人VEGF165基因转染成人成肌细胞的实验研究

目的：将人血管内皮生长因子165（hVEGF165）导人原代离体成肌细胞,观察该细胞hVEGF分泌情况,探讨成人自体转基因成肌细胞移植的可行性。方法：采用两步消化法对成人骨骼肌组织消化获取相对较纯的成肌细胞,通过差速贴壁法进行进一步的纯化。以脂质体转染法将pcDNA3．1-hVEGF165导入成肌细胞,通过RT—PCR、ELISA和Western-blot进行hVEGF165定量检测,MTT测定和Mile’s实验检测VEGF165的生物学活性。结果：转基因细胞经RT—PCR扩增出一条VEGF的特异性泳带,ELISA显示转基因细胞培养上清VEGF浓度分别达到18．92±1．77rig／mL、19．04±2．15ng／mL,Western blot检测转基因成肌细胞上清均检测到VEGF蛋白特异性的杂交带,MTT显示转基因细胞上清明显促内皮细胞增殖,Mile’s实验显示转基因细胞上清明显增加毛细血管通透性。结论：质粒pcDNA3．1-hVEGF165能成功转入成人成肌细胞,转基因细胞能分泌有生物活性的VEGF165蛋白。     

TGF-β1基因转染大鼠脂肪干细胞向软骨细胞分化的实验研究

目的TGF-β1基因转染大鼠的ADSCs(adipose stromal cells,ADSCs),诱导其向软骨细胞分化,为软骨组织工程学种子细胞提供新方法。方法分离、培养大鼠的ADSCs,免疫荧光法进行鉴定;TGF-β1基因转染ADSCs,对转染后细胞进行筛选;MTT法测定筛选后细胞的增殖活性;RT-PCR、Western blot对筛选后细胞的表达进行检测。结果成功分离、培养大鼠的ADSCs;细胞表面标志物CD29和CD44表达阳性,CD106和CD34表达阴性;基因转染后细胞的增殖力变强;转染TGF-β1的ADSCs在目的基因TGF-β1、SOX9、Aggrecan、Collagen II mRNA的表达和软骨特异性基质-Collagen II的分泌增强,明显高于对照组和转染空载体组;结果TGF-β1基因转染后ADSCs具有了软骨细胞的表型特征,可以用作软骨组织工程的种子细胞。     

VEGF重组质粒pcDNA/V的构建及其在大鼠急性心肌缺血模型中的表达

构建人血管内皮细胞生长因子(VEGF165)真核表达载体,并研究其在细胞水平和大鼠急性心肌梗死动物模型中的表达。利用RT-PCR方法,从人扁桃体组织中扩增人VEGF165基因,构建真核表达载体pcDNA/V。应用脂质体介导的基因转移技术,将pcDNA/V转染至人胚肾细胞(293细胞)中,经G418筛选获得稳定表达的重组质粒细胞克隆。ELISA、Westernblot检测证实重组质粒pcDNA/V能在293细胞中高效表达外源VEGF基因,鸡胚绒毛尿囊膜血管生成实验证实表达产物具有促血管生成的活性。进一步的体内表达研究,建立大鼠急性心肌梗死模型,将重组质粒pcDNA/V、空质粒pcDNA3.1( )分三点注射于梗死交界处心肌内,四周后取材。经免疫组化染色检测,pcDNA/V组在梗死交界区有VEGF阳性表达;电镜观察显示,pcDNA/V组在梗死交界处心肌细胞间有大量毛细血管内皮细胞增生。实验结果表明成功克隆了人VEGF165基因,构建了其真核表达载体。体内、外表达研究证实重组质粒的表达产物具有促血管生成的生物学活性,为VEGF基因治疗缺血性心肌病的研究提供实验基础。     

