pCDNA3.1-HEGF真核表达质粒转染牙髓干细胞的研究

目的将人表皮生长因子基因转染入牙髓干细胞,并检测其蛋白的表达,为下一步的牙髓干细胞的增殖和分化取得前期的实验基础。方法利用前期实验构建的pcDNA3.1-HEGF,经脂质体转染质粒pcDNA3.1-HEGF入牙髓干细胞,转基因细胞培养48h后作Q-PCR和Western-blot分析。结果成功把pCDNA3.1-HEGF真核表达质粒转染到牙髓干细胞,而且通过检测其表达增强,转hEGF基因细胞经Q-PCR检测,与对照组相比,该基因上调表达了6-21倍,扩增反应产物溶解温度较均一,目的基因具有很好的特异性,反应体系良好,Western-blot检测到HEGF表达明显升高。结论质粒pcDNA3.1-HEGF在脂质体介导下成功转染牙髓干细胞,而且表达增强。     

表达血管内皮生长因子-siRNA及双自杀基因yCDglyTK的联合基因载体构建及其应用研究

构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-si...     

病毒负性调节因子在内皮细胞稳定表达细胞株的建立

建立HIV-1的调节基因Nef基因在内皮细胞稳定表达的细胞株ECV304-Nef,为研究Nef对血管内皮细胞生物学活性的影响奠定试验基础。构建真核表达载体pcDNA3.1(+)-Nef,将其质粒和pcDNA3.1(+)质粒(阴性对照)分别转染血管内皮细胞ECV304,G418筛选。通过RT-PCR检测NefmRNA在细胞中的表达;细胞免疫荧光法检测Nef蛋白的表达及定位;Western blotting检测Nef蛋白的特异性表达,获得稳定表达的细胞株。构建的重组质粒pcDNA3.1(+)-Nef经BamHI和EcoRI双酶切鉴定,得到的片段大小与理论值相符,分别为载体的5400bp和目的基因的621bp。测序结果显示碱基序列与GenBank(登录号:K03455)序列相同。转染细胞经G418筛选后获得稳定表达Nef的ECV304细胞株,RT-PCR显示转染pcD-NA3.1(+)-Nef质粒的ECV304细胞出现621bp条带,对照组无目的条带出现;荧光显微镜下观察转染pcDNA3.1(+)-Nef质粒的ECV304细胞表达的Nef蛋白主要定位于细胞质中。Western blotting结果显示,转染pcDNA3.1(+)-Nef质粒的ECV304细胞约27kD处检测到目的条带,表明pcDNA3.1(+)-Nef表达正确。     

SREBP-1基因过表达促进人肾小管上皮细胞甘油三酯形成

目的 SREBP-1重组质粒转染人肾小管上皮细胞(HKC)检测SREBP-1基因表达和细胞内脂滴的关系。方法体外培养人肾小管上皮细胞并随机分为空白对照组、pcDNA3.1空质粒对照组和pcDNA3.1-SC1重组质粒转染组,采用阳离子脂质体法将SREBP-1特异性质粒pcDNA3.1-SC1及pcDNA3.1空质粒转染到细胞内并培养48小时,半定量RT-PCR和Westernblot分析目标基因表达丰度的变化,并采用油红O染色检测细胞内脂滴。结果 pcDNA3.1-SC1重组质粒转染的细胞内SREBP-1mRNA表达呈现明显升高,扩增条带积分光密度值分别是空白对照组和阴性对照组的6.158倍和4.194倍,SREBP-1蛋白也出现明显上调,条带积分光密度值为3.092±0.254。空白对照组和pcDNA3.1阴性对照组细胞内均未见有红染脂滴颗粒,而pcDNA3.1-SC1重组质粒转染组中出现了清晰的红染颗粒。结论 SREBP-1表达可增加人肾小管上皮细胞脂肪合成证实HKC细胞中SREBP-1表达和脂滴形成之间存在有直接关系。     

