一比值影响不大．表明ｐＣＭＶＡ可在ＨｅＬａ细胞中随着细胞传代而传递下去,并保持结构与功能完整     

反义核酸真核表达载体的构建及其对重组HeLa细胞H654中IVS-2-654C→T突变β-珠蛋白基因剪接缺陷的抑制作用

在原工作的基础上 ,将非细胞系统中证明有效的反义片段通过高效真核表达载体 pCDNA3导入能稳定表达IVS -2 -6 5 4C→T突变 β 珠蛋白基因的重组HeLa细胞 (H6 5 4)中 .结果显示 ,转染后第 3天 ,H6 5 4细胞内正常剪接的β -珠蛋白mRNA水平 ( β/( β + β *) )明显上升 ,从 0 0 7上升到 0 2 2 ,并维持到第 1 5天 ,最高达到0 43.实验表明构建的反义片段真核表达载体在细胞中能稳定转录反义RNA并有效抑制IVS -2 -6 5 4C→T突变β 珠蛋白基因的异常剪接 ,为开展反义核酸治疗剪接缺陷型遗传性疾病提供了更直接的科学依据     

导入反义蜡质基因改良水稻稻米的食味品质和营养品质

经PCR、Southern blot和稻米GUS检测,蜡质基因反义片段与gus构成的融合基因已整合到8株水稻基因组中,并能正确表达。通过稻米GUS染色追踪分析获得转反义蜡质基因纯合后代。将转基因植株的稻米送农业部稻米及制品质量监督检验测试中心进行糙米蛋白质含量和直链淀粉含量测定。结果显示：（1）在转反义蜡质基因纯合的超2-10粳稻后代中,大多数单株糙米在直链淀粉含量降低的同时,蛋白质含量有不同程度提高,糙米蛋白质含量和直链淀粉含量呈显著负相关关系;（2）对照糙米直链淀粉平均含量和糙米蛋白质平均含量分别为13.4%和9.5%。在转基因植株中,稻米直链淀粉含量最低的为11.4%,而蛋白质含量也相对最高,为13.5%。本试验结果表明在水稻中导入反义蜡质基因不仅能够降低水稻稻米的直链淀粉含量,还有可能提高水稻稻米的蛋白质含量。     

芳香烃龙胆酸降解途径蛋白质组学的研究

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶（GDOI）及其下游片段被克隆。产碱假单胞菌NCIB9867（P25X）的突变株-SNZ28 GDOI被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALDI-TOF和Q—TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌（Ralstonia species）龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDOII的存在。     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

2017年中国植物科学若干领域重要研究进展

2017年中国植物科学继续保持高速发展态势, 重大成果频出, 具体表现在中国植物学家在国际顶级学术期刊发表的文章数量平稳上升。中国植物科学领域的研究工作者成果精彩纷呈, 如新型广谱抗病机制的发现、水稻广谱抗病遗传基础及机制和疫霉菌诱发病害成灾机制研究等。2017年中国生命科学领域十大进展评选中, 有两项植物科学领域的研究成果入选。水稻生物学、进化与基因组学和激素生物学等领域学科发展突出。另外, 值得一提的是, 长期从事高等植物与代谢途径调控分子网络研究和水稻品种设计育种的李家洋院士的研究成果“水稻高产优质性状形成的分子机理及品种设计”荣获2017年国家自然科学一等奖。这一具有重大国际影响的开创性贡献标志着中国植物科学在该领域的国际科学前沿居于引领和卓越地位。该文对2017年中国本土科学家在植物科学若干领域取得的重要研究成果进行了系统梳理, 旨在全面追踪和报道当前中国植物科学领域发展的最新前沿动态, 与广大读者共同分享我国科学家所取得的辉煌成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义。综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望。     

