小鼠输精管内转染法建立人组织型纤溶酶原激活剂(tPA)乳腺定位表达生物反应器的研究

外源基因受精卵内显微注射目前仍然是种常用的建立转基因动物的方法,但此方法操作复杂,成本高。因此利用精子作为载体将外源基因带入卵细胞内建立转基因动物成了极具吸引力的方法,并且已有一些成功的报道。虽然该方法简单,但总体来说可重复性较差。我们在建立乳腺定位表达人组织型纤溶酶原激活剂(tPA)小鼠模型中,对此方法做了改进,即将脂质体包裹的外源基因在小鼠输精管及附睾管内转染精子,而非以往的在体外转染,结果:(1)五只转染小鼠,术后10天内共使10只正常母鼠受精;(2)共繁殖出79只首建者小鼠,存活42只;(3)用PCR检测首建者小鼠外源基因的整合,阳性率为7/42(16.67%);(4)用凝胶平板溶圈试验检测5只PCR阳性的雌性首建者小鼠泌乳期乳汁中的活性tPA,3/5表达,表达量在12—20IU/ml之间(约相当于48—80ng/ml);(5)PCR检测4只子一代小鼠外源基因的遗传情况,阳性率为2/4。     

NOVEL MARKERS FOR CONSTITUTIVE SECRETION USED TO SHOW THAT TISSUE PLASMINOGEN ACTIVATOR IS SORTED TO THE REGULATED PATHWAY IN TRANSFECTED PC12 CELLS

The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human-secreted placental alkaline phosphatase (SEAP) and bacterial β-lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

胞内钙与蛋白激酶C对人脐静脉内皮细胞组织因子途径抑制物及组织因子表达的影响

为探讨细胞内钙及蛋白激酶C在血管内皮细胞释放组织因子（TF）,组织因子途径抑制物（TFPI）中的作用,本文采用钙离子载入剂A23187与蛋白激酶C激动剂佛波脂（PMA）刺激原代培养人脐静脉内皮细胞（HUVEC）,观察各实验组内皮细胞冻融液中TF活性及条件培养液中TFPI活性的变化,以一期凝固法测TF活性,二期生色法测定TFPI活性。结果显示：A23187,PMA及A23187＋PMA组TF活性较对照明显升高（P＜0.001）,三组中A23187和A23187＋PMA组活性明显低于PMA组（P＜0．05）,但前两组间无显著性差异（P＞0．05）;A23187组TFPI活性与对照无明显差别,但PMA与A23187＋PMA组TFPI活性较对照及A23187组明显升高（P＜0.01）。结果提示：细胞内钙升高及PKC激活促进血管内皮细胞TF的合成,以PKC为强;PKC促进血管内皮细胞释放TFPI,而钙对此无明显作用。     

Production of human tissue-type plasminogen activator (htPA) using in vitro cultured transgenic pig mammary gland cells

Abstract

Tissue plasminogen activator (tPA) is a protein involved in the breakdown of blood clots. We have previously produced a human tPA (htPA)-overexpressing transgenic pig using a mammary gland-specific promoter. In this study, we have established a transgenic pig mammary gland cell line that produces recombinant htPA. The mammary gland cells grew well and retained their character over long periods of culture. There was no difference in the extent of apoptosis in transgenic cells compared to wild-type mammary gland cells. In addition, the transgenic mammary gland cells expressed and secreted htPA into the conditioned media at a concentration similar to that in milk. This transgenic cell line represents a simple and ethical method for recombinant htPA production.     

Complexity untwined: The structure and function of cucumber (Cucumis sativus L.) shoot phloem

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and ¹⁴C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-sequencing. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis, we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP.     

