雨生红球藻FACHB-712营养细胞和厚壁孢子低温保藏效果及优化

以高产虾青素的雨生红球藻（Haematococcus pluvialis Flotow）FACHB-712藻株为材料,研究2种细胞形态（营养细胞和厚壁孢子）在低温保藏下的复苏率及其差异原因。结果显示,采用两步法（先预冻降温后再投入液氮中）冻存其营养细胞,在不同冻存条件下,其存活率均低于5%,以10%甘油作为保护剂、冻存速率为0.5℃/min、预冻温度为-40℃、保留30 min,然后再投入液氮罐（-196℃）中保藏,其存活率可达到13.3%。采用两步法冻存厚壁孢子,其复苏存活率高达66.13%,复苏萌发后细胞的生长特性、虾青素含量与液氮保藏前无明显差异（P > 0.05）。对液氮保藏前后藻细胞形态和超微结构观察结果表明,超低温保藏后,营养细胞的结构受到较大损伤,而厚壁孢子受到的损伤相对较小。当添加不同保护剂后,直接将厚壁孢子分别冻存在-20℃、-80℃低温及液氮中,发现-80℃低温冻存处理组的复苏存活率相对较高,可达27%。研究表明采用两步法先预冻降温后再投入液氮中冻存厚壁孢子,是长期保藏雨生红球藻FACHB-712的最佳方法,也可采用一步法将厚壁孢子冻存于-80℃冰箱中。     

低浓度二甲基亚砜保护SSMC7721细胞冻存后活力

目的研究二甲基亚砜作为冻存保护剂对SSMC7721细胞冻存复苏后细胞生长相对活力和凋亡的影响。方法选择传代培养处于对数生长期SSMC7721细胞,体积分数2%、5%、10%和20%DMSO分别冻存10d、30d后复苏,采用倒置显微镜形态学观察、MTT法测定细胞相对活力、流式细胞仪检测细胞凋亡,综合分析不同浓度DMSO冻存不同时间复苏对SSMC7721的影响。结果各浓度DMSO冻存SSMC7721对细胞生长和凋亡均有影响。20%DMSO对SSMC7721细胞影响明显,细胞培养不易贴壁逐渐脱落,浓度低于5%时,细胞生长状态良好;10%和20%DMSO细胞相对活力急剧下降;随着DMSO浓度增加和冻存时间延长,细胞凋亡率明显上升,存活率明显下降,5%DMSO冻存的凋亡率10.24%,20%DMSO冻存的凋亡率93.49%。结论 2%~5%DMSO冻存肝癌SSMC7721细胞,复苏后培养有较好的细胞相对活力,能有效减少细胞凋亡,起到的冻存保护剂作用。     

基于电容测定的罗汉果细胞超低温保藏快速方法

建立了一种基于活细胞电容值定量测定的植物细胞超低温保藏的快速评价方法,优化了罗汉果细胞超低温保藏方法。通过采用活细胞传感仪测定冻存后细胞的存活率并结合细胞生活力(细胞线粒体活性/TTC)对罗汉果细胞的低温保藏过程进行优化,确定了罗汉果细胞较为适宜的冷冻保护剂组分为基本培养基中添加10%的蔗糖和10%的DMSO。预处理剂的考察实验表明,采用0.2 mol/L蔗糖的预处理剂处理细胞时冻存后细胞存活率和细胞活力较高;采用0.2 mol/L蔗糖预处理剂处理细胞时,随着预处理时间的增加,细胞存活率先增加后降低,预处理时间为9 h时,细胞存活率和细胞活力最高。保藏后的细胞复苏实验结果表明:细胞存活率与采用活细胞电容值得到的细胞存活率具有很好的一致性,同时经过冻存的细胞复苏培养后,仍保留了原始细胞的形态和合成甜苷V的特性,说明该冻存方法适用于罗汉果细胞的超低温保藏。因此基于活细胞传感仪测得的电容值进行细胞冻存过程细胞活性的快速评价方法具有较好的可行性和可靠性。     

