高产人参寡糖素培养细胞克隆系的筛选

人参（ＰａｎａｘｇｉｎｓｅｎｇＣ．Ａ．Ｍｅｙ．）培养细胞经细胞平板克隆,获得近３００个克隆系。克隆系在细胞生长速率和寡糖素含量及产率上均存在显著差异,且寡糖素产率和细胞生长之间有明显的相关性。经１１代连续继代培养观察及过氧化物酶同工酶谱特征分析,筛选到一株寡糖素产率高且稳定的克隆系ＰＧ－１８０,其平均生长速率是０．４９５ｇ于重／Ｌ·天,为原始株系的１．３９倍,平均寡糖素含量为１４．６９％干重,是亲本的１．６５倍,平均寡糖素产率是２．１８３ｇ／Ｌ,为原始株系的２．３２倍。比较克隆系ＰＧ－１８０和原始株系细胞悬浮培养时间进程发现,由人参培养细胞生产寡糖素的最佳细胞收获期为３周左右。     

高产人参寡糖素培养细胞变异克隆系的筛选

用２ｍｍｏｌ／Ｌ的ＭＮＮＧ处理经过滤的人参悬浮培养细胞１小时后,细胞存活率下降显著,细胞克隆植板率只是对照组的１０．１２％。经细胞平板克隆共获得克降系１５１株,其中很多克隆系转移培养中生长缓慢,甚至不生长而死亡,经分析可供测定的克隆系生长的寡糖素含量的差异,对１１株寡糖素含量较高克隆系经连续１０代继代培养观察,选出一株稳定高产人参寡糖素优良克隆系ＰＧＭＢ－３７,其平均生长速率是０．５５８ｇＤＷＬ＾     

缺氧性肺动脉高压大鼠肺组织中血小板源性生长因子和c—my…

本研究分析了大鼠肺组织中血小板源性生长因子Ａ链、Ｂ链和ｃ－ｍｙｃ原癌基因ｍＲＮＡ。正常肺组织可表达１．７ｋｂ的ＰＤＧＦ－ＡｍＲＮＡ和３．５ｋｂ的ＰＤＧＦ－ＢｍＲＮＡ,还有少量２．２ｋｂｃｍＲＮＡ．在缺氧过程中,ＰＤＧＦ－Ｂ链ｍＲＮＡ和ｃ－ｍｙｃｍＲＮＡ迅速增加,至缺氧１４ｄ时,分别为正常的３倍和５倍。而ＰＤＧＦ－ＡｍＲＮＡ在缺氧７ｄ时增高,而后又略有降低。结果表明：缺氧的肺组织局生成的ＰＤＧＦ激活     

利用DDRT-PCR技术分析神经生长因子低亲和力受体诱导的细胞凋亡过程中基因表达的差异

转染了Ｐ７５ＮＧＦＲ的Ｒ２神经细胞系Ｒ２Ｌ１在去血清的培养时可以诱导细胞凋亡的发生．此凋亡可以被ＲＮＡ合成抑制剂放线菌素Ｄ和蛋白质合成抑制剂环己酰胺所抑制．利用ＤＤＲＴ－ＰＣＲ技术比较了去血清培养的发生凋亡的Ｒ２Ｌ１细胞与有血清培养的不发生凋亡的Ｒ２Ｌ１细胞以及去血清培养的不发生凋亡的Ｒ２Ｐ细胞基因表达的差异．克隆了数个特异或差异表达的短ｃＤＮＡ片段,经Ｎｏｒｔｈｅｒｎ杂交证实其中两个片段ＬＩＡＲＥＳＴ－１和ＬＩＡＲＥＳＴ－２表达量在凋亡细胞中显著高于不发生凋亡的细胞中,ＧｅｎＢａｎｋ检索表明此二片段为新的ｃＤＮＡ序列并给予登录号Ｕ４７３１５和Ｕ４７３１６．另有一个ｃＤＮＡ片段ＬＩＡＲＣＤ－３在凋亡细胞中受到了明显的抑制,经检索为一已知的与前强啡肽原上游调控区结合的ＤＮＡ结合蛋白ｃＤＮＡ编码区的一部分,首次被证实它与Ｐ７５ＮＧＦＲ诱导的神经细胞凋亡调控关联     

细胞培养生产人参寡糖素降低成本的途径

在人参（Ｐａｎａｘｇｉｎｓｅｎｇ）细胞悬浮培养中,以无离子水代替重蒸馏水,细胞生长速率和寡糖素产率分别降低２．３％和２．９％。用白糖代替蔗糖,细胞生长速率和寡糖素产生率分别降低１．７４％和１．２３％。综合上述两方面结果,以无离子水和白糖分别替代原培养基中的重蒸馏水和蔗糖组成替代培养基,用替代培养基培养人参培养细胞,其生长速率可达０．５０９ｇＤＷ／Ｌ．ｄ．寡糖素产率可达１．４４３ｇ／Ｌ,和原培养基相     

缺氧性肺动脉高压大鼠肺组织中血小板源性生长因子和c－myc基因表达增强

本研究分析了大鼠肺组织中血小板源性生长因子Ａ链、Ｂ链（ＰＤＧＦ－Ａ,ＰＤＧＦ－Ｂ）和ｃ－ｍｙｃ原癌基因ｍＲＮＡ在正常和缺氧时的含量变化。正常肺组织可表达１．７ｋｂ的ＰＤＧＦ－ＡｍＲＮＡ和３．５ｋｂ的ＰＤＧＦ－ＢｍＲＮＡ,还有少量２．２ｋｂｃ－ｍｙｃｍＲＮＡ。在缺氧过程中,ＰＤＧＦ－Ｂ链ｍＲＮＡ和ｃ－ｍｙｃｍＲＮＡ迅速增加,至缺氧１４ｄ时,分别为正常的３倍和５倍。而ＰＤＧＦ－ＡｍＲＮＡ在缺氧７ｄ时增高,而后又略有降低。结果表明：缺氧的肺组织局部生成的ＰＤＧＦ激活了ｃ－ｍｙｃ原癌基因,这对于缺氧性肺动脉高压的形成具有重要作用。     

u-PA/t-PA嵌合型纤溶酶原激活剂(ut-PA)的构建、表达、纯化和性质研究

将尿激酶原（ｐｒｏ－ＵＫ）ｃＤＮＡ和组织型纤溶酶原激活剂（ｔ－ＰＡ）Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核甘酸诱导的大片段定点删除和一次寡核苷酸诱导的多位点突变,得到ｕ－ＰＡ（Ｌｅｕ１４４－Ｇｌｙ４０８）／ｔ－ＰＡ（Ｓｅｒ１－Ｔｈｒ２６３）（ｕｔ－ＰＡ）融合基因．将ｕｔ－ＰＡ融合基因克隆到表达载体ｐＣＭ－βｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞,筛选稳定表达株．收集无血清表达上清,经苯甲脒柱纯化得到ｕｔ－ＰＡ纯品,ＳＤＳ－ＰＡＧＥ和纤维蛋白自显影显示ｕｔ－ＰＡ有两种分子量形式,分子量分别为６８ｋＤ和６１ｋＤ．纤维蛋白亲和性试验表明,ＬＵＫ（低分子量尿激酶）对纤维蛋白没有亲和性,而含有ＬＵＫ的ｕｔ－ＰＡ则对纤维蛋白表现出很强的亲和性,但ｕｔ－ＰＡ的亲和性略低于亲本ｔ－ＰＡ．     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

外源激素在诱导风信子花被外植体不同部位细胞再生花芽中的作用

外源激素诱导风信子（Ｈｙａｃｉｎｔｈｕｓ ｏｒｉｅｎｔａｌｉｓＬ．）同一发育时期花被外植体不同部位细胞再生花芽的实验表明∶１． 诱导花被外植体细胞再生花芽,外源激素是必需的;２． 仅有细胞分裂素就可以诱导花芽再生,生长素并不是必需的;３． 花被外植体上的不同部位的细胞再生花芽时,需要不同浓度的外源激素． 单独加６－ＢＡＰ或玉米素２ ｍ ｇ／Ｌ可以诱导花被下部的细胞再生花芽;６－ＢＡＰ或玉米素２ ｍ ｇ／Ｌ和２,４－Ｄ ０．１ ｍ ｇ／Ｌ的组合有利于花被中部的细胞再生花芽;６－ＢＡＰ或玉米素２ ｍ ｇ／Ｌ和２,４－Ｄ １．０ ｍ ｇ／Ｌ的组合能促进花被上部的细胞分化花芽     

亲和层析纯化HepG_2细胞分泌的PAI－1

本文报道用抗ＰＡＩ－１单克隆抗体（ＭｃＡｂ）亲和层析建立了纯化ＰＡＩ－１的简便方法。经免疫亲和层析,ＳｅｐｈａｃｒｙｌＳ２００凝胶过滤,从ＨｅｐＧ２细胞培液中分离到糖基化和非糖基化两种形式的ＰＡＩ－１,回收率为８４％,ＰＡＩ－１比活性６．１×１０４ＩＵ／ｍｇ。糖基化ＰＡＩ－１分子量为５０ｋＤ,比活性５．８×１０４ＩＵ／ｍｇ。非糖基化ＰＡＩ－１分子量４３ｋＤ,占总ＰＡＩ－１３０％,仍具有ＰＡＩ－１活性。用ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ亲和层析进一步纯化得到ＳＤＳ－ＰＡＧＥ纯的糖基化ＰＡＩ－１。     

Screening of Clone Lines with High Yield of Oligosaccharins from Culture Cells of Panax ginseng

Cell plating clone technique was employed to screen clone lines with high yield of oligosaccharins from culture cells of Panax ginseng C. A. Mey. Near 300 clone lines were obtained. The results from some clone lines analysed implied that these clone lines were significantly different in cell growth rate, oligosaccharins content and yield. Furthermore, there was a distinct correlation between oligosaccharins productivity and cell growth. A more stable high-yield oligosaccharin clone line PG-180 had been selected according to the characteristics of growth rate, oligosaccharin yield and peroxidases isozyme patterns during successive subculturing of 11 generations of clone lines. The mean growth rate of clone line PG-180 was 0. 495 g dry wt/L · d, and was 1.39 folds higher than to the original strain. Its mean content and yield of oligosaccharins were 14. 69 % dry wt and 2.183 g/L, which were 65 % and 132% respectively higher than those of the original strain. In comparing the time course of cell suspension culture between clone line PG-180 and the original strain, the optimal period for high oligosaccharin production from P. ginseng culture cells was approximately three weeks.     

