犊牛肝细胞的分离与原代培养

以初生犊牛作肝细胞供者,采用稍加改良的两步胶原酶灌流法和一步灌流结合组织块消化法分离获取肝细胞,并进行原代培养;以台盼蓝染色法测细胞活力,在倒置显微镜下观察肝细胞形态变化,采用Beckman全自动生化分析仪检测较好培养体系不同时间培养上清液中白蛋白、乳酸脱氢酶(LDH)、尿素的含量。结果显示,相比较于一步灌流结合组织块消化法,胶原酶消化法所获取的肝细胞形态完整、贴壁良好、活性高、功能强;LDH漏出量、白蛋白分泌及尿素合成等指标在1周内呈现规律性变化,第3和第4天时LDH漏出量最低,白蛋白分泌及尿素合成功能正常,表明所分离的肝细胞在培养第3￣4天功能最佳。     

PEG胁迫对不同培养方式下金发草愈伤组织再生能力的影响

在静置液体培养和振荡液体培养方式下,研究了聚乙二醇(PEG)胁迫对金发草[Pogona the rumpaniceum(Lam.)Hack]愈伤组织的再生能力和游离脯氨酸(F-Pro)积累的影响。发现金发草愈伤组织具有较高的耐PEG胁迫能力,培养方式与PEG胁迫对其再生抑制上存在时间与程度两个方面的表现。振荡培养方式主要表现为延迟再生时间,而PEG胁迫则主要表现为降低再生频率。两种培养方式都能使愈伤组织在PEG胁迫下发生F-Pro积累,F-Pro含量随PEG浓度的增加和培养时间的增长而升高。去胁迫后,大部分愈伤组织都能够恢复再生能力,F-Pro可能在抑制和恢复过程中作用较复杂。因此,在利用愈伤组织筛选抗旱植株时,PEG浓度应在300g.L-1以上,处理时间3周以上,静置培养更有利于抗旱突变体的筛选。     

组织灌流与贴壁法体外培养奶牛乳腺组织的比较研究

采用组织灌流和组织块贴壁法体外培养奶牛乳腺组织,通过LDH活力测定、台盼蓝染色、琼脂糖凝胶电泳、透射电镜技术比较两种培养方法对奶牛乳腺组织活性及组织超微结构的影响,建立奶牛乳腺组织的培养体系。结果表明,以1mL/h的速度灌流培养的奶牛乳腺组织在DMEM 10%小牛血清培养基中12～60h内保持良好的组织活性和超微结构,贴壁培养的奶牛乳腺组织在DMEM 10%小牛血清的培养基中60～108h内保持良好的组织活性和超微结构,两种培养方法各有优缺点。     

紫茉莉悬浮细胞培养体系的建立

为建立紫茉莉(Mirabilis jalapa L.)悬浮细胞培养体系,以紫茉莉无菌苗叶片诱导的愈伤组织为材料,筛选紫茉莉悬浮细胞的适宜培养体系。结果表明,紫茉莉愈伤组织在MS+2,4-D 1 mg L-1+KT 0.5 mg L-1的液体培养基中悬浮继代培养3~4次,能得到稳定的悬浮细胞系。培养基的pH值为5.5~5.9,蔗糖浓度为30 g L-1更适合悬浮细胞的生长。紫茉莉悬浮细胞的生长曲线大致呈S型。最佳继代培养时间是10 d,培养液的体积为40 mL时,接种量为7.5 mL,可以较好地保持悬浮细胞系。1 L培养液中可提取分泌蛋白(0.42±0.15) g。这些有助于对悬浮细胞提取分泌蛋白的研究。     

不同培养时间对鸡卵泡颗粒细胞孕酮、雌激素分泌水平及FSHR、LHR基因表达的影响

目的：研究培养不同时间鸡卵泡颗粒细胞孕酮和雌激素的分泌水平,促卵泡素受体（FSHR）和促黄体素受体（LHR）的基因表达水平,推断体外培养时间对颗粒细胞激素分泌及相关受体基因表达的影响。方法：通过细胞体外培养的方法,分别于0 h、24 h、48 h、72 h、96 h收集鸡卵泡颗粒细胞上清液,采用ELISA法测定细胞上清液内的孕酮及雌激素分泌水平,并采用荧光定量PCR技术检测颗粒细胞内FSHR和LHR基因表达情况。结果：在培养初期0 h~48 h孕酮和雌激素分泌量显著降低（P < 0.05）,随着培养时间增加到72 h两种激素的分泌量又开始增加,并达到培养初期水平,培养至96 h细胞内孕酮和雌激素分泌量再次降低;颗粒细胞FSHR和LHR mRNA的表达水平则随着培养时间的增加而降低（P < 0.05）。结论：体外培养的卵泡颗粒细胞内孕酮和雌激素的分泌量随体外培养时间的延长呈先降低后升高的趋势,可能与体外培养细胞的生长状态相关,从整体上看随着培养时间的延长,细胞内孕酮和雌激素的分泌量均降低,可能与两种促性腺激素受体FSHR和LHR基因表达量下降相关。     

