基于拉曼光谱的鼻咽癌细胞株总蛋白放射敏感性研究

本文以鼻咽癌细胞株CNE2为放射敏感性的研究对象,经不同剂量X射线照射及不同时间培养后分别提取总蛋白,用共聚焦显微拉曼光谱仪检测其拉曼光谱。统计分析表明:被测样品的拉曼光谱中观察到一些可以归属于蛋白质物质的较为明显的基团频率振动峰;不同剂量的X射线照射后,总蛋白质的平均拉曼光谱与对照组谱形基本一致,但与对照组间的光谱存在着对应峰信号强度的不同。实验提示:照射后谱峰强度的增大或减小,提示着相关物质含量有所改变。分析照射后癌细胞总蛋白拉曼光谱的变化情况,结合数学统计方法,以探寻放射敏感性的特征拉曼标志,可以作为研究肿瘤放射敏感性的手段之一。     

Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones

Following locus-specific genome editing of mouse embryonic stem cells (ESCs), the identification of correctly targeted clones remains a challenge. We applied multiplex ligation-dependent probe amplification (MLPA) to screen for homologous recombination-based genomic integration of a knockout construct in which part of a gene is deleted. All candidate ESCs thereby identified were subsequently validated by conventional methods. Thus, MLPA represents a highly reliable as well as cost- and time-efficient alternative to currently applied methods such as Southern blotting and polymerase chain reaction (PCR)-based approaches. It is also applicable to knockin recombination strategies and compatible with the CRISPR/Cas9 system and other genome editing strategies.     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

大鼠输精管平滑肌细胞的体外培养及形态学特征(英文）

建立大鼠输精管平滑肌细胞的培养方法。取大鼠输精管,剥离外膜和内膜,用组织块法进行体外培养。用抗α-SMA(anti α-smooth muscle actin)免疫组化染色的方法鉴定培养的细胞。结果显示,在倒置显微镜下观察细胞形态多样,表现为长梭形或星形,细胞伸出突起互相接触,彼此融合,部分区域细胞多层重叠,部分区域细胞单层高低起伏,呈"峰-谷"状生长。免疫组化染色鉴定呈阳性反应,用该方法所分离、培养的输精管平滑肌细胞纯度达99%以上。应用组织块法培养大鼠输精管平滑肌细胞,操作简单,结果稳定。     

小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础     

火针层孔菌（桑黄）粗多糖对荷瘤小鼠的免疫调节研究

研究了火针层孔菌不同粗多糖对荷瘤小鼠的免疫调节作用。荷瘤小鼠随机分为四组:胞外粗多糖组、菌丝体粗多糖组、子实体粗多糖组和生理盐水阴性对照组。给药10d后测定荷瘤小鼠脾NK细胞活性和脾淋巴细胞的增殖率。结果显示火针层孔菌粗多糖组与阴性对照组相比能够提高小鼠脾NK细胞活性和脾淋巴细胞的增殖率(P<0.01),表明火针层孔菌液体发酵粗多糖和子实体粗多糖对荷瘤小鼠免疫功能均有调节作用。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

应用固定化里氏木霉糖化玉米秆纤维素的研究

采用多孔聚酯材料固定里氏木霉（TrichodermareeseiRutC30）菌丝细胞,将固定化细胞在生长限制条件下重复分批培养,使纤维酶的合成与玉米秆纤维原料的酶解糖化耦合在一个反应器中同时进行。在30℃、初始pH4.8、摇瓶转速150r／min的条件下,连续重复进行12次分批培养试验。每批玉米秆用量为60g／L,培养周期4．5d,共54d。培养液中含滤纸酶活力平均为0.70IU／ml,还原糖26．41g／L,糖化率达到理论值的89.11％。固定化菌丝形态正常,菌量保持在10g／L左右。在间歇添料条件下,玉米秆原料的总量可提高到120g／L,7d后还原糖浓度达52.81g／L,糖化率为89．20％。利用固定化里氏木霉同时产酶和糖化植物纤维原料,工艺简便、成本低廉、易于连续自动化操作,是一条有效利用可再生纤维素资源的新途径。     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein