转基因红花中角质细胞生长因子KGF-1的表达

通过构建重组表达质粒载体p139035S-KGF1和根癌农杆菌介导在红花（Carthamus tinctorius）中表达角质细胞生长因子（KGF-1）。从侵染到诱导生根共需要14周,转化率达0.1%。红花子叶在潮霉素筛选培养基上培养4-5周后便可获得丛生芽,再生芽移入含潮霉素的伸长生根培养基,培养4-8周可诱导生根。通过PCR、Southern blot、RT-PCR及Western blot检测证明目的基因KGF-1已经整合到红花细胞的染色体中,实现了KGF-1外源蛋白在红花中的成功表达,为开发KGF-1蛋白新的生产途径奠定了基础。     

微型月季愈伤组织诱导及植株再生

选取微型月季(Rosa hybrida)幼嫩离体叶片(叶龄12天)为外植体, 以MS为基本培养基, 研究不同配比的植物生长调节剂对愈伤组织诱导、不定芽诱导和生根培养的影响。结果表明: (1) 诱导愈伤组织的最佳培养基配比为1.0 mg·L^–16-BA+3.0 mg·L^–12,4-D, 诱导率为100%; (2) 6-BA在微型月季不定芽诱导中起着关键作用, 诱导不定芽产生的植物生长调节剂配比为1.0 mg·L^–16-BA +(0.05–0.5) mg·L^–1NAA+(0.02–0.2) mg·L^–1TDZ, 其中最适配比1.0 mg·L^–16-BA+0.1 mg·L^–1NAA+0.02 mg·L^–1TDZ的愈伤组织再分化率达到92.6%; (3) 生长素对微型月季的生根有重要影响, 以1/4MS+0.1 mg·L^–1NAA为生根培养基可获得100%的生根率。实验最终建立了微型月季组织培养和高频离体再生体系。     

盐地碱蓬幼嫩花序的组织培养及植株再生

选用盐地碱蓬(Suaedasalsa)幼嫩花序为外植体,建立了快速而高效的离体培养体系。在附加1.0mg.L-16-BA和0.4mg.L-1IAA的MS培养基上培养25天可诱导出不定芽,诱导频率达到82.1%;不定芽在此培养基上可快速扩增和长期继代培养。不定芽转至MS培养基中培养2￣3周生根形成完整植株。     

红花绣线菊的组织培养和快速繁殖(简报)

以红花绣线菊当年生幼嫩茎段为材料,MS 6-BA 0.5mg/L(单位下同) NAA 0.05培养基诱导不定芽;1/2MS 6-BA 1.5 NAA 0.05培养基继代增殖培养,25d继代一次,繁殖系数约为4.6;以1/2MS IBA 0.5培养基生根培养;移栽到泥炭与珍珠岩(1∶1)混合的基质中,成活率85%以上。     

玫瑰香叶的试管繁殖

1 植物名称玫瑰香叶(Pelargonium roseum)。 2 材料类别带芽的茎段。 3 培养条件基本培养基为MS。(1)诱导芽增殖培养基为MS+6-BA 0.2～1.0 mg/L(单位下同)+NAA0.02～1.0;(2)诱导生根培养基为1/2 MS+IBA 0.1。均采用固体培养,pH 5.8。培养温度25±1℃。每天光照12 h,光照强度2000 lx。 4 生长与分化情况带芽的茎段接种一周后可见芽     

盐地碱蓬幼嫩花序的组织培养及植株再生

选用盐地碱蓬(Suaeda salsa)幼嫩花序为外植体, 建立了快速而高效的离体培养体系。在附加1.0mg.L-16-BA和0.4 mg.L-1 IAA的MS培养基上培养25天可诱导出不定芽, 诱导频率达到82.1%; 不定芽在此培养基上可快速扩增和长期继代培养。不定芽转至MS培养基中培养2~3周生根形成完整植株。     

提高成年云南山楂树试管培养苗生根率的方法

用成年云南山楂(Crataegus scabrifolia(Franch)Rehd.树的无菌增殖芽苗作为生根试验材料。结果表明：接种在含生长素(IBA、NAA)0.01-1.0mg/1的MS/2培养基中,芽苗的平均生根率为50.4%;在高浓度(150-250mg/1)生长素溶液中浸泡芽苗基部30分钟,然后接种到无生长素的MS/2培养基中,其生根率为60.1%;用高浓度生长素液蘸芽苗基部的生根率为83.5%;芽苗在含IBA1-5mg/1的培养基中培养2-4天,然后转入MS/2培养基中诱导生根,生根率可达92%以上,根伸长正常。黑暗条件明显抑制生根,每日16小时至24小时光照对芽苗生根有益。     

红厚壳茎段丛生芽诱导与植株再生北大核心CSCD

红厚壳(Calophyllum inophyllum)为藤黄科红厚壳属多年生木本植物,有很高的药用价值。该研究以红厚壳带节茎段为外植体,探讨生长调节剂对腋芽萌发及丛生芽诱导、伸长和试管苗生根的影响。研究结果表明,外植体腋芽萌发和丛生芽诱导效果最好的培养基是MS+NAA1.0+TDZ0.5,在此条件下培养21天后,转入添加0.5 g·L–1活性炭且无生长调节剂的MS培养基,可有效促进不定芽的伸长。将带不定芽的外植体先在附加1.0 mg·L–1NAA的1/2MS培养基上进行生根诱导4周,之后转入附加1.0 g·L–1活性炭的无激素培养基进行根的伸长培养,这样的两步生根法能有效促进红厚壳生根。     

红厚壳茎段丛生芽诱导与植株再生

红厚壳(Calophyllum inophyllum)为藤黄科红厚壳属多年生木本植物,有很高的药用价值。该研究以红厚壳带节茎段为外植体,探讨生长调节剂对腋芽萌发及丛生芽诱导、伸长和试管苗生根的影响。研究结果表明,外植体腋芽萌发和丛生芽诱导效果最好的培养基是MS+NAA1.0+TDZ0.5,在此条件下培养21天后,转入添加0.5 g·L–1活性炭且无生长调节剂的MS培养基,可有效促进不定芽的伸长。将带不定芽的外植体先在附加1.0 mg·L–1NAA的1/2MS培养基上进行生根诱导4周,之后转入附加1.0 g·L–1活性炭的无激素培养基进行根的伸长培养,这样的两步生根法能有效促进红厚壳生根。     

酸枣叶片不定梢形成及玻璃化的影响因素

以酸枣无菌苗叶片为外植体,研究了培养条件对不定梢再生及不定梢玻璃化的影响.结果表明,叶片在加有细胞分裂素TDZ的诱导培养基(培养基Ⅰ)上连续培养,可诱导不定芽形成,但不能进一步发育成不定梢;而在诱导培养基Ⅰ上培养2周后转移到不加TDZ的培养基Ⅱ上,可获得不定芽伸长的不定梢.培养基Ⅱ的基本培养基组成影响不定芽(梢)的玻璃化症状:MS培养基产生玻璃化的不定芽(梢),而WPM培养基产生正常不定芽梢;光培养条件的变化对玻璃化症状的发生没有影响.不定芽(梢)玻璃化的发生可能与培养基中铵或硝酸铵的浓度有关,在不定芽伸长发育阶段,培养基中高浓度的铵导致了玻璃化苗的发生.     

Keratinocyte growth factor-2 (FGF-10) promotes healing of experimental small intestinal ulceration in rats

Keratinocyte growth factor-2 (KGF-2, repifermin) is a homolog of KGF-1 with epithelial mitogenic activities. We investigated the therapeutic role of KGF-2 in intestinal ulceration and its mechanisms of protection. KGF-2 (0.3-5 mg/kg) was administered before or after induction of small intestinal ulceration by indomethacin (Indo) in prevention and treatment protocols. In acute studies, KGF-2 was injected for up to 7 days before or daily for 5 days after Indo. In a 15-day chronic study, KGF-2 was injected intravenously daily beginning before or 7 days after Indo. Injury was evaluated by blinded macroscopic and microscopic inflammatory scores, epithelial BrdU staining, tissue IL-1beta, PGE(2), and hydroxyproline concentrations, and collagen type I RNA expression. In vitro effects of KGF-2 were evaluated by epithelial cellular proliferation, restitution of wounded monolayers, PGE(2) secretion, and expression of COX-2 and collagen mRNA. Intravenous KGF-2 significantly decreased acute intestinal injury by all parameters and significantly decreased chronic ulceration. Pretreatment, daily infusion, and delayed treatment were effective. KGF-2 promoted in vitro epithelial restitution with only modest effects on epithelial cell proliferation, stimulated COX-2 expression in cultured epithelial cells, and upregulated in vitro and in vivo PGE(2) production. KGF-2 did not affect in vivo fibrosis, although it induced collagen expression in cultured intestinal myofibroblasts. These results suggest that KGF-2 inhibits intestinal inflammation by stimulating epithelial restitution and protective PGs.     

Mass production of pure gibberellin A1 by Phaeosphaeria sp. L487 and the fungal preparation of [U-13C]gibberellin A1.

The mass production of pure gibberellin A1 (GA1) by shake-culturing Phaeosphaeria sp. L487 was investigated. Its GA1 production was markedly influenced by natural nitrogen sources and NH4NO3. When the fungus was cultured in an 8% glucose-1.5% oatmeal-0.1% NH4NO3-0.5% KH2PO4-0.1% MgSO4 x 7H2O medium for 3 weeks, the amount of GA1 in the culture filtrate was up to ca. 200 microg/ml: the addition of safflower oil to the culture medium two weeks after inoculation prolonged the GA1-production period to produce 300 microg/ml. Further preparation of [U-13C]GA, as a tool for the analysis of a complex of GA1 and its binding protein was attempted by using the fungus. The fungal culture in a [U-13C]glucose-oatmeal medium gave 6 mg of crystalline 13C-enriched GA1. Its 13C-enrichment of ca. 75% and 1J(CC) values were determined by NMR spectrometry.     

角质细胞生长因子-2与核糖体蛋白L22相互作用的发现及在哺乳动物细胞中的验证

通过聚合酶链式反应从人胎肝cDNA文库中钓取KGF-2 cDNA,构建诱饵蛋白载体pAS2-1-KGF-2并对其自身转录激活活性进行鉴定,利用酵母双杂交系统筛选人胎肝cDNA文库,挑选双阳性克隆.DNA序列分析和同源检索显示,所获侯选蛋白为人核糖体蛋白L22(RPL22).将KGF-2和侯选蛋白分别克隆至哺乳动物细胞双杂交的BD、AD质粒中,共同转染COS-7细胞,通过CAT分析验证了KGF-2和侯选蛋白之间的相互作用.为阐明KGF-2作用的分子机制提供有益线索.     

激肽释放酶转基因大鼠模型的建立

目的建立KLK1转基因大鼠模型。方法腹腔注射PMSG-HCG（150-150IU/kg）超排不同周龄（4-8周）SD大鼠比较超排效果的差异,以可用于注射胚胎数作为评判标准确定最佳超排周龄。构建pBC-klk1转基因构件,经酶切、纯化后通过显微注射方法导入SD大鼠受精卵原核并移植到同期受孕的SD受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过RT-PCR和免疫组化方法检测klk1基因表达。结果4-8周SD大鼠超排后分别获得受精卵14±14.8、30±15.2、13.3±13.7、13±14.7、8±5.7,5周龄大鼠超排效果最好,与其他周龄大鼠相比有显差异,P〈0.05;显微注射1538枚卵,移植685（44.53%）枚卵于31只受体输卵管中,12（12/31,38.7%）只怀孕,共产仔62只,经PCR检测获得6只阳性鼠,Southern检测3只阳性。对Southern检测阳性转基因大鼠子代进行RT-PCR检测和免疫组化分析证明klk1基因在肾脏、胰腺和乳腺内表达。结论成功建立klk1基因表达的转基因大鼠模型,该模型是高血压病研究的理想动物模型。     

红花注射液对气虚血瘀证模型大鼠基因调控作用的研究

目的:采用基因芯片技术,分别构建气虚血瘀证大鼠和红花注射液给药处理后气虚血瘀证大鼠的差异基因表达谱,比较并分析,筛选出红花能够治疗气虚血瘀证的关键基因群,并推测其起治疗作用的基因组调控机制。方法:15只SD大鼠随机分为模型组、给药组、空白对照组。模型组和给药组采用疲劳游泳和饥饿饲养处理。造模一周后,给药组尾静脉注射红花注射液(100mg/kg/d),模型组给予相同体积生理盐水;对照组不做任何处理。造模进行两周后处死大鼠,取血检验血流变指标并评价造模情况;另抽取足够的血分离mRNA并逆转录杂交基因芯片;扫描信号分析确定受红花注射液调控的基因;并通过基因数据库查询相关基因功能,结合相关文献分析初步探讨红花作用的机制。结果:两周后经过检验和观察发现模型组大鼠在不同切率下的全血粘度增加,并且其体征表现出虚弱和瘀血的状态、体重下降,确定造模成功;给药组大鼠则相对于模型组的各项检测指标和状态有所改善,确认药物有疗效。在差异基因的比较中,空白组相对于给药组上调基因252条,下调基因54条;给药组相对于模型组上调基因196条,下调基因32条;两次差异表达基因中有16条相同基因,这些差异基因涉及到炎症损伤、免疫调节反应等方面。结论:红花注射液对于气虚血瘀证有治疗作用,在基因层次上是通过抗炎症损伤机制实现的。     

人角质化细胞生长因子-2基因的克隆、表达及产物的纯化、鉴定

用高表达菌株BL21codon plus compentent cells表达重组人角质化细胞生长因子(Hkgf-2)蛋白并初步纯化和检测其活性。通过RTPCR从流产胎儿肺组织中钓取hKGF-2cDNA,将其克隆入pBV220载体质粒。在大肠杆菌BL-21codon plus compent cells中表达hKGF-2蛋白。采用亲和层析和离子交换层析分离纯化,以细胞增殖实验测定表达蛋白的生物活性。结果显示,hKGF-2蛋白在BL21中得到高效表达;hKGF-2蛋白能刺激NIH3T3细胞的增殖,具有显著的促有丝分裂活性。     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

角质细胞生长因子2对细胞生长、移行及创伤愈合的作用

角质细胞生长因子2(KGF-2)是成纤维细胞生长因子超家族的一员,由间质细胞合成并分泌,能特异促进上皮细胞增殖、分化与迁移,对脊椎动物多种组织和器官的发育起重要调控作用．通过PCR从人的肾组织cDNA文库中克隆分离获得了KGF-2的cDNA,表明该因子在成人肾中有表达．采用大肠杆菌表达并纯化重组蛋白用于生物学功能研究的结果显示：KGF-2在体外不仅能够促进角质细胞的生长和增殖,而且对其凋亡具有抑制作用,还对细胞的移行具有影响．在动物实验中,KGF-2能促进皮肤切除产生的伤口愈合,提示该蛋白质可以作为创伤治疗或辅助用药的候选分子．