藏红花细胞悬浮培养动力学研究

基于已经建立的藏红花细胞悬浮培养体系,研究了其悬浮培养动力学.结果表明,悬浮培养时细胞的生长周期约为20 d,在20 d时生物量达到最大,为12.3 g DW/L,但是藏红花素的合成周期大约为28 d,在28 d时藏红花素的含量和产量达到最大,分别为95.8 mg/g和0.92 g/L.藏红花素的积累与细胞的生长之间的关系为半生长偶联型,为其生物反应器放大培养提供了参数等理论依据.     

茉莉茎段遗传转化体系初步研究

以双瓣茉莉(Jasminum sambac)一年生茎段为外植体,探索茉莉花遗传转化体系。结果显示,预培养7 d后进行农杆菌侵染,然后置于含卡那霉素(Kan) 80～100 mg·L-1的筛选培养基上培养可获得成活率较高的抗性茎段。GFP转化预培养7 d的茉莉茎段再培养3 d及30 d后,以及转化预培养20 d茉莉茎段愈伤再培养14 d后愈伤组织中均能检测到GFP荧光。说明利用该转化系统进行茉莉遗传转化是可行的。     

鱼腥藻7120三类液泡三种内含物的比较分析

分别从培养4d,24d和KCl诱导的材料分离液泡,对这3种液泡进行了蛋白质、还原糖和藻青蛋白测定,结果表明,3种物质含量呈现规律性变化。培养4d的材料液泡中各物质含量低,培养24d的材料液泡中物质含量升高,KCl诱导的液泡中含量下降,液泡中各种物质的相对含量在3种液泡中依次升高。这一结果说明,培养4d,液泡处于初期阶段,培养24d,液泡处于充分发育阶段,KCl诱导液泡为衰老阶段。随着细胞发育,液泡的生理作用提高。     

神经干细胞三维空间壳聚糖生物材料中培养

目的:体外培养神经干细胞,并将其种植在三维空间壳聚糖材料中,体外培养一段时间,使壳聚糖材料内尽量分布足够多的细胞.方法:将NSCs种植在4不同孔径直径16通道壳聚糖材料中,分别培养7d和14d.DAPI标记细胞.荧光镜下观察细胞在不同孔径直径材料中的分布.MTT法检测不同孔径直径壳聚糖材料内细胞的活性.结果:DAPI荧光显示,培养7d时.细胞仍然成团贴附在材料的通道内,少有细胞迁移至壳聚糖材料内,而培养14d可见细胞较均匀的分布在材料内,同时观察到,孔径直径为0-75μm和75-125μ m两种壳聚糖材料,容纳细胞数较孔径直径为125-200μ m和200-300μm少.MTT结果显示,200-300μ m孔径直径的壳聚糖材料内细胞活性为各组最高,间接提示其内所含细胞数最多,而培养7d和14d两种培养方式对同种孔径直径材料内所含细胞教并无影响.结论:壳聚糖可降解生物材料能显示出良好生物相容性;体外培养NSCs于孔径为200-300μm的壳聚糖材料内14d,其存活细胞多且分布较均匀.     

三裂叶野葛毛状根的生长及其培养基营养物质的消耗变化

研究了发根农杆菌（Agrobacterium rhizogenes）ATCC15834遗传转化产生的三裂叶野葛（Pueraria phaseoloides）毛状根在液体培养过程中生长及其部分营养物质消耗的关系．结果表明：三裂叶野葛毛状根液体培养0～4d内处于生长迟滞期、8～16d为快速生长期、16d后进入生长平台期．培养基的PO4^2-、硝态氮和铵态氮在毛状根液体培养过程中被逐渐吸收和消耗,培养16d时培养基中的PO4^3-被消耗殆尽,其浓度仅为培养基起始PO4^3-浓度的0．26％;培养基的铵态氮和硝态氮则在培养20d时才消耗殆尽;而培养基中的Ca^2＋浓度在培养过程中逐渐降低．但在培养20d时仍未被完全消耗,其浓度约为起始浓度的30．5％．培养基的pH值随培养时间的延长而不断降低,培养20d后pH值由5．62降低到4．09;而毛状根的颜色也随培养基pH值的降低和培养时间的延长逐渐由白色变成浅黄色和浅褐色．该结果为今后设计合适的培养基以开展野葛毛状根的大规模液体培养来生产葛根素提供了可能性．     

雨生红球藻培养过程中色素动态变化与光合生理特性研究

以雨生红球藻Haematococcus pluvialis CG-06为实验材料,分析测定在正常培养周期内藻细胞主要色素的变化动态、光合生理特性,以及培养基中硝态氮的含量。结果表明,雨生红球藻在绿色细胞阶段的主要色素包括:叶绿素、叶黄素、β-胡萝卜素,培养至红色细胞阶段增加了角黄素、海胆酮、虾青素单酯及双酯等次生类胡萝卜素。硝态氮浓度在培养初期下降迅速,第3 d降至4.875 mg/L,下降了85.3%,至第7 d下降为0.169 mg/L。雨生红球藻培养至第7 d时,细胞中开始检测出虾青素,含量为0.159 mg/g,此时虾青素合成速度较快,至第11 d虾青素含量上升为1.68 mg/g,在虾青素合成初期β-胡萝卜素的含量下降。藻细胞的光合速率、呼吸速率和NPQ在培养前期比较稳定,第7 d细胞光合速率开始下降,而呼吸速率和NPQ则上升,在整个培养周期中,藻细胞的Fv/Fm变化不明显。     