油樟悬浮培养及次生代谢产物的诱导

以油樟(Cinnamomum longepaniculatum)叶片为外植体在附加6-BA和NAA不同激素浓度组合的MS培养基上筛选质地疏松、生长旺盛的愈伤组织, 分别接种在MS、B5、WPM三种液体培养基中进行细胞悬浮培养, 并检测诱导产生的次生代谢产物。结果表明: 2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA能诱导质地疏松、生长旺盛的愈伤组织, B5基本培养基中细胞长势最好; 愈伤组织在B5+2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA中悬浮培养, 继代2次后形成均一的单细胞; 从油樟悬浮培养物中检测出50%以上的成分是苯甲醇。     

对生玉米离体培养再生体系的建立

以对生玉米的雌、雄幼穗为外植体,研究了不同质量浓度激素及其组合对愈伤组织诱导、再分化苗和试管苗生根的影响,建立了对生玉米离体培养再生体系。结果表明：长度为15-17 mm的雌、雄幼穗能够诱导出质量较好的愈伤组织,但只有来自于雄幼穗的愈伤组织才能再生成苗。适合愈伤组织诱导的培养基为Ms+1．5-2．0 mg·L-12,4-D+0．5 mg·L-16-BA+0．5 mg·L-1 NAA+500 mg·L-1脯氨酸+1000 mg·L-1水解酪蛋白+30 g·L-1蔗糖;适合愈伤组织再分化培养基为MS+1．5-2．0 mg·L-1 6-BA+0．1 mg·L-1 NAA+500 mg·L-1水解酪蛋白+30 g·L-1 蔗糖;适合试管苗生根培养基为1／2 MS+0．25-0．5 mg·L-1 IBA+20 g·L-1蔗糖。     

巫山淫羊藿愈伤组织细胞的悬浮培养条件优化的初步研究

通过研究接种量、激素配比、糖浓度、培养基种类对巫山淫羊藿悬浮培养细胞生长及其愈伤组织黄酮类含量的影响,建立了巫山淫羊藿细胞悬浮培养的技术体系.结果表明:巫山淫羊藿愈伤组织细胞悬浮培养在B5基本培养基中并附加1.0 mg·L-12,4-D和0.2 mg· L-1BA,蔗糖浓度40 g·L-1,接种量每30 mL为鲜重2 ...     

铅暴露与排放对中华鲟幼鱼血液中碱性磷酸酶、乳酸脱氢酶及肌酸激酶活力的影响

采用水溶液静态置换法,从中华鲟受精卵发育至96h开始,进行了16周的Pb暴露试验和6周的Pb排放试验,研究了不同浓度Pb2+水溶液(0、0.2、0.8和1.6mg.L-1)对中华鲟幼鱼血液中碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)和肌酸激酶(CK)活力的影响。Pb暴露后结果显示:幼鱼血液中的ALP活力总体表现为随Pb暴露剂量增加而下降的趋势,其中1.6mg.L-1组极显著低于其他各组(P0.01);幼鱼血液中的LDH和CK活力均表现为随Pb暴露剂量增加而升高的趋势,其中LDH活力只在1.6mg.L-1组显著升高(P0.05);而CK对Pb浓度则比较敏感,0.8mg.L-1组达到对照组的8倍(P0.01),1.6mg.L-1组则高达对照组的20倍(P0.01)。Pb排放后结果显示:幼鱼血液中的ALP活力总体仍表现为随Pb暴露剂量增加而下降的趋势,但各暴露组之间比较差异不显著(P0.05);幼鱼血液中LDH活力各组之间均无显著差异(P0.05)。积累和排放对比显示,血液中的ALP活力在0.8和1.6mg.L-1组有所回升但无显著差异(P0.05);而LDH活力在1.6mg.L-1组极显著降低(P0.01),表现出明显恢复。初步认为:经1.6mg.L-1Pb2+暴露后的中华鲟幼鱼会发生代谢异常;血液CK变化较ALP和LDH更为明显,是Pb污染的敏感指标。     

体外培养成骨细胞的影响因素

成骨细胞是骨形成和骨代谢的核心部分,成骨细胞体外培养是研究骨代谢和成骨机制的重要手段。本从物理因素,微量元素,生长因子和激素等四个方面综述了体外培养成骨细胞的影响因素,以期有助于研究有效的体外培养成骨细胞的方法应用于组织工程学的研究。     

