含人纤溶酶原K5基因的腺病毒载体制备和功能研究

应用PCR将人纤溶酶原信号肽序列引入K5 cDNA基因,与真核表达载体pcDNA3重组,形成重组质粒pcDNA3K5,与穿梭质粒pShuttle 重组得pShuttleK5,经与腺病毒DNA重组,PCR鉴定正确,即为pAdK5。脂质体法将其转染293细胞后,制备细胞裂解液;噬斑分析法测定病毒滴度为5×10⁸ pfu/mL。将病毒以不同的感染系数(MOI)感染人脐静脉内皮细胞株ECV304和人乳腺癌细胞株MDA-MB-231,MTT法检测两者的增殖情况：ECV304细胞增殖受抑制,而MDA-MB-231细胞增殖未受明显影响。将感染病毒的ECV304细胞接种于ECMatrix^TM胶,显示内皮细胞分化和毛细血管管腔形成受抑制。表明所构建的含人纤溶酶原K5基因的重组复制缺陷型腺病毒具有抑制ECV304细胞增殖、分化和管腔形成的作用而对MDA-MB-231细胞的生长则无影响。     

反义c-myc重组腺病毒载体的构建及其对大鼠胸腺淋巴细胞的增殖抑制作用

目的:观察反义c-myc重组腺病毒载体对大鼠胸腺淋巴细胞的增殖抑制作用.方法:构建大鼠反义及正义c-myc细菌质粒,并将所得细菌质粒与E1缺失腺病毒质粒导入293细胞系,经共转染得到正义及反义重组腺病毒载体.MTS法检测重组腺病毒载体对大鼠淋巴细胞增殖的抑制作用,RT-PCR检测重组腺病毒载体对c-myc mR-NA表达的影响.结果:反义c-myc重组腺病毒载体可抑制大鼠淋巴细胞增殖,降低淋巴细胞c-myc mRNA的表达.结论:反义c-myc重组腺病毒载体可抑制大鼠淋巴细胞增殖.     

腺病毒载体介导CEA基因元件调控自杀基因治疗

构建以CEA启动子控制HSV-TK基因表达的复制缺陷型腺病毒载体(AdCEATK).纯化的重组腺病毒滴度达1×10¹²pfu/ml.CEA阴性的HeLa细胞感染AdCMVTK后对丙氧鸟苷(GCV)很敏感,而感染了AdCEATK后不被GCV杀伤.与此相反CEA阳性的LoVo细胞中AdCMVTK和AdCEATK都有很好的表达活性,说明CEA启动子有良好的细胞专一性.AdCEATK/GCV系统还有明显的旁杀伤效应.此载体将有助于实现对CEA阳性肿瘤的专一性自杀基因治疗.     

人骨保护素(OPG)重组腺病毒的制备及其生物活性研究

采用RT-PCR法得到人OPG的编码区cDNA,克隆至穿梭质粒pShuttle,构建重组有OPG编码区cDNA的腺病毒DNA,经Pac I 酶切线性化,在脂质体介导下转染HEK293细胞,制备重组腺病毒并测定病毒滴度约为5×10⁶～1.5×10⁷ pfu/mL。体外感染小鼠成肌细胞C2C12,Western blot及ELISA检测证实有OPG蛋白的表达,并可在细胞培养上清中持续表达6周。感染OPG重组腺病毒的C2C12细胞生长状态良好、细胞周期无明显变化。将重组腺病毒加入体外培养的小鼠骨髓细胞的培养基中,诱导形成的破骨细胞数量及在象牙片上形成的吸收陷窝的数量显著减少（P<0.01）。      