微生物对尾矿中钾、硅元素释放性能研究

本研究探究了AMCC10037、AMCC10079、AMCC10080和AMCC10096 4株菌在尾矿处理过程中不同培养状态下的细菌密度、p H值及硅、钾元素的释放性能,采用ICP-AES测定硅、钾元素的释放浓度。结果表明,在振荡方式培养下,菌株AMCC10096在32 d内对钾的最大释放浓度为22.54 mg/L;AMCC10079在32 d内对元素硅的最大释放浓度为19.12 mg/L;在静置培养方式下,菌株AMCC10079在32 d内对钾的最大释放浓度为24.76 mg/L;菌株AMCC10079在32 d内对元素硅的最大释放浓度为20.28 mg/L;在尾矿处理过程中,选用静置培养的方法在32 d时间内更易促进钾、硅元素的释放,为尾矿的回收利用开拓了新的方向。     

番茄青枯雷尔氏菌强致病力菌株变异的研究

为研究番茄青枯雷尔氏菌强致病力菌株的变异,探索了继代培养、在NB培养基上不同时间培养、不同pH处理7d和15d、不同温度处理1h后对强致病力菌株变异的影响。结果表明:随着继代培养的培养代数增加,平均弱化指数成增大趋势,在第10代出现了无致病力菌株;在NB培养基上培养15d时,强致病力菌株已完全转化为不确定菌株和无致病力菌株,在培养30d时,强致病力菌株几乎完全转化为无致病菌株;pH7.0时,处理7d和15d后,强致病力菌株比例均为最大,分别为93.33%和92.22%,pH5.8时,强致病力菌株比例最低,分别为46.67%和31.11%;用不同温度处理强致病力菌株发现,温度50℃时,菌株死亡,温度40℃时,活菌数显著低于其他(4~30℃)处理,强致病力菌株比例为4~40℃所有处理中最低。     

刺齿贯众及贯众配子体发育的培养观察

用腐叶土为基质培养刺齿贯众和贯众的孢子,在光学显微镜下观察其配子体的发育过程.结果表明.刺齿贯众和贯众的配子体发育特征极为相似:孢子均为单裂缝,具周壁,培养5～7 d后萌发;原丝体形态多样;35.d后发育为原叶体,心形,具毛状体;60 d后性器出现,多数为雌雄异株;85～90d后配子体发育出幼孢子体.     

体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响

微囊化基因工程细胞移植治疗肿瘤是一种新兴的肿瘤治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。体外培养和冷冻保存是生物微胶囊制备过程中两个重要的环节,因此需要考察体外培养和冷冻保存对微囊化重组基因细胞生长和蛋白表达的影响。以重组CHO细胞为模型,考察了体外培养时间和冷冻保存对微囊化细胞在动物体内生长和内皮抑素表达的影响及体外培养时间对微囊化细胞冷冻保存的影响。结果表明:体外培养时间对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性具有较大的影响,体外不培养和培养4d的微囊化细胞在小鼠腹腔内生长良好、内皮抑素表达量高,并且微囊稳定性好,而体外培养8d的微囊化细胞在移植后的第26天破裂。体外培养时间对微囊化细胞冷冻保存也具有较大的影响,体外培养4d和8d的微囊化细胞在液氮中冷冻保存40d,复苏后细胞生长良好、内皮抑素表达量高,而冻存前未经过体外培养的微囊化细胞,复苏后细胞几乎全部死亡。综上所述,生物微胶囊在体外比较适宜的培养时间为4d。并且冷冻保存对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性没有显著的影响。     

Expansion of cultures of human tracheal epithelium with maintenance of differentiated structure and function

We have developed a technique for expanding primary cultures of human tracheal epithelium while minimizing loss of differentiated structure and function. Cells were seeded at 2 x 10(4) cells/cm2 into T75 flasks and trypsinized when approximately 80% confluent. The dispersed cells were then passaged at the same plating density into further T75 flasks or seeded at 5 x 10(5) cells/cm2 on porous-bottomed inserts and maintained with an air-interface. Differentiation of cells on inserts was assessed from transepithelial electrical resistance (an index of tight junction formation), short-circuit current (an index of transepithelial salt transport), cell numbers, total cell protein, and histology. Unpassaged cells (P0) and cells passaged once (P1) took about a week to become 80% confluent on T75 flasks, with 10-fold and 5-fold increases in cell numbers, respectively. Confluence was achieved in approximately 3 days following plating to inserts. Functionally and structurally, P1 and P2 cells (cells passaged twice) were little different from P0 cells. Thus, within a little over 2 weeks, the numbers of confluent cell sheets can be increased 50-fold with minimal change in function. However, there was a marked decline in differentiation by cells passaged three times (P3), and not all cell preparations could be taken to P4 (cells passaged four times).     