红豆杉的愈伤组织诱导及培养研究（初报）

以红豆杉雄球花、茎切段等为外植体诱导产生了愈伤组织。适宜愈伤组织诱导及其生长的培养基为１／２ＭＳ培养基附加２,４—Ｄ２．０ｍｇ／Ｌ和ＮＡＡ０．１ｍｇ／Ｌ。在不同浓度激素配比的培养基上均未观察到愈伤组织发生再分化。     

高原鼠兔在胚后发育过程中热代谢和褐色脂肪组织(BAT)的变化

高原鼠兔(Ochotona curzoniae)新生个体不能维持稳定的体温,仅具有初步的热调节能力,主要利用小气候环境和BAT产热克服低温压力,随着个体发育高原鼠兔的产热能力和隔热性都得到发展,但隔热能力比产热能力优先发育,同时BAT的重要性相对下降。体重达到成体水平后,最低代谢率高于同体重的Kleiber期望值,热传导率低于同体重的Bradley-Deavers期望值。另外,高原鼠兔通过行为热调节对生理调节加以补充。     

Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.     

~(35)S标记探针检测人类单拷贝基因

应用改进的缺口翻译方法,将α-~(35)S-dATP参入DNA分子,获得了>10~8dpm/μgDNA的高比度~(35)S标记探针。用其做Southern吸印杂交探测人类单拷贝基因组织,放射自显影4～10天,能够得到满意的力谱。与~(32)P相比,~(35)S具有半衰期长,无辐射伤害,价格便宜等优点。     

CONDITIONAL PRODUCTION OF A FUNCTIONAL FISH GROWTH HORMONE IN THE TRANSGENIC LINE OF NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Plasmid phr‐YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a RUBISCO SSU 2 promoter, was transferred into the protoplast of marine microalga Nannochloropsis oculata (Droop) D. J. Hibberd by electroporation. Four transgenic clones were obtained in which the transferred phr‐YPGHc was integrated into the genome and existed stably at least until the 50th generation. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in the amount of 0.42 to 0.27 μg · mL^?1 from the 50 mL of medium. We incubated artemia with the wildtype and transgenic N. oculata for 6 h and then fed these microalgae‐treated artemia to red‐tilapia larvae. After feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater (i.e., statistically significant: P < 0.05) than that of larvae that were fed artemia incubated with nontransgenic microalgae: 316% versus 104% in weight gain, and 217% versus 146% in body length increase, respectively. Therefore, the N. oculata enables production of functional GH, and we propose that it might be an excellent bioreactor material.     

促黄体素释放激素类似物(LH-RH-A)对人蜕膜组织核糖核酸和蛋白质合成的抑制作用

高剂量LH-RH-A可明显抑制人蜕膜组织在离体下对~3H-尿嘧啶和~3H-亮氨酸的摄取;低剂量LH-RH-A无明显影响。在人蜕膜组织中也发现有高亲合力(K_?=3.83±1.36×10~?M~(-1))的LH-RH-A特异受体。上述发现表明,高剂量LH-RH-A可通过直接抑制RNA和蛋白质的合成而调节蜕膜组织的功能。     

TPA对原代白血病细胞的诱导分化作用

本文报告了TPA对32例不同类型白血病细胞的体外分化诱导结果。TPA(1.6×10~-7M)可诱导急性非淋巴细胞(ANLL)白血病细胞迅速出现单核巨噬细胞分化标志:细胞贴壁、胞浆丝状伪足形成,具有类似巨噬细胞的形态改变及相应的细胞化学反应特征。急性淋巴细胞白血病(ALL)和桨细胞白血病(PCL)细胞不发生上述变化,表现为细胞聚集成闭现象。慢性淋巴细胞白血病(CLL)出现桨细胞样形态转化。初发与复发病例的诱导反应相类似。TPA体外诱导分化实验,有助于了解病人白血病细胞的分化潜能,对于鉴别粒单系和淋巴系两类白血病,尤其对于用常规方法分型困难的低分化白血病有一定的临床诊断意义。     

辛德毕斯病毒非结构蛋白nsP2在细胞内的分布

应用免疫电镜技术,直观地显示出辛德毕斯病毒的nsP2蛋白存在于细胞核中以及它在核内的分布.将含有nsP2蛋白的一段SbV的cDNA转染细胞,结果表明,单独表达的nsP2仍可进入细胞核中.     