腺病毒载体介导PDX-1在骨髓间充质干细胞中的表达

为研究PDX-1基因在骨髓间充质干细胞中的表达情况及生物学功能的发挥,构建了含PDX-1基因的重组腺病毒载体. 酶切PDX-1基因并连入穿梭质粒pAdTrack-CMV.用电穿孔法使穿梭质粒pAdTrack-CMV-PDX-1与病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组.利用脂质体介导重组腺病毒载体转染293细胞,包装出完整的腺病毒.分离、培养、扩增骨髓间充质干细胞.用重组腺病毒感染间充质干细胞.用荧光显微镜、RT-PCR、免疫荧光染色等方法检测PDX-1、胰岛素基因及蛋白质的表达,用放射免疫分析法检测转基因细胞分泌胰岛素情况.结果表明:通过测序、PCR、酶切等鉴定PDX-1基因已正确插入穿梭质粒中,并与病毒骨架质粒重组.重组腺病毒滴度为6.3×10⁷ PFU/ml.通过荧光显微镜观察证实重组腺病毒可高效感染骨髓间充质干细胞,经RT-PCR、免疫荧光染色证实转染pAd-PDX-1后培养7天的细胞中有PDX-1及胰岛素基因的表达.这些转基因的细胞向胞外分泌的胰岛素量为(15.21±3.50) mIU/L.     

Microdystrophin基因重组腺病毒的构建及转染mdx骨髓间充质干细胞的表达

构建含有人microdystrophin基因的重组腺病毒,来感染dystrophin基因敲除小鼠mdx的骨髓间充质干细胞(ＭＳＣ)进行基因修饰,为同种异体基因修饰的干细胞移植治疗ＤＭＤ疾病奠定基础。用NotⅠ酶切含microdystrophin基因的pBSK-MICRO质粒,获得microdystrophin基因。片段回收后定向插入腺病毒穿梭质粒pShuttle-CMV,获得重组质粒pShuttle-CMV-MICRO。PmeⅠ线性化重组质粒pShuttle-CMV-MICRO,去磷酸化后回收后与腺病毒骨架质粒pAdeasy-1共电转化BJ5183感受态细胞。同源重组后用选择性培养基筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察293细胞病变及PCR扩增目的基因等方法鉴定重组的腺病毒。然后将病毒上清转染DMD模型鼠mdx小鼠的骨髓间充质干细胞,通过RT-PCR以及间接免疫荧光检测microdystrophin的转录及蛋白表达。成功构建了含有microdystrophin基因的重组腺病毒,病毒滴度为5.58×10¹²vp/mL。间接免疫荧光检测可见microdystrophin蛋白在mdx小鼠MSCs中高效表达。该重组腺病毒载体的构建及成功转染到mdx MSCs内表达为下一步用microdystrophin基因修饰的mdx MSCs进行同种异体移植治疗ＤＭＤ疾病奠定了基础。     

Microdystrophin基因重组腺病毒的构建及转染mdx骨髓间充质干细胞的表达

构建含有人microdystrophin基因的重组腺病毒,来感染dystrophin基因敲除小鼠mdx的骨髓间充质干细胞(ＭＳＣ)进行基因修饰,为同种异体基因修饰的干细胞移植治疗ＤＭＤ疾病奠定基础。用NotⅠ酶切含microdystrophin基因的pBSK-MICRO质粒,获得microdystrophin基因。片段回收后定向插入腺病毒穿梭质粒pShuttle-CMV,获得重组质粒pShuttle-CMV-MICRO。PmeⅠ线性化重组质粒pShuttle-CMV-MICRO,去磷酸化后回收后与腺病毒骨架质粒pAdeasy-1共电转化BJ5183感受态细胞。同源重组后用选择性培养基筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察293细胞病变及PCR扩增目的基因等方法鉴定重组的腺病毒。然后将病毒上清转染DMD模型鼠mdx小鼠的骨髓间充质干细胞,通过RT-PCR以及间接免疫荧光检测microdystrophin的转录及蛋白表达。成功构建了含有microdystrophin基因的重组腺病毒,病毒滴度为5.58×10¹²vp/mL。间接免疫荧光检测可见microdystrophin蛋白在mdx小鼠MSCs中高效表达。该重组腺病毒载体的构建及成功转染到mdx MSCs内表达为下一步用microdystrophin基因修饰的mdx MSCs进行同种异体移植治疗ＤＭＤ疾病奠定了基础。     

用构建的新型BmNPV载体在家蚕高效表达人β-干扰素

构建了家蚕核多角体病毒(Bombyx mori nuclear polyhedrosis virus,BmNPV)新型载体pBm92,该载体将多角体蛋白基因的起始密码ATG改变为ATT,然后在多角体蛋白基因的+12位后连接有5个外源基因的克隆位点。将HulFN-β基因克隆在多角体蛋白基因的+12位后,构建了pBmIFN+12;同时构建了HuIFN－β克隆在－3位后的转移载体pB．mIFN－3。将两种转移载体DNA分别与BmNPV基因组DNA共转染Bm—N细胞。利用重组病毒不产生多角体蛋白的特征,筛选重组病毒。用HuIFN－β基因探针与重组病毒DNA进行杂交鉴定。重组病毒BmIFN+12感染Bm-N细胞,其上清IFN活性为2．0×10⁶Iu／ml,将BmIFN+12注射5龄家蚕虫体,表达水平为5．O×10⁷Iu／ml,是HulFN-β基因克隆在多角体蛋白基因的-3位后获得的重组病毒表达量的2—4倍。构建的新型BmNPv载体能够在家蚕高效地表达HuIFN-β。家蚕虫体生产的rHulFN-β蛋白具有天然HuIFN-β的抗原性。     

特异性eIF-4EsiRNA重组腺病毒构建及其对人卵巢癌细胞SKOV-3转移相关能力的影响

目的：构建表达真核细胞起始因子-4E（eukaryoticinitiationfactor4E,eIF-4E）特异性siRNA的重组腺病毒载体,观察其对人卵巢癌细胞SKOV-3体外转移能力的影响。方法：应用基因重组技术将eIF．4EsiRNA序列构建于腺病毒载体pLP－Ade－X,经包装细胞包装后得到高滴度重组腺病毒pLP—Ade-4EsiRNA（psiE）。将腺病毒psiE感染SKOV．3细胞,用定量PCR进行elf．4E基因表达检测,然后应用transwell小室法观察对细胞侵袭和运动能力的影响,同时检测感染后细胞内VEGF、MMP－2、MMP－9蛋白表达。结果：病毒检测结果与预期相符,real—timePCR可检测到感染重组腺病毒psiE后SKOV．3细胞没有eIF-4E基因表达;病毒感染后transwell小室法检测到SKOV-3细胞的侵袭和运动能力均受到显著的抑制（均为P〈0．01）;此外病毒感染的SKOV．3细胞中VEGF、MMP-2、MMP-9蛋白表达降低。结论：封闭eIF-4E基因表达对人卵巢癌细胞SKOV．3的侵袭和运动都有抑制作用,其作用机制可能与VEGF、MMP-2、MMP-9表达相关。     

腺病毒载体介导的重组蛋白sTNFRII-gAD快速表达和制备

利用腺病毒载体高效感染哺乳动物细胞及表达外源基因的特性,建立一种快速高效表达及制备重组蛋白的方法,并用该方法获得sTNFRII-gAD (可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白) 蛋白纯品。首先用携带EGFP基因的腺病毒载体rAd5-EGFP以不同的腺病毒用量 (MOI) (0~1 000) 感染BHK21c022细胞,观察比较其转导效率和细胞毒性。用AdMax腺病毒载体系统制备携带融合基因sTNFRII-gAD的重组复制缺陷型5型腺病毒rAd5-sTNFRII-gAD。用rAd5-sTNFRII-gAD以不同的MOI (0~1 000) 感染BHK21c022细胞,收取上清进行Western blotting分析,比较上清中sTNFRII-gAD蛋白的表达量。在此基础上,用rAd5-sTNFRII-gAD以MOI 100感染大量培养的BHK21c022细胞,在无血清培养条件下反复多次收取培养上清,经过硫酸铵浓缩、分子筛柱层析、透析等步骤浓缩和纯化sTNFRII-gAD融合蛋白,并体外测定该融合蛋白拮抗TNFa的活性。结果获得了携带sTNFRII-gAD融合基因的重组腺病毒rAd5-sTNFRII-gAD;用rAd5-EGFP感染BHK21c022细胞结果表明,随着腺病毒用量的增高,表达EGFP蛋白的BHK21c022细胞数量和亮度明显增加;MOI在0~100之间被感染的BHK21c022细胞未表现出明显的细胞毒性,MOI为1 000时可观察到细胞变圆和少量死亡现象。Western blotting分析结果表明,随着腺病毒用量的增高,培养上清中sTNFRII-gAD融合蛋白表达量明显增加,以MOI为1 000时最高。在此基础上,我们用MOI为100的rAd5-sTNFRII-gAD感染5个转瓶培养的BHK21c022细胞以制备sTNFRII-gAD融合蛋白。每个转瓶加无血清培养液100 mL,每48 h收获上清1次并换液,反复6次收取培养上清共约3 L,经过纯化获得了约11 mg的sTNFRII-gAD融合蛋白。体外活性测定实验表明,获得的sTNFRII-gAD融合蛋白能有效拮抗TNFα对L929细胞的杀伤作用。腺病毒载体/BHK21细胞表达系统是一个简便、高效、通用的表达系统,利用该系统成功制备了有生物学活性的sTNFRII-gAD融合蛋白。该表达系统的特点是生产规模易于放大,适应无血清培养,可以反复多次收获目标蛋白。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Physiological characteristics of various species of strains of carboxydobacteria

