小鼠RNA干扰RelA基因慢病毒载体的构建与鉴定

目的:构建小鼠Rel A基因的RNA干扰慢病毒载体,转染小鼠成骨样细胞并鉴定。方法:针对小鼠Rel A基因序列,设计特异性的sh RNA序列,应用基因重组技术插入慢病毒载体GV-248。得到的重组质粒转化感受态大肠杆菌DH5α,筛选得到阳性克隆并扩大培养。所得质粒进行测序分析确定载体构建成功。重组质粒载体及包装辅助质粒转染293T细胞,得到目的病毒并测定相应病毒滴度。慢病毒转染MC3T3-E1细胞后,Real-time PCR及Western blot检测MC3T3-E1细胞Rel A基因及成骨相关基因ALP、OCN、RANKL的表达。结果:成功构建小鼠Rel A基因的RNA干扰慢病毒载体,感染MC3T3-E1细胞后,Rel A基因的表达明显受到抑制,同时RANKL基因表达水平明显下降,ALP、OCN基因表达水平明显上升。结论:成功构建了小鼠Rel A基因的RNA干扰慢病毒载体。当小鼠成骨细胞Rel A基因表达被干扰,NF-κB通路被抑制后,小鼠成骨细胞成骨相关基因ALP、OCN的表达明显上升,成骨功能增强;同时RANKL的表达明显下降,其介导的破骨细胞骨吸收功能减弱。     

Noggin基因沉默表达载体的构建及效果评价

目的构建慢病毒介导的Noggin RNAi干扰序列,并分析这些干扰序列对Noggin基因的沉默效果。方法针对目的基因Noggin的m RNA设计四条干扰序列,并将这些序列连接到Lenti-KD慢病毒载体,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒。将重组病毒感染MC3T3-E1细胞,利用puromycin进行筛选,获得稳定表达细胞系。通过实时荧光定量PCR和Western blot技术分析不同干扰序列的干扰效果。结果实时荧光定量PCR结果显示,四种干扰序列对Noggin基因的表达都有一定的沉默效果,但只有sh Noggin-1(P0.01)对其表达影响显著。Western blot结果显示,四种干扰序列中只有sh Noggin-1(P0.01)对Noggin的表达蛋白具有显著的降低作用。结论获得了一种Noggin基因的干扰序列,该序列能够干扰Noggin基因m RNA的稳定性,从而影响蛋白的表达。该干扰序列可以用于部分敲除Noggin基因,从而用于研究Noggin基因的功能。     

骨样细胞MLO-Y4与成骨样细胞MC3T3-E1生物学特性的比较

分别采用倒置显微镜观察法、细胞计数法、RT-PCR法、磷酸对硝基苯酚法(PNPP法)和ELISA法来比较小鼠骨样细胞MLO-Y4与小鼠成骨样细胞MC3T3-E1的细胞形态、增殖、相关基因的表达和分泌功能的差异。结果显示MC3T3-E1细胞呈长梭形,具有少量短的突触;而MLO-Y4细胞呈星状或树枝状且具有很多长的突触。MC3T3-E1细胞的增殖能力强于MLO-Y4细胞,两者的倍增时间分别是18 h和20 h。MC3T3-E1细胞中原癌基因c-fos和骨桥蛋白基因OPNmRNA的表达明显高于MLO-Y4细胞,而骨钙素基因OCmRNA的表达则是MC3T3-E1细胞远低于MLO-Y4细胞,白细胞分化抗原44基因CD44 mRNA在两种细胞中的表达差异不明显。ALP的分泌在MC3T3-E1细胞中高于MLO-Y4细胞,NO的分泌在两种细胞中没有显著性差异,M-CSF在MLO-Y4细胞中的分泌较高。由此可见骨样细胞MLO-Y4与成骨样细胞MC3T3-E1在形态、ALP和MCSF分泌及c-fos、OPN和OCmRNA表达方面差异明显。     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的:构建并鉴定YAP基因短发夹干扰RNA(shRNA)慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法:荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果:在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论:成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

慢病毒siRNA靶向干扰YAP基因胃癌细胞株的建立

目的：构建并鉴定YAP基因短发夹干扰RNA（shRNA）慢病毒载体,建立稳定干扰YAP基因表达的胃癌细胞株SGC7901。方法：荧光定量PCR检测YAP基因在多种胃癌细胞株中的表达情况。构建重组靶向YAP基因的shRNA慢病毒表达质粒PGC-shRNA-YAP,用脂质体转染的方法将载体导入胃癌细胞。经杀稻瘟菌素筛选后,建立稳定表达siRNA的细胞株。荧光定量PCR检测干扰效率。结果：在胃癌细胞株SGC7901中,YAP基因显示高表达。测序验证PGC-shRNA-YAP重组质粒构建成功。将重组质粒稳定转染入胃癌细胞株SGC7901后能明显抑制YAPmRNA表达水平。结论：成功构建了PGC-shRNA-YAP慢病毒重组质粒,建立了靶向稳定干扰YAP基因表达的siRNA胃癌细胞株SGC7901。     

乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒的构建及鉴定

目的：构建乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。方法：乙酰肝素酶cDNA全长扩增和最佳siRNA干扰片段筛选分别采用PCR和Real-time PCR方法,慢病毒系统载体分别使用pWPI和siRNA pSico系统,采用限制性内切酶快速连接方法联接目的基因和最佳最佳siRNA干扰片段,表达载体鉴定均采用核苷酸序列测定,HPSE重组慢病毒表达质粒和siRNA片段细胞转染采用脂质体转染法。结果：成功扩增乙酰肝素酶全长并连接入pWPI载体构建成重组表达载体HPSE-pWPI,重组质粒测序结果显与HPSE基因的同源性达99%。转染293T后有HPSE基因的表达。筛选出最佳siRNA干扰片段为HPSE-1222并成功插入pSico载体,构建成重组表达载体HPSE-siRNA pSico,重组载体测序显示与构建的shRNA结构序列完全一致。结论：成功采用慢病毒载体系统构建了乙酰肝素酶重组慢病毒转基因和siRNA干扰质粒,为探讨HPSE在在肿瘤浸润转移过程中的分子机理奠定基础。     

EGFL7 基因RNAi慢病毒表达载体的构建及鉴定

目的：构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）重组慢病毒表达载体。方法：参照小分子干扰RNA 的设 计原则,应用OligoDesigner 3.0 软件设计三条靶向人EGFL7 基因的RNA干扰序列（hEGFL7-RNAi）,并将其分别插入含有绿色 荧光蛋白（GFP）的慢病毒载体pLV3 中,获得重组质粒,与包装质粒pRsv-REV、pMDlg-pRRE 和pMD2G共同转染293T细胞,包 装产生重组慢病毒,培养72 h后,应用qRT-PCR 检测慢病毒感染人脐静脉内皮细胞（HUVEC）后靶基因mRNA 的水平,以评价 其基因沉默效果。结果：筛选出3 条人EGFL7 基因的RNAi 序列,分别包装出重组慢病毒,其滴度分别为1× 108、2× 108和5× 108TU/mL,将其转染入HUVEC 后,EGFL7mRNA表达均受到明显抑制(P<0.05)。结论：成功筛选出三条针对人EGFL7基因的 RNAi有效靶序列,并成功构建重组慢病毒表达载体,证明该序列可沉默HUVECs 中EGFL7 基因的表达。     

NGF基因重组慢病毒载体的构建及其在人脐带血间充质干细胞的表达

目的:构建神经生长因子(NGF)的慢病毒表达载体,并观察其转染人脐带间充质干细胞后的表达情况。方法:采用实时定量PCR(RT-PCR)方法获取NGF基因编码片段,并将构建的慢病毒载体质粒与包装质粒和包膜质粒共转染293T细胞,包装生产慢病毒。应用相同滴度的慢病毒转导等量间充质干细胞(MSCs),观察转染后细胞的生长形态及生长曲线,再采用RT-PCR、Western Blot方法检测NGF m RNA、蛋白质的表达水平。结果:经PCR、酶切和测序结果证明成功构建NGF基因重组慢病毒载体。同时NGF基因重组慢病毒载体能够成功转染人脐带间充质干细胞,转染率达95.35%,转染后干细胞在NGF m RNA及蛋白质的表达方面较对照组明显升高,同时经倒置显微镜观察及生长曲线实验证实转染后干细胞的生长与对照组相比无明显差异。结论:重组NGF的慢病毒表达载体能够高效的转染人脐带间充质干细胞,基因转染后干细胞的增殖分化能力与未转染细胞差异无统计学意义,可作为一种高效的干细胞转染方法。     

EGFL7基因RNAi慢病毒表达载体的构建及鉴定（英文）

目的:构建人类表皮生长因子域7(EGFL7)基因RNA干扰(RNAi)重组慢病毒表达载体。方法:参照小分子干扰RNA的设计原则,应用Oligo Designer 3.0软件设计三条靶向人EGFL7基因的RNA干扰序列(h EGFL7-RNAi),并将其分别插入含有绿色荧光蛋白(GFP)的慢病毒载体pLV3中,获得重组质粒,与包装质粒pRsv-REV、pMDlg-pRRE和pMD2G共同转染293T细胞,包装产生重组慢病毒,培养72 h后,应用qRT-PCR检测慢病毒感染人脐静脉内皮细胞(HUVEC)后靶基因mRNA的水平,以评价其基因沉默效果。结果:筛选出3条人EGFL7基因的RNAi序列,分别包装出重组慢病毒,其滴度分别为1×10~8、2×10~8和5×10~8TU/mL,将其转染入HUVEC后,EGFL7mRNA表达均受到明显抑制(P0.05)。结论:成功筛选出三条针对人EGFL7基因的RNAi有效靶序列,并成功构建重组慢病毒表达载体,证明该序列可沉默HUVECs中EGFL7基因的表达。     

小鼠resistin基因慢病毒表达载体构建及在KMB17细胞中的表达

[目的]构建携带小鼠resistin基因的慢病毒表达载体,并高效转导靶细胞。[方法]从小鼠脂肪组织提取总RNA,经PCR扩增、酶切、连接、转化后构建重组质粒;通过293T细胞包装为重组慢病毒颗粒,然后感染KMB17细胞,检测小鼠resistin基因在靶细胞中的表达。[结果]PCR扩增到预期的342 bp大小的resistin基因目的片段;构建的重组质粒经酶切得到了342 bp大小的目的片段,测序鉴定为小鼠resistin基因序列;重组慢病毒包装可观察到很强的绿色荧光,说明具有较高的质粒转染效率;重组慢病毒感染KMB17后可检测到小鼠resistin基因转录水平和蛋白水平的表达,证明resistin基因得到有效转导并可在靶细胞中高水平表达。[结论]成功构建了携带小鼠resistin基因的慢病毒表达载体并可在KMB17靶细胞中获得高效表达。     

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.     

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。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

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.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

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.