渗透与非渗透性抗冻剂联用技术对铜绿微囊藻的超低温保藏研究

铜绿微囊藻(Microcystis aeruginosa Kütz)是一种在全世界分部很广的淡水水华蓝藻。在实验室长期的研究工作中, 为了使铜绿微囊藻能维持稳定的生理学特征, 通常使用超低温保藏技术长期冻存藻细胞。研究发现, 同时使用渗透性和非渗透性的抗冻剂比只使用传统的渗透性保护剂能显著提高Microcystis aeruginosa超低温保藏的存活率。以三株铜绿微囊藻为材料进行二步法超低温保藏, 对4 种抗冻剂(甲醇、二甲亚砜、丙三醇、聚乙烯吡咯烷酮), 两种降温速率(-1 /℃min、-0.5℃/min), 第一步温度设置(-30℃、-40℃、-80℃)进行筛选; 用流式细胞仪和细胞计数检测存活率, 并监测冻后相关生理参数、PSII、细胞色素、生长曲线等以确保该方法可保持藻株的活性和生理状态。结果表明, 5%的二甲亚砜和30%的聚乙烯吡咯烷酮(PVP)同时使用时能达到最好效果, 并能保持生理活性与冻前一致。       

豹猫体细胞建系及其生物学特性分析

研究体外培养豹猫(Prionailurus bengalensis)细胞的形态、细胞贴壁率、冷冻存活率、生长曲线及细胞核型等生物学特性,为深入开展豹猫基因组学及濒危野生动物保护提供依据。本实验选择豹猫3种组织,即剑状软骨、心和肺,采用组织块贴壁培养法进行体细胞的原代培养;酶消化法完成细胞的传代培养;程序降温完成细胞的冷冻保存;通过细胞计数法计数细胞冷冻存活率;绘制细胞生长曲线;采用常规染色体标本制备技术,对豹猫的染色体核型及G带进行分析。细胞原代培养结果显示,剑状软骨组织在培养第3天出现纤维样细胞、培养6~7 d铺满培养瓶;心组织在培养第5天出现上皮样细胞、12 d铺满培养瓶;肺组织在培养4 d后出现成纤维细胞、8~9 d铺满培养瓶。3种来源体细胞均显示成纤维细胞特征,剑状软骨源细胞最易贴壁、肺源细胞次之、心源细胞最弱。对比3种不同来源体细胞从6代(P6)至12代(P12)冷冻存活率,剑状软骨源细胞冷冻存活率显著下降(冻存前91.0%~97.6%,冻存后76.8%~85.5%,P0.05),心源细胞冷冻存活率亦显著下降(冻存前82.7%~88.1%,冻存后43.7%~80.5%,P0.05),肺源细胞冷冻存活率有下降趋势,但无显著差异(冻存前83.4%~96.8%,冻存后73.9%~93.3%,P0.05)。生长曲线分析显示,3种体细胞均呈"S"型,其中剑状软骨源细胞增殖最快、肺源细胞次之、心源细胞最慢。核型分析结果显示,3种不同来源的成纤维体细胞染色体数目均为2n=38,18对为常染色体,形态类型为6m+10sm+2st,1对为性染色体,X染色体形态类型为m。本研究建立了3种组织来源的豹猫成纤维细胞建系技术及体细胞系,揭示了该物种成纤维细胞的基本生物学特性,为动物遗传信息研究及豹猫保护提供了重要的实验材料和基础信息。     

紫球藻的培养与利用（综述）

紫球藻为单细胞红藻,其细胞富含红藻多糖、多不饱和脂肪酸(主要为AA和EPA）及藻胆蛋白等高价值生物活性物质,在医药和精细化工领域有广泛的应用前景。本文综述紫球藻的生物学特性、生物活性物质、培养与培养特性等方面的研究概况。     

紫外线对紫球藻的生物学效应

采用紫外线辐照紫球藻,处理剂量分别为10s、20s、30s、60s、90s、120s和180s。结果表明,10s剂量对紫球藻有促长作用,20s及30s对藻细胞生长和各项生理生化指标影响不显著,30s以上处理对其生长以及代谢产物的合成与分泌有抑制作用,且照射时间越长,抑制越强烈,紫球藻对紫外线的耐受极限为120s。结果还表明,高剂量(20~90s)照射能增加单细胞代谢产物含量;经紫外线辐照后的藻细胞第二代培养物在生长速度、叶绿素a含量、胞外多糖和β-胡萝卜素产量等与对照组相似。     

马来丝虫微丝蚴超低温冻存技术的研究

本文应用超低温(-196℃)冻存马来丝虫微丝蚴成功。试验结果再次说明聚乙烯吡咯酮(PVP)低温保护剂的冻存效果优于二甲亚砜(DMSO)。表面作用剂吐温-80有增进两种保护护的冻存效力。低温保存中的冷平衡,在本试验中似乎没有影响。     