Seven strains of aerobic carbon monoxide-oxidizing bacteria (carboxydebacteria) when growing on CO as sole source of carbon and energy had doubling times which ranged from 12–42 h. The activity profiles obtained after discontinuous sucrose density gradient centrifugation indicated that the CO-oxidizing enzymes are soluble and the hydrogenases are membrane-bound in all strains examined. The CO-oxidizing enzymes of Pseudomonas carboxydohydrogena, Pseudomonas carboxydoflava, Comamonas compransoris, and the so far unidentified strains OM2, OM3, and OM4 had a molecular weight of 230,000; that of Achromobacter carboxydus amounted to 170,000. The molecular weights of the CO-oxidizing and H₂-oxidizing enzymes turned out to be identical. The cell sonicates were shown to catalyze the oxidation of both CO and H₂ with methylene blue, thionine, phenazine methosulfate, toluylene blue, dichlorophenolindophenol, cytochrome c or ferricyanide as electron acceptors. Methyl viologen, benzyl viologen, FAD⁺, FMN⁺, and NAD(P)⁺ were not reduced. The spectrum of electron acceptors was identical for all strains tested. Neither free formate, hydrogen nor oxygen gas were involved in the CO-oxidation reaction. Methylene blue was reduced by CO at a 1:1 molar ratio. The results indicate that CO-oxidation by carboxydobacteria is catalyzed by identical or similar enzymes and that the reaction obeys the equation CO+H₂OCO₂+2H⁺+2e^- as previously shown for Pseudomonas carboxydovorans.Dedicated to Otto Kandler remembering almost three decades of enjoyable cooperation     

Modulation of allostery of pyruvate kinase by shifting of an ensemble of microstates

Since the introduction of the concepts of allostery about four decades ago, much advancement has been made in elucidating the structure-function correlation in allostery. However, there are still a number of issues that remain unresolved. In this review we used mammalian pyruvate kinase (PK) as a model system to understand the role of protein dynamics in modulating cooperativity. PK has a triosephosphate isomerase (TIM)(α/β)₈ barrel structural motif. PK is an ideal system to address basic questions regarding regulatory mechanisms about this common (α/β)₈ structural motif. The simplest model accounting for all of the solution thermodynamic and kinetic data on ligand-enzyme interactions involves two conformational states, inactive E^T and active E^R. These conformational states are represented by domain movements. Further studies provide the first evidence for a differential effect of ligand binding on the dynamics of the structural elements, not major secondary structural changes. These data are consistent with our model that allosteric regulation of PK is the consequence of perturbation of the distribution of an ensemble of states in which the inactive E^T and active E^R represent the two extreme end states. Sequence differences and ligands can modulate the distribution of states leading to alterations of functions. The future work includes: defining the network of functionally connected residues; elucidating the chemical principles governing the sequence differences which affect functions; and probing the nature of mutations on the stability of the secondary structural elements, which in turn modulate allostery.