瞬时外向钾通道Kv4.3真核表达载体的构建

[目的]构建Kv4.3真核表达载体,观察其在真核细胞中表达情况。[方法]提取小鼠前额叶皮质RNA,通过RT-PCR和常规PCR技术扩增获得Kv4.3目的基因,利用EcoRⅠ和XbaⅠ限制性内切酶和T4 DNA连接酶将其克隆在pcDNA3.1载体中,并将构建好的重组质粒转染至HEK293细胞中,免疫印迹方法检测Kv4.3蛋白表达。[结果]成功扩增了Kv4.3基因(2 000 bp处有明显条带),先后经酶切和酶连后的重组体转化培养,提取质粒,再通过双酶切观察到2 000 bp处有Kv4.3目的条带和5 400 bp处有pcDNA3.1载体条带。将此重组质粒送去测序,结果显示基因测序结果与GenBank中Kv4.3序列一致。免疫印迹结果发现在HEK293细胞中,转染的重组质粒能够表达Kv4.3蛋白。[结论]成功构建了Kv4.3真核表达载体,其在HEK293细胞中可以稳定表达。     

Survivin-2B增强甲氨蝶呤抑制BEL-7402

目的:研究高表达Survivin-2B对化疗药物甲氨蝶呤抑制肝肿瘤细胞株BEL-7402作用的影响.方法:构建真核表达重组质粒Survivin-2B/pcDNA3.1(-),利用增强型绿色荧光蛋白pEGFP-C1真核质粒测定脂质体转染条件及效率,采用该条件瞬时转染Survivin-2B/pcDNA3.1(-)、Survivin/pcDNA3.1(-)以及pcDNA3.1(-)阴性对照质粒至肝肿瘤细胞株Bel-7402,MTT法检测100mg/ml甲氨蝶呤作用时,各组细胞抑制情况的差异.结果:成功构建Survivin-2B/pcDNA3.1(-)重组质粒,脂质体转染48h转染效率为62％,甲氨蝶呤对高表达Survivin-2B的Bel-7402细胞抑制率最高.结论:Survivin-2B与甲氨蝶呤同时作用可以提高对Bel-7402细胞的抑制率,为肿瘤治疗提供了新思路.     

怀化医学高等专科学校医学形态学实验中心

目的:探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法:将p16 cDNA亚克隆至pcDNA3.1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721。用MTT法和Western blot分析转染细胞的生长情况。结果:成功构建重组表达质粒pcDNA3.1-p16,转染pcDNA3.1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论:重组pcDNA3.1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

汉滩病毒S基因及其5'端真核表达载体的构建及在细胞中的表达

体外研究汉滩病毒(HTNV)S基因及其5'端表达的意义,为核蛋白T细胞表位的研究奠定基础.设计2套引物,用PCR方法从PBV220-S22原核质粒中扩增出S基因全读码框(37-1326bp)及S基因5'端(37-501bp),用TA克隆将其克隆入pcDNA3.1/V5-His-TOPO载体中,成功构建pcDNA3.1-S及pcDNA3.1-S-N真核表达载体,并通过脂质体转染至Vero细胞中,进行了瞬时表达.间接免疫荧光成功检测到pcDNA3.1-S及pcDNA3.1-S-N在Vero细胞中的表达.pcDNA3.1-S及pcDNA3.1-S-N真核表达载体有较高的转染效率,目的基因能在宿主细胞中表达,有利于研究HTNV-S基因在T细胞表位研究中的意义.     

Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达

目的:构建同源异性框基因Rhox5的真核表达质粒,转染NIH3T3细胞,建立稳定过表达Rhox5的细胞系。方法:PCR方法扩增Rhox5的全长cDNA序列,PCR产物双酶切后和人工合成的HA抗原表位标签共同克隆至pcDNA3.1(-)哺乳动物细胞表达载体中,构建pcDNA-Rhox5-HA融合表达质粒。脂质体法将经过测序成功的pcDNA-Rhox5-HA融合质粒和pcDNA3.1空载体分别转染NIH3T3细胞,潮霉素B筛选后建立阴性对照pcDNA3.1 in NIH3T3和稳定过表达Rhox5的Rhox5-HA in NIH3T3细胞系。RT-PCR和western blotting方法检测Rhox5-HA在稳定转染细胞系中的表达情况。结果:成功构建了pcDNA-Rhox5-Myc重组质粒,获得稳定过表达Rhox5的NIH3T3细胞系。RT-PCR和Western blotting结果表明,构建的稳定细胞系中成功表达Rhox5-HA融合蛋白。结论:Rhox5基因真核表达质粒的构建及其在NIH3T3细胞中的稳定表达为进一步体外研究Rhox5蛋白单独的功能及其与其他分子间功能性相互作用奠定了实验基础。     