SSTR5 ablation in islet results in alterations in glucose homeostasis in mice

Somatostatin (SST) peptide is a potent inhibitor of insulin secretion and its effect is mediated via somatostatin receptor 5 (SSTR5) in the endocrine pancreas. To investigate the consequences of gene ablation of SSTR5 in the mouse pancreas, we have generated a mouse model in which the SSTR5 gene was specifically knocked down in the pancreatic beta cells (betaSSTR5Kd) using the Cre-lox system. Immunohistochemistry analysis showed that SSTR5 gene expression was absent in beta cells at three months of age. At the time of gene ablation, betaSSTR5Kd mice demonstrated glucose intolerance with lack of insulin response and significantly reduced serum insulin levels. Insulin tolerance test demonstrated a significant increase of insulin clearance in vivo at the same age. In vitro studies demonstrated an absence of response to SST-28 stimulation in the betaSSTR5Kd mouse islet, which was associated with a significantly reduced SST expression level in betaSSTR5Kd mice pancreata. In addition, betaSSTR5Kd mice had significantly reduced serum glucose levels and increased serum insulin levels at 12 months of age. Glucose tolerance test at an older age also indicated a persistently higher insulin level in betaSSTR5Kd mice. Further studies of betaSSTR5Kd mice had revealed elevated serum C-peptide levels at both 3 and 12 months of age, suggesting that these mice are capable of producing and releasing insulin to the periphery. These results support the hypothesis that SSTR5 plays a pivotal role in the regulation of insulin secretion in the mouse pancreas.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.     

Changes in aquatic vegetation in Rhine floodplain streams in Alsace in relation to disturbance

Abstract. Changes are described in aquatic vegetation in oligotrophic, groundwater-fed Rhine floodplain streams in Alsace (eastern France), resulting from disturbance. Disturbance factors include changes in nutrients, either permanent ones - effluent from a waste water treatment plant or trout hatcheries - or periodic ones: flooding. Regular inputs of high levels of phosphate and ammonia modified the macrophyte vegetation in these streams. The floristic composition, which was characteristic of oligotrophic waters upstream of the eutrophicated sector, changed to that of a eutrophic situation as originally found downstream. Periodic disturbance by floods which normally occur once a year, irregularly eutrophicates the small streams, causing the development of a mixture of eutrophic and oligotrophic species. Six macrophyte communities are distinguished, indicating different trophic levels. The aquatic vegetation is adapted to the variations of phosphate and ammonia levels. Hence, aquatic macrophytes can be used as bio-indicators of fluctuations in water nutrient levels in relation to the type of disturbance.     

Morphine-potentiated platelet aggregation in in vitro and platelet plug formation in in vivo experiments

The detailed mechanisms underlying morphine-signaling pathways in platelets remain obscure. Therefore, we systematically examined the influence of morphine on washed human platelets. In this study, washed human platelet suspensions were used for in vitro studies. Furthermore, platelet thrombus formation induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium was used for an in vivo thrombotic study. Morphine concentration dependently (0.6, 1, and 5 microM) potentiated platelet aggregation and the ATP release reaction stimulated by agonists (i.e., collagen and U46619) in washed human platelets. Yohimbine (0.1 microM), a specific alpha(2)-adrenoceptor antagonist, markedly abolished the potentiation of morphine in platelet aggregation stimulated by agonists. Morphine also potentiated phosphoinositide breakdown and intracellular Ca(2+) mobilization in human platelets stimulated by collagen (1 microg/ml). Moreover, morphine (0.6-5 microM) markedly inhibited prostaglandin E(1) (10 microM)-induced cyclic AMP formation in human platelets, while yohimbine (0.1 microM) significantly reversed the inhibition of cyclic AMP by morphine (0.6 and 1 microM) in this study. The thrombin-evoked increase in pH(i) was markedly potentiated in the presence of morphine (1 and 5 microM). Morphine (2 and 5 mg/g) significantly shortened the time require to induce platelet plug formation in mesenteric venules. We concluded that morphine may exert its potentiation in platelet aggregation by binding to alpha(2)-adrenoceptors in human platelets, with a resulting inhibition of adenylate cyclase, thereby reducing intracellular cyclic AMP formation followed by increased activation of phospholipase C and the Na(+)/H(+) exchanger. This leads to increased intracellular Ca(2+) mobilization, and finally potentiation of platelet aggregation and of the ATP release reaction.