玫瑰茄培养细胞中花色苷积累的代谢调节

调整培养基组成是提高玫瑰茄（ＨｉｂｉｓｃｕｓｓａｂｄａｒｉｆｆａＬ．）培养细胞花色苷产量的有效途径之一。改变Ｂ５培养其中钙、锰、铜等离子的浓度,增加了培养细胞花色苷的积累量,其中又以锰离子最为有效。其浓度为６＊１０＾４ｍｏｌ／Ｌ时,花色苷磁量达０．３４１ｇ／Ｌ,是对照的２．８２倍。     

乙酰胆碱对培养T细胞功能的作用

本文研究不同浓度（１０＾－１０－１０＾－４ｍｏｌ／Ｌ）乙酰胆碱（ＡＣｈ）对离体培养的大鼠脾脏Ｔ淋巴细胞增殖的影响,并探讨其作用机制。实验结果表明：１０＾－９－１０＾－４ｍｏｌ／Ｌ可显著增强Ｔ细胞由刀豆素Ａ（Ｃ－Ａ）诱导的增殖反应,以１０＾－７－１０＾－６ｍｏｌ／Ｌ时最强。淋巴细胞先用ＡＣｈ刺激１ｈ或者刺激１ｈ后洗弃ＡＣｈ,再用ＣｏｎＡ诱导６ｈ的Ｔ细胞的增殖。１０＾－７－１０－６ｍｏｌ／Ｌ阿托品可阻     

组胺H2受体激动剂4—甲基组胺及其拮抗剂雷尼替丁对HL—60白…

在ＨＬ－６０白血病细胞的体外培养中,组胺Ｈ２受体激动剂４－甲基组胺（１０＾－８－１０＾－４ｍｏｌ／Ｌ）对ＨＬ－６０细胞的增殖具有轻微的抑制作用,而组胺Ｈ２受体拮抗剂雷尼替丁（１０＾－８－１０＾－４ｍｏｌ／Ｌ）对ＨＬ－６０细胞的增殖具有较强的抑制作用。但用１０＾－６ｍｏｌ／Ｌ的雷尼替丁预处理ＨＬ－６０细胞则可部分地拮抗４－甲基组胺（１０＾－８－１０＾４ｍｏｌ／Ｌ）对ＨＬ－６０白血病细胞的抑制作用。     

钙通道阻滞剂对肾上腺素能受体激动剂促血管平滑肌细胞增殖的抑制

本工作观察到１０＾－６－１０＾－５ｍｏｌ／Ｌ去甲明上腺素（ＮＥ）用１０＾－７－１０＾－５ｍｏｌ／Ｌ异丙基肾上腺素（ＩＳＯ）可明显促进离体培养的血管平滑肌细胞（ＶＳＭＣ）的增殖和ＤＮＡ的合成,并呈剂量依赖效应,该效应可为相应的受体阻断剂ｐｈｅｎｔｏｌａｍｉｎｅ（１０＾－６ｍｏｌ／ｌ）和ｐｒｏｐｒａｎｏｌｏｌ（１０＾－５ｍｏｌ／Ｌ）所抑制;ｎｉｆｅｄｉｐｉｎｅ（１０＾－６ｍｏｌ／Ｌ）和ｖｅｒａｐａｍｉ     

亚硝酸盐对培养心肌细胞周期的影响

本文用流式细胞仪测定了ＮａＮＯ２对体外培养的Ｗｉｓｔａｒ乳鼠心肌细胞周期的影响。结果表明,１０＾－６ｍｏｌ／Ｌ ＮａＮＯ２引起Ｓ期细胞明显减少（Ｐ〈０．０５）;１０＾－８ｍｏｌ／Ｌ ＮａＮＯ２对其 显著（Ｐ〉０．０５）。应用「３Ｈ」ＴｄＲ掺入法测定了ＮａＮＯ２对心肌细胞增殖的作用。实验发现,１０＾－６ｍｏｌ／Ｌ ＮａＮＯ２明显抑制细胞增殖,而１０＾－９－１０＾７ｍｏｌ／Ｌ的ＮａＮＯ２则促进细胞增殖     

玫瑰茄县浮细胞合成花青素的光效应研究

光照对悬浮培养的玫瑰茄细胞生物量无影响。随着光照强度增大,玫瑰茄细胞合成花青素的量增加,光照强度３１．０ｗ／ｍ＾２为饱和光照强度,超过该强度,玫瑰茄细胞合成花青素的量不再进一步增加;可见光中蓝光（４２０￣５３０ｎｍ）是促进玫瑰茄细胞合成花青素最有效单色光,光强为３０．０ｗ／ｍ＾２,接种量为０．２ｇ湿细胞的５０ｍｌ培养液经１６ｄ培养,花青素产量为８．９７ｍｇ／５０ｍｌ,高出相同光照强度全色光下的６．     