pcDNA3.1-Canstatin-3Flag真核表达载体的构建及转染HepG2细胞中的表达

目的:构建pcDNA3.1-Canstatin-3Flag载体并稳定转染肝癌HepG2细胞,检测canstatin在mRNA水平的表达。方法:胎盘中提取总RNA,RT-PCR法获得canstatinDNA,克隆至pcDNA3.1(-)载体中,并测序,重组质粒pcDNA3.1-Canstatin-3Flag转染肝癌HepG2细胞,G418筛选出稳定转染细胞,RT-PCR检测canstatin mRNA表达。结果:1.成功构建出pcDNA3.1-Canstatin-3Flag重组质粒;2.获得稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞;3.发现转染后的肝癌HepG2细胞canstatin在mRNA水平比未转染细胞有明显的增强。结论:获得了稳定转染pcDNA3.1-Canstatin-3Flag的肝癌HepG2细胞,为后期canstatin在肝癌中的研究提供了支持。     

Epidermal Tissue Homeostasis

Abstract. Toad epidermis is a suitable model for studies on tissue homeostasis because cell pool size, influx into and efflux from the cell pool can be easily determined. the cell pool size was obtained by cell counting on photomicrographs, the influx (cell birth rate) was assessed by the metaphase-arrest technique, and the efflux (cell loss by moulting) assessed by counting the number of cells in the corneal layer and recording of intermoult periods. the importance of the methods for assessing these parameters per square unit of skin surface is emphasized.
These parameters were studied in eight groups of ten adult male toads sacrificed at various hours of the day. There were minor variations in the cell birth rate, fluctuating around a mean of 26 cells/mm²/hr (obtained at the metaphase collection period from 11.00-14.00 hours). By summation of the cell productions during the eight metaphase collection periods of 3 hr, and extrapolation to an intermoult period (time between two moults), a calculated cell production of about 6340 cells/mm² in 10.3 days was obtained, whereas the cell loss at each moult was only 2370 cells/mm². Thus the cell production rate exceeds the rate of cell loss through moults by a factor of 2.7. Arguments are presented that the 'surplus' of cells produced cannot be permanently accommodated within the living epidermis. Consequently a cell deletion rate beyond that by moulting of about 4000 cells/mm² in 10.3 days or 16 cells/mm²/hr can be calculated.
These results are discussed in relation to current concepts of tissue homeostatic mechanism(s). the results are consistent with the hypothesis that controlled cell deletion may be a tissue homeostatic mechanism complementary to controlled cell divisions.     

Epidermal arachidonate lipoxygenase

Guinea pig skin was found to display a high lipoxygenase activity, evidenced by the formation of a hydroxyeicosatetraenoic acid (HETE) from exogenous [14C]arachidonic acid. The lipoxygenase activity was localized to the epidermal layer of the skin, was completely inhibited by eicosatetraynoic acid (ETYA) and slightly enhanced by indomethacin. Susceptibility to inactivation by sulfhydryl-directed reagents indicated that an essential sulfhydryl is present in a hydrophobic region of the molecule. The enzyme exhibited a broad pH activity optimum and a Km of 2.48 . 10(-5) M. The cytosolic enzyme has been partly purified by ammonium sulfate precipitation and two steps of of column chromatography and exhibited an apparent high molecular weight. The lipoxygenase and hydroperoxidase activities were resolvable from one another. The physiological and pathophysiological roles of the enzyme remain to be elucidated.     

Epidermal Stem Cells

Epidermis contains a compartment of stem cells but currently there is no common criterion to recognize individual stem cells with any confidence. Epidermis appears to contain stem cells of different levels of maturity and it is very likely that the main repository of epidermal stem cells is located in the hair follicle from which cells can emigrate into epidermis and also give rise to follicular and sebaceous keratinocytes. Epidermis consists of proliferative units containing stem and transit-amplifying cells, but the exact size of a proliferative unit cannot be measured accurately. The available data suggest that populations of stem and transit-amplifying cells are not discrete but represent a continuum from cells with a high self-renewal capacity and a low probability of differentiation to those with low self-renewal capacities and high commitments to differentiation. Stem cells occupy a special niche that provides a microenvironment, including an adhesion of stem cells to the basal membrane and their paracrine interactions with neighbor epidermal and mesenchymal cells. The fate of an epidermal stem cell depends on its prehistory and microenvironment.