Hepatoblast and mesenchymal cell-specific gene-expression in fetal rat liver and in cultured fetal rat liver cells

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture. Tümen Mansuroglu and József Dudás contributed equally to this work.     

Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.     

牛肾细胞传代稳定性研究

为了掌握牛肾细胞在体外连续传代过程中的增殖特点,将原代牛肾细胞培养形成良好单层后,按4×104个细胞/cm2连续进行传代,测定毎代次收获的细胞数,并记录每代细胞培养时形成良好单层的时间。结果显示,原代牛肾细胞传代至24代时,培养96h仍能形成良好单层,且细胞形态基本保持一致,遗传物质稳定,为建立牛肾细胞库奠定了基础。     

Establishment of long term cultures of neural stem cells from adult sea bass,Dicentrarchus labrax

Long term cell cultures could be obtained from brains of adult sea bass (Dicentrarchus labrax) up to 5 days post mortem. On three different occasions, sea bass brain tissues were dissected, dispersed and cultured in Leibovitz's L-15 media supplemented with 10% fetal bovine serum. The resulting cellular preparations could be passaged within 2 or 3 weeks of growth. The neural cells derived from the first trial (SBB-W1) have now been passaged over 24 times within two years. These cells have been cryopreserved and thawed successfully. SBB-W1 cells are slow growing with doubling times requiring at least 7 days at 22 °C. These long term cell cultures could be grown in suspension as neurospheres that were immunopositive for nestin, a marker for neural stem cells, or grown as adherent monolayers displaying both glial and neural morphologies. Immunostaining with anti-glial fibrillary acidic protein (a glial marker) and anti-neurofilament (a neuronal marker), yielded positive staining in most cells, suggesting their possible identity as neural stem cells. Furthermore, Sox 2, a marker for neural stem cells, could be detected from these cell extracts as well as proliferating cell nuclear antigen, a marker for proliferating cells. SBB-W1 could be transfected using pEGFP-N1 indicating their viability and suitability as convenient models for neurophysiological or neurotoxicological studies.     

Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst

A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n = 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.     

小鼠主动脉平滑肌细胞的分离培养及鉴定

目的:建立分离培养小鼠原代主动脉血管平滑肌细胞(VSMC)的方法并检测其生长特性。方法:剥离小鼠主动脉中膜层,分别采用组织块培养法及胶原酶消化法分离培养小鼠主动脉来源的原代VSMC,免疫荧光法检测细胞的纯度和分化状态;3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法测定小鼠主动脉VSMC传代细胞的生长、增殖特性。结果:组织块培养法培养组织块8d后,细胞从组织块边缘爬出,18 d后细胞汇合度达到80%以上后传代;胶原酶消化法分离培养的细胞生长7 d后,汇合度可达80%,此时进行传代;2种方法获得的细胞进行免疫荧光染色,结果显示细胞传至第3代时纯度在95%以上,传至第8代时分化状态并没有改变;MTT法显示细胞生长3~5 d时处于指数生长期。结论:本研究建立了2种可靠稳定的分离和培养小鼠主动脉VSMC的方法,VSMC纯度高,多次传代后细胞特征稳定。     

Human embryonic stem cells passaged using enzymatic methods retain a normal karyotype and express CD30

Human embryonic stem cells (hESCs) are thought to be susceptible to chromosomal rearrangements as a consequence of single cell dissociation. Compared in this study are two methods of dissociation that do not generate single cell suspensions (collagenase and EDTA) with an enzymatic procedure using trypsin combined with the calcium-specific chelator EGTA (TEG), that does generate a single cell suspension, over 10 passages. Cells passaged by single cell dissociation using TEG retained a normal karyotype. However, cells passaged using EDTA, without trypsin, acquired an isochromosome p7 in three replicates of one experiment. In all of the TEG, collagenase and EDTA-treated cultures, cells retained consistent telomere length and potentiality, demonstrating that single cell dissociation can be used to maintain karyotypically and phenotypically normal hESCs. However, competitive genomic hybridization revealed that subkaryotypic deletions and amplifications could accumulate over time, reinforcing that present culture regimes remain suboptimal. In all cultures the cell surface marker CD30, reportedly expressed on embryonal carcinoma but not karyoptically normal ESCs, was expressed on hESCs with both normal and abnormal karyotype, but was upregulated on the latter.     

HIV-1 SH01分离株在MT4细胞的超微结构特征和形态发生学研究

用外周血单个核细胞混合培养法分离到ＨＩＶ－１ ＳＨ０１株,具有典型的ＨＩＶ颗粒的形态学特征,核心颗粒呈锥形,可见芽生释放的全过程。偶尔可在胞装空泡内见到ＨＩＶ颗粒,同时还有细胞碎片和溶酶体结构,故此类空泡实际为ＨＩＶ吞噬泡。另一少见的现象是溶酶体摄取并消化ＨＩＶ颗粒,在ＨＩＶ－１ ＳＨ０１株感染７天或持续感染的ＭＴ４细胞中均可见到,后者尤为普遍。在ＨＩＶ－１ ＳＨ０１株持续感染的ＭＴ４转化细胞中,