人骨髓间充质干细胞向血管内皮细胞诱导分化的研究

目的:分析人骨髓间充质干细胞(hMSCs)和脐静脉内皮细胞(hUVECs)的基因表达差异,探讨体外基因转染诱导内皮分化的可行性以及作为血管组织工程种子细胞来源的应用前景。方法:分别从人骨髓和脐静脉分离间充质干细胞(hMSCs)和内皮细胞(hUVECs),扩增培养后进行流式细胞仪、免疫细胞化学,免疫荧光鉴定和超微结构观察。通过BiostarH-40S表达谱芯片分析,选择两者的差异表达基因,导入hMSCs,经RT-PCR、ELISA鉴定该基因的转染和表达,并分析hMSCs的内皮分化程度。结果:hMSCs表达内皮细胞的多种特异性mRNA,经VEGFl65基因瞬时转染后RT-PCR有明显条带,ELISA定量检测VEGF165蛋白表达为(707.9±11.3)ng/L,同时CD44表达明显下调38.80%,CD31则明显上调达56.82%,FI-1,FVⅢAg和CD34的表达也有不同程度升高。结论:hMSCs具有内皮分化潜能,体外基因转染诱导hMSCs产生功能性内皮细胞和组织工程化血管具有广阔前景。     

PEG-PEI共聚物介导VEGF165基因转染及对内皮细胞生长的影响

为了考察PEG-PEI共聚物作为基因载体介导VEGF165基因的能力,合成不同接枝量的PEG-PEI共聚物,考察共聚物的细胞毒性,同时采用PCR技术获得上下游含有HindⅢ和BamHⅠ酶切位点的目的基因VEGF165,与pEGFP-C1构建重组质粒pEGFP-VEGF165,将PEG-PEI作为基因载体,与pEGFP-VEGF165通过自组装成DNA复合物,使其转染脐静脉内皮细胞(HUVEc),测定发荧光细胞百分数获得转染率,利用ELISA、RT-PCR检测VEGF的表达,用MTT法考察VEGF165转染HUVEc后对内皮细胞生长的影响.结果显示,形成PEG-PEI共聚物后可显著降低PEI的细胞毒性.作为基因载体介导pEGFP-VEGF165转染HUVEc后,在荧光显微镜下可见强绿色荧光蛋白表达,转染率与接枝PEG的量及N/P有关,PEG-PEI(5-25-1)在N/P=30时转染率达到最大值,比PEI显著提高.转染后血管内皮生长因子(VEGF)蛋白表达及mRNA水平均有显著提高,且可有效地刺激内皮细胞增殖.研究表明,PEG-PEI共聚物可做为基因载体,有效地介导pEGFP-VEGF165基因的传递.     

TGF-β1基因真核表达载体的构建及在BMSCs中的表达

目的研究真核表达载体pCDNA3.1( )-TGF-β1在骨髓间充质干细胞(BMSCs)中的表达.方法基因克隆构建pCDNA3.1( )-TGF-β1真核表达载体,转染大鼠BMSCs,G418筛选获得稳定转染的细胞,RT-PCR、ELISA和免疫细胞化学检测其表达,MTT法检测其增殖活性.结果成功构建含TGF-β1基因的真核表达载体;RT-PCR、ELISA、免疫细胞化学证实了TGF-β1基因在BMSCs中至少可以表达一个月;MTT法提示转染TGF-β1基因可以促进BMSCs增殖.结论 pCDNA3.1( )-TGF-β1转染BMSCs可获得稳定表达.     

反义CD151基因转染对大鼠血管平滑肌细胞迁移的影响

目的观察pcDNA3.1真核表达载体介导的反义CD151基因转染对培养的大鼠动脉平滑肌细胞(VSMCs)迁移的影响。方法构建携带全长正义和反义CD151的真核表达载体pcDNA3.1-CD151和pcDNA3.1-anti-CD151重组质粒,转染体外培养的VSMCs,以RT-PCR和Western blot方法检测CD151的表达,用Boyden趋化小室方法观察细胞迁移。结果与载体对照组、脂质体对照组和空白对照组3组均值比较,转染48h后,反义CD151组mRNA表达降低58%,蛋白表达降低51%,正义CD151组的VSMCs CD151mRNA表达增加171%,蛋白表达增加133%;趋化迁移的细胞数,反义CD151组为37.9±6.3,正义CD151组为86.5±12.4;载体对照组、脂质体对照组和空白对照组分别为60.3±7.1、61.8±7.6和67.3±9.6。反义CD151组显著低于其余各组(P<0.01),正义CD151组显著高于其余各组(P<0.01)。结论pcDNA3.1真核表达载体介导的反义CD151转染,通过抑制CD151的表达,能够显著抑制大鼠VSMCs的迁移。     