EPO对缺血/再灌注损伤大鼠心肌细胞Mfn2蛋白表达及心肌细胞凋亡的影响

目的观察重组人促红细胞生成素（recombinant human erythropoietin,rhEPO）对缺血/再灌注损伤大鼠心肌细胞Mitofusin2（Mfn2）蛋白表达的影响及其抗心肌细胞凋亡的作用。方法选取成年SD大鼠35只,随机分为正常组（Normal）,假手术组（Sham）,缺血再灌注组（I/R）,缺血再灌注EPO治疗组（I/R＋EPO）。各组分别于再灌注3h和24h后,剪取心脏缺血/再灌注损伤区域,用脱氧核苷酸末端转移酶介导的缺口末端标记法（TUNEL）检测心肌细胞凋亡,免疫组化法检测Mfn2蛋白的表达。结果再灌注3h和24h后,与正常组和假手术组相比,I/R组Mfn2蛋白的表达和心肌细胞凋亡均显著增加;与I/R组相比,I/R＋EPO组Mfn2蛋白的表达和心肌细胞凋亡均显著降低。结论EPO可以下调缺血再灌注损伤后心肌细胞Mfn2蛋白的表达,抑制心肌细胞的凋亡。     

人类免疫缺陷病毒1型(HIV-1)核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

在大肠杆菌中,利用新构建的含Ｔ７噬菌体ｇ－１０核糖体结合位点（ＲＢＳ）,以及λ噬菌体ＰＲ启动子的新型原核表达载体,通过表达ｇａｇ－ｐｏｌ基因片段,获得了具有天然序列的人类免疫缺陷病毒１型（ＨＩＶ－１）核心蛋白ｐ２４（ＣＡ）的高效表达。克隆的ｇａｇ－ｐｏｌ基因片段在其阅读框架移位区域插入了４ｂｐ碱基,其表达的病毒蛋白酶在阅读框架上与ｇａｇ一致,从而实现了对ｇａｇ－ｐｏｌ融合蛋白的有效加工,产生成熟的核心蛋白ｐ２４及其它产物。重组ｐ２４以可溶形式存在,可以被抗ｐ２４的单克隆抗体特异识别。测定的Ｎ－端７个氨基酸序列与从病毒纯化的ｐ２４完全一致,在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到９５％以上的纯度。ＥＬＩＳＡ分析表明,纯化的ｐ２４可以作为特异性很强的试剂而用于ＨＩＶ感染的诊断及病情的预后,并可用于ｐ２４的生化及结构分析。     

Structural modulation of factor VIIa by full-length tissue factor (TF1-263): implication of novel interactions between EGF2 domain and TF

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF_1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF_1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.     

维胺酸对体外转化人支气管上皮细胞及体内构建人支气管上皮组织的抑制作用

癌前改变是肿瘤演变过程中的关键阶段。许多研究显示维甲类化合物对动物肿瘤及体外恶性细胞系具有抑制作用,但尚未见其对肺癌前病变作用的实验室研究报道。人类肺癌的绝大部分起源于支气管上皮,为研究维胺酸对体外转化人支气管上皮M细胞系以及在大鼠气管构建后移植到裸鼠体内生长的具有癌前病变特点的人支气管上皮组织的抑制作用,采用上皮细胞无血清培养技术,人支气管上皮组织大鼠气管内构建／裸鼠皮下移植生长技术,流式细胞学分析,免疫组化、凋亡细胞原位末端标记以及病理学检查等研究方法发现,维胺酸可抑制体外培养的转化人支气管上皮细胞的增殖,使S期细胞比例下降,以及细胞增殖标志Ki-67、mpm-2阳性反应细胞比例下降;明显诱导细胞凋亡。裸鼠腹腔注射给予维胺酸也可使大鼠气管内构建后移植到裸鼠体内生长的癌前期人支气管上皮组织的生长率明显降低,病变程度明显减轻;同样可以诱导细胞凋亡。研究结果提示,维胺酸对体外培养的转化人支气管上皮细胞系及大鼠气管构建／裸鼠体内移植生长的人支气管上皮组织均有明显的抑制作用,是有希望的肺癌化学预防药物。