Mechanisms of action of CCK to activate central vagal afferent terminals

Cholecystokinin [CCK] is a peptide released as a hormone by the proximal gut in response to the presence of peptones and fatty acid in the gut. Considerable evidence suggests that CCK inhibits feeding behavior and gastric function by acting as a paracrine modulator of vagal afferents in the periphery, especially in the duodenum. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. More recent studies have suggested that CCK may act directly within the CNS to activate central vagal afferent terminal inputs to the solitary nucleus. We have developed an in vitro calcium imaging method that reveals, for the first time, the direct effects of this peptide on vagal terminals in the solitary nucleus. In vitro imaging reveals that CCK provokes increases in intracellular calcium in vagal afferent terminals as a consequence of a complex interaction between protein kinase A [PKA] and phospholipase C [PLC] transduction mechanisms that open L-type calcium channels and causes endoplasmic reticular [ER] calcium release. The subsequent activation of PKC may be responsible for initiating calcium spiking which is dependent on a TTX-sensitive mechanism. Thus, imaging of the isolated but spatially intact hindbrain slice has allowed a more complete appreciation of the interdependent transduction mechanisms used by CCK to excite identified central vagal afferent fibers and varicosities.     

大花蕙兰组织培养技术研究

以大花蕙兰Cymbidium hybridum侧芽为外植体,以N6、B5、CC、MS为基本培养基,设计无机大量元素、无机微量元素、有机物三因素四水平正交试验,探究大花蕙兰丛生芽增殖的最佳基本培养基、生长调节剂浓度配比以及诱导生根的最佳生长调节剂浓度。结果显示,大花蕙兰丛生芽增殖的最佳培养基组合为B5无机大量元素+ CC无机微量元素+MS有机物+蔗糖30 g·L-1 +琼脂7 g·L-1 +活性炭0.5 g·L-1(pH 5.8～6.0),诱导丛生芽增殖的适宜生长调节剂浓度配比为6-BA 4.0 mg·L-1 + NAA 0.2 mg·L-1,诱导生根的最佳生长调节剂浓度为NAA 1.0 mg·L-1。     

向日葵组织培养研究进展

向日葵(Helianthus annuus)是世界主要的油料作物之一.近几年来有关向日葵的研究很多,其中向日葵组织培养研究越来越受到重视.本文从向日葵外植体培养、植株再生影响因素、组培过程中存在的问题及解决方法等方面进行了综述.     

猕猴桃的组织培养和遗传转化研究进展

综述了国内外猕猴桃组织培养和遗传转化研究进展,内容包括花药培养、胚培养、胚乳培养、子叶、叶、茎段等器官培养、原生质体培养以及遗传转化等,并对生物技术在猕猴桃研究中存在的问题以及今后在猕猴桃中的应用前景进行了讨论。     

蝴蝶兰组织培养研究进展(综述)

本文从蝴蝶兰外植体的选择．不同基本培养基、激素及添加物对其增殖与分化的影响,外植体褐变的防治以及生根壮苗方法等,概述蝴蝶兰组培快繁方面的研究进展,为其组培技术研究提供参考。     

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678. © 2008 Wiley Periodicals, Inc.     

Regulation of gut hormone secretion. Studies using isolated perfused intestines

The incretin hormones glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted from enteroendocrine cells in the intestine along with other gut hormones (PYY, CCK and neurotensin) shown to affect metabolism and/or appetite. The secretion of many gut hormones is highly increased after gastric bypass operations, which have turned out to be an effective therapy of not only obesity but also type 2 diabetes. These effects are likely to be due, at least in part, to increases in the secretion of these gut hormones (except GIP). Therefore, stimulation of the endogenous hormone represents an appealing therapeutic strategy, which has spurred an interest in understanding the regulation of gut hormone secretion and a search for particularly GLP-1 and PYY secretagogues.The secretion of the gut hormones is stimulated by oral intake of nutrients often including carbohydrate, protein and lipid. This review focuses on stimulators of gut hormone secretion, the mechanisms involved, and in particular models used to investigate secretion. A major break-through in this field was the development of methods to identify and isolate specific hormone producing cells, which allow detailed mapping of the expression profiles of these cells, whereas they are less suitable for physiological studies of secretion. Isolated perfused preparations of mouse and rat intestines have proven to be reliable models for dynamic hormone secretion and should be able to bridge the gap between the molecular details derived from the single cells to the integrated patterns observed in the intact animals.     

植物组织培养中的玻璃化现象及其预防

玻璃化是植物组织培养中常见的一种现象。本从形态解剖和生理生化方面比较了玻璃化苗和正常植株的差异。围绕水势平衡、激素平衡和矿质元素平衡探讨了玻璃化发生的内在机制,在此基础上总结了防止玻璃化发生的可能途径：改善光照,选用透气封口膜,降低细胞分裂素的浓度等,并提出多学科的合作将是防止玻璃化发生的必由之路。     

珍稀濒危植物组织培养研究进展

本文总结了我国一、二级保护植物的组织培养研究现状,分析了已有组织培养报道的珍稀植物在外植体选择和分化途径上的特点,并从基本培养基、激素及附加营养成分等方面分析了珍稀植物组织培养的影响因素;提出了在珍稀濒危植物组织培养研究和实际应用中存在的问题.