高表达CD151细胞系的构建

目的构建高表达CD151细胞系Tca8113-CD151,为研究CD151在肿瘤细胞迁移及肿瘤转移中的作用和相关机制提供实验模型.方法构建pcDNA3.1-CD151-HA重组体,采用磷酸钙共沉淀法转染Tca8113细胞,经G418筛选抗性克隆.通过RT-PCR和Western blot分别检测这些克隆CD151基因的转录和翻译.结果成功构建真核表达重组体pcDNA3.1-CD151-HA;Tca8113-CD151细胞系在mRNA和蛋白质水平高表达外源CD151.结论构建了稳定高表达CD151的细胞系Tca8113-CD151,为探讨CD151基因在肿瘤转移中的作用及其相关机制奠定了实验基础.     

脂质体介导VEGF基因对成骨细胞增殖、合成骨钙素以及细胞周期的相关研究

目的:研究脂质体介导血管内皮生长因子(VEGF)基因对成骨细胞增殖、合成骨钙素以及细胞周期的影响。方法:通过脂质体介导的基因转染方法,将携带外源性VEGF重组pcDNA3-hVEGF质粒导入体外培养的成骨细胞,酶联免疫吸附测定法(ELISA)检测转染后细胞中VEGF浓度变化,以判断转染效果;采用细胞计数法检测转染重组质粒的成骨细胞的增殖活性;流式细胞术检测转染重组质粒的成骨细胞周期的变化;ELISA检测转染重组质粒的成骨细胞骨钙素浓度变化。结果:与对照组相比,转染组成骨细胞中VEGF的浓度显著增加,与对照组间差异具有统计学意义(P0.05);转染重组质粒的成骨细胞的增殖能力较对照组显著增强,差异具有统计学意义(P0.05),与对照组相比,转染重组质粒的成骨细胞周期(G2/M+S)%明显增加,差异具有统计学意义(P0.05);转染重组质粒的成骨细胞合成的骨钙素浓度较对照组显著升高,差异具有统计学意义(P0.05)。结论:脂质体介导成骨细胞增加血管内皮生长因子的水平,可促进成骨细胞增殖,增加成骨细胞骨钙素的浓度,从而提高成骨细胞的功能。     

hAng-1真核表达载体的构建及其在MSCs中的表达

目的:通过基因重组技术,构建人血管生成素-1(human angiopoietin 1,hAng-1)真核表达载体体系,并将其转染至大鼠的骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)内进行培养,进而验证hAng-1的表达.方法:将hAng-1编码序列(互补脱氧核糖核酸)通过酶切,插入至pcDNA3.1(+)质粒的多克隆位点,构建质粒pcDNA 3.1 (+)/hAng-1真核表达质粒;重组质粒经脂质体介导转染鼠MSCs.应用逆转录聚合酶联反应(RT-PCR)、蛋白免疫印迹法(Western blot)等方法检测hAng-1的表达情况.结果:pcDNA 3.1 (+)/hAng-1真核表达质粒转染鼠MSCs后,应用流式细胞仪检测,转染效率约为15％.同时应用RT-PCR能够检测出目的基因mRNA,Western blot能够检出hAng-1的蛋白表达.结论:本实验通过基因重组技术,构建的pcDNA3.1 (+)/hAng-1真核表达载体能够在转染的鼠MSCs中表达,且表达较为持续,为hAng-1基因应用于基因治疗的研究奠定了基础.     

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.     

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.     

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.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.