粉叶小檗愈伤组织单细胞克隆

以粉叶小檗愈伤组织为材料 ,用B5液体培养基进行悬浮培养建立悬浮细胞系。经 3～ 4次继代培养即可得到悬浮的单细胞。悬浮细胞通过细胞平板克隆 (一般B5培养基平板克隆 ,优化培养基平板克隆和条件培养基平板克隆 ) ,经 5代连续继代培养观察和薄板层析 -分光光度法分析 ,发现用优化培养基进行平板克隆植板率最高 ,且克隆最易成功 ,并且还筛选到一株小檗碱产率高且稳定的克隆CV 5 7,其平均生长速率为 14 .4 12mg .Fw/L .d ,为原始株系的 1.91倍 ,平均小檗碱含量为 2 .17%干重 ,是原始株系的 2 .2 6倍。     

高含量薯芋皂素植株的细胞克隆

盾叶薯芋(Dioscorea zingibernisis Wright)细胞克隆系的建立采用平板培养法。试验了18种不同的培养外界环境因素对植板率的影响:如培养基pH值,在培养基中添加谷氨酰胺、丝氨酸、柠檬酸、葡萄糖和偶合低聚糖等。其中,以添加偶合低聚糖的植板率最高。获得31个克隆细胞系。经大量培养,测定细胞生长,筛选出18个优良克隆系。以代号为Rc81的克隆系在接种量为0.7g/L(折合干重,下同),培养28d可获得15.1g/L。     

模拟微重力环境因子对人参细胞生长和人参皂苷含量的影响

与在正常重力条件培养下的对照相比,经回转器水平回转处理的人参细胞鲜重和干重均增加,人参皂苷含量提高10％左右。在去Ca2 培养基上生长的人参愈伤组织细胞,经回转器水平回转3周后,人参皂苷含量约为正常重力条件下培养细胞的2倍。另外,在试验范围内,如果培养基中起始钙离子浓度越高,则其培养的人参细胞中人参皂苷含量越低。     

Selection of Cell Lines with High Productivity of Shikonin Derivatives by Protoplast Culture of Lithospermum erythrorhizon Cells

We selected high-yield cell lines, using protoplast culture of Lithospermum erythrorhizon cells. Three cell lines having different shikonin productivities were used as parent cells for the selection, and cell lines with high productivity were obtained efficiently in every case. The best cell line had 6.45 g shikonin/g inoculum/23 days of production which was almost 1.5 times higher than that of the original cell line. The productivities of protoplast-derived cell lines were distributed widely and their average productivity was similar to the original one. The subculture of such a protoplast-derived cell line for eight months showed that its shikonin productivity was stabler than the original cell line.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate     

Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium.

The effect of polyvinyl formal (PVF) culture surface on the growth of 10 mammalian continuous cell lines, including Swiss 3T6, NCTC clone 929 L, BHK-21 clone 13, CHO-K1, PK 15, A 431, HeLa, MDCK, LLC-MK2 and Vero in protein-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with trace elements and L-ascorbic acid 2-phosphate, was investigated. Most of the cell lines showed only some initial proliferation on PVF similar to the polystyrene (PS) surface of commercially available culture flasks. In contrast, proliferation of monkey kidney cell line Vero was by far greater on PVF than on PS or poly-D-lysine treated culture surface. In addition, Vero cells on PVF could be subcultured in the protein-free medium without any significant decrease of growth rate in successive passages. These results showed that PVF provides a culture surface which selectively promotes continuous growth of Vero cells in protein-free, chemically defined medium.     

人参发根的诱导及其适宜培养条件的研究

利用发根农杆菌A₄菌株在人参根外植体上直接诱导产生发根。在1/2MS固体培养基上建立起发根离体培养系,经连续多代的培养,发根仍保持旺盛生长状态。PCR扩增结果表明,发根农杆菌R_I质粒的rolC基因已在人参发根基因组中整合并得到表达。液体培养基中发根生长速度约为固体培养的2倍。经对发根中人参皂苷含量及比生长速率的测定,筛选出高产发根系R9923。利用HPLC法测定了R9923发根系中单体皂苷Rg1、Re、Rf、Rb1、Rc、Rb2和Rd的含量,人参总皂苷含量达15.2mg/g。确定1/2MS培养液（30g/L蔗糖）、摇床转速110r/min、每２周更换一次培养液、继代培养时间4周,为人参发根生长适宜条件。探讨了培养容积、发根初始接种量以及分级放大培养工艺对发根大规模生产过程中生物产量和皂苷含量的影响。