2.5次微分伏安法对生物品中丙二醛的测定

以０．１ｍｏｌ／ＬＮＨ４Ｃｌ溶液为介质,用２．５次微分伏安法测定了丙二醛,线性范围为１．０＊１０＾－６至１．０＊１０＾－３ｍｏｌ／Ｌ,检测限达１．０＊１０＾－７ｍｏｌ／Ｌ。并测定了细胞培养液介质中新生ＳＤ大鼠室肌细胞样品的丙二醛。     

缓激肽对大鼠背根神经节分离神经元ATP激活电流的调制作用

在新鲜分离大鼠背根神经节（ＤＲＧ）的５６个细胞标本上,应用全细胞膜片箝技术进行记录。胞外加缓激肽（ＢＫ,１０＾－６￣１０＾－４ｍｏｌ／Ｌ）引坊的ＤＲＧ细胞膜反应结果如下：（１）７１．４％的细胞为内向电流,其电流反应的幅值具有明显的浓度信赖性;（２）１２．５％的细胞为外向电流;（３）１６．１％的细胞未引起可检测的膜反应,单独给予ＡＴＰ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）在大多数受栓细胞（５４／５６）     

玫瑰茄悬浮细胞合成花青素的光效应研究

光照对悬浮培养的玫瑰茄细胞生物量无影响。随着光照强度增大,玫瑰茄细胞合成花青素的量增加,光照强度３１．０ｗ／ｍ２为饱和光照强度,超过该强度,玫瑰茄细胞合成花青素的量不再进一步增加;可见光中蓝光（４２０～５３０ｎｍ）是促进玫瑰茄细胞合成花青素最有效单色光,光强为３０．０ｗ／ｍ２,接种量为０．２ｇ湿细胞的５０ｍｌ培养液经１６ｄ培养,花青素产量为８．９７ｍｇ／５０ｍｌ,高出相同光照强度全色光下的６．５３ｍｇ／ｍｌ;黄光和绿光分别有一定的促进作用。当黑暗下的培养时间不超过８ｄ,后期经过不少于８ｄ的光照可以诱导出和全程光照相当的花青素产量,分别为６．６４和６．７２ｍｇ／５０ｍｌ（总培养时间不少于１６ｄ）。当黑暗下培养时间超过１２ｄ,由于营养成分消耗,光照延长,花青素产量也无法提高,添加１０ｍｌ新鲜培养基再进行光诱导,花青素产量可以提高（６．７５ｍｇ／５０ｍｌ）。     

内皮素对失血性休克鼠脑微循环的影响及尼莫地平,川芎…

利用大鼠颅骨开窗观察软脑膜微循环的方法研究了内皮素（ＥＴ－１）１０＾－１０－１０＾－７ｍｏｌ／Ｌ对软脑膜微循环的影响以及失血性体克时软脑膜对ＥＴ－１的反应性。并用１０＾－７ｍｏｌ／Ｌ造成失血性休克后脑血管痉挛的模型,观察尼莫地平、川芎嗪、６５４－２对内皮素引起血管痉挛的治疗作用。１０＾－９,１０＾－８和１０＾－７ｍｏｌ／Ｌ３种浓度ＥＴ－１可使脑膜小动脉、细动脉强烈收缩,收缩率分别为２７．７％、４６     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

若干因子对鸡冠花悬浮培养中花色素苷积累的影响

本文研究几种植物生长调节剂和蔗糖浓度对鸡冠花细胞悬浮培养中花色素苷积累的影响。结果表明,细胞分裂素KT使花色素苷积累明显高于6-BA,且KT在2 μmol/L时积累量最高;2,4-D在2 μmol/L时对花色素苷积累效果明显,其它浓度的2,4-D和NAA对花色素苷积累效果不明显。高浓度蔗糖有利于花色素苷积累;MS+2,4-D(2 μmol/L)+KT(2 μmol/L)+蔗糖(292 mmol/L)为鸡冠花悬浮细胞培养生产花色素苷的最佳培养基。研究中还发现,在黑暗条件培养下无花色素苷积累,推断光是诱导花色素苷积累的主要因素。随着继代次数的增加,花色素苷含量明显增高,但到第4代时基本稳定。     

Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae)

Melastoma malabathricum, belongs to the Melastomaceae family, is an important medicinal plant widely distributed from Madagascar to Australia, that is used in traditional remedies for the treatment of various ailments. Besides its medicinal properties, it has been identified as a potential source of anthocyanin production. The present study was carried out to investigate the effect of sucrose and methyl jasmonate and feeding time on cell biomass yield and anthocyanin production in cell suspension culture of M. malabathricum. Addition of different concentrations of sucrose into the cell culture of M. malabathricum influenced cell biomass and pigment accumulation. The addition of methyl jasmonate was found to have no effect on cell biomass but the presence of higher amount (12.5-50 mg/L) had caused a reduction in anthocyanin production and accumulation. MS medium supplemented with 30 g/L sucrose and 3.5 mg/L of MeJA added on cero day and 3rd day produced high fresh cell mass at the end of nine days of culture but did not support the production of anthocyanins. However, cells cultured in the medium supplemented with 45 g/L sucrose without MeJA showed the highest pigment content (0.69 +/- 0.22 CV/g-FCM). The cells cultured in MS medium supplemented with 30 g/L sucrose with 3.5 mg/L MeJA added on the 3rd and 6th day of culture, showed the lowest pigment content (0.37-0.40 CV/g-FCM). This study indicated that MeJA was not necessary but sucrose was needed for the enhancement of cell growth and anthocyanin production in M. malabathricum cell cultures.     

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11mg (g-fresh cell weight)(-1), which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance.     

Characterization of Intravacuolar Pigmented Structures in Anthocyanin-Containing Cells of Sweet Potato Suspension Cultures

Intravacuolar pigmented structures occurred in anthocyanin-producingcultured cells of sweet potato (Ipomoea batatas) were characterized.Formation of the pigmented structures in sweet potato cellswas induced by transfer of callus cultured in 2,4-D containingagar medium into 2,4-D free liquid medium under continuous illumination.These structures were found in the vacuoles. The pigmented structureswere isolated from the protoplasts by precipitation in 60% (w/w)sucrose after centrifugation. Electron microscopic observationsof the anthocyanin-containing cultured cells showed these structureshad neither membrane boundary nor internal structures, and werefound as strongly osmiophilic globules in vacuoles. Numeroussmall osmiophilic globules were observed in central vacuolesat the early stage of anthocyanin accumulation, but not foundin cytoplasm. Similar pigmented structures in vacuoles werealso formed by treatment with neutral red. These observationsindicate that these pigmented structure is the high densityand insoluble globules highly concentrated with anthocyanin,which was synthesized in cytoplasm and transported to the centralvacuoles. ⁴Present address: Department of Cell Biology, National Institutefor Basic Biology Myodaijicho, Okazaki, 444 Japan     

Effects of abiotic stress on biomass and anthocyanin production in cell cultures of Melastoma malabathricum

Plant cell cultures could be used as an important tool for biochemical production, ranging from natural coloring (pigments) to pharmaceutical products. Anthocyanins are becoming a very important alternative to synthetic dyes because of increased public concern over the safety of artificial food coloring agents. Several factors are responsible for the production of anthocyanin in cell cultures. In the present study, we investigate the effects of different environmental factors, such as light intensity, irradiance (continuous irradiance or continuous darkness), temperature and medium pH on cell biomass yield and anthocyanin production in cultures of Melastoma malabathricum. Moderate light intensity (301 - 600 lux) induced higher accumulation of anthocyanins in the cells. The cultures exposed to 10-d continuous darkness showed the lowest pigment content, while the cultures exposed to 10-d continuous irradiance showed the highest pigment content. The cell cultures incubated at a lower temperature range (20 ± 2 oC) grew better and had higher pigment content than those grown at 26 ± 2 oC and 29 ± 2 oC. Different medium pH did not affect the yield of cell biomass but anthocyanin accumulation was highest at pH 5.25 - 6.25.     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Induction of anthocyanin accumulation in rose suspension-cultured cells by conditioned medium of strawberry suspension cultures

A conditioned medium (CM) prepared from suspended cultures of strawberry, Fragaria ananassa, which stimulated anthocyanin accumulation in cultured strawberry cells, was applied to the suspension-cultured cells of rose, Rosa hybrida sp which did not normally produce anthocyanin. When the rose cells were transferred into the CM, it induced anthocyanin formation and accumulation in the rose cells. It is suggested that the CM may be effective for inducing anthocyanin accumulation in cultured cells of other species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.