人外周血淋巴细胞长期冻存后结构和功能的研究

从肝素和 ACD 抗凝血中分离的淋巴细胞,用 DMSO 或甘油作为低温保护剂,保存在液氮内,在冻前和冻后10、20、40和365天时分别检测细胞的功能活性。当冻存液内含10%DMSO 和40%人 AB 血清时,冻存效果最好。冻存一年时,淋巴细胞的回收率和存活率均大于92.5%。冻存细胞的 E 和 ME 花环形成率、ANAE 阳性率、LDH 同工酶谱和细胞超微结构与冻前相比,均无明显变化,冻存细胞的淋转能力虽在冻存初期有所下降,以后就处于稳定状态。     

紫球藻多糖浓度增加对其他逆境适应性的改变

紫球藻 (Porphyridiumsp .)是一种海水单细胞红藻 ,是多种天然产物的来源。在其培养繁殖过程中 ,能够合成藻胆蛋白、高不饱和脂肪酸、硫酸酯多糖等生物活性物质 ,具有广阔的应用前景。盐胁迫会导致紫球藻的结合态多糖浓度的增加 ,由此可能产生相应的耐盐性的提高 ,并有利于对其他逆境的适应。该项研究采用外加紫球藻多糖或采用盐逆境诱导紫球藻多糖的积累 ,然后解除盐逆境的胁迫的方法获得多糖含量有显著提高的紫球藻试材 ,再给与其他的逆境 :如光抑制 ,低温处理 ,并测定主要的生理生化参数。试验结果表明 ,外加 0 .0 5 %紫球藻多糖的藻细胞光合效率 ,在光抑制条件下 ,低于不加多糖的对照 ,但在低温 ( 4℃ )时 ,高于对照。外加多糖对PSⅡ没有显著影响。紫球藻在去盐后的 48h恢复培养时间内 ,多糖的含量以及光抑制和低温条件下的光合效率都逐渐恢复到对照的水平。但是 ,去盐恢复培养的紫球藻PSⅡ效率在光抑制条件下却高于加盐及未加盐的两种对照 ,显示盐诱导的紫球藻多糖可能增加了PSⅡ对光抑制的忍耐程度。     

Permeabilization and in situ adsorption studies during growth and coumarin production in hairy root cultures of Cichorium intybus L

Effect of addition of a permeabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied. Continuous permeabilization of the hairy root cultures of C. intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2% (v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % v/v) to hairy root cultures of C. intybus, showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1% media filtrate (MF) of cultures of Phytopthora parasitica var. nicotiana treatment. It was observed that treatment with DMSO (0.2% v/v) and 1% MF of P. parasitica showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD-7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD-7 along with 1% MF of P. parasitica showed enhanced growth, coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO/XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO/XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. intybus by using DMSO at 0.2% (v/v) level coupled with 1% MF of P. parasitica maintained viability of tissues and produced coumarins at higher level.     

A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum

Chenopodium rubrum photoautotrophic cell suspensions were grown in plastic tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed cell volume, chlorophyll content and oxygen production. Such microtiter dishes are suitable systems for the serial assay of growth inhibition and various physiological effects (i.e. chlorophyll fluorescence, cell viability, oxygen production) of photoautotrophic cells as caused by herbicides and fungal phytotoxins. The applicability of the test system is discussed.Abbreviations pcv packed cell volume - fr.w. fresh weight - rpm revol. per minute - DMSO dimethyl sulfoxide - PMS phenazine methosulfate - NBT nitro-blue tetrazolium chloride     

Novel cryoprotectant significantly improves the post-thaw recovery and quality of HSC from CB

BACKGROUND: Hematopoietic stem cells (HSC) have traditionally been frozen using the cryoprotectant DMSO in dextran-40, saline or albumin. However, the process of freezing and thawing results in loss of HSC numbers and/or function. METHODS: This study investigated the use of CryoStor for the freezing of HSC from cord blood (CB). CB donations (n = 30) were collected under an Institutional Ethics Committee-approved protocol, volume reduced and frozen using three different methods of cryoprotection. Aliquots were frozen with either 10% DMSO in dextran-40, 10% DMSO in CryoStor or 5% DMSO in CryoStor. Prior to freezing samples were separated for nucleated cell (NC) and CD34+ counts and assessment of CD34+ viability. Aliquots were frozen and kept in vapor phase nitrogen for a minimum of 72 h. Vials were rapidly thawed at 37 degrees C and tested for NC and CD34+ counts and CD34+ viability and colony-forming unit (CFU) assay. RESULTS: Cells frozen with CryoStor in 10% DMSO had significantly improved NC (P < 0.001), CD34+ recovery, viable CD34+ (P < 0.001) and CFU numbers (P < 0.001) compared with dextran in 10% DMSO. CryoStor in 5% DMSO resulted in significantly improved NC (P < 0.001) and CFU (P < 0.001). Discussion: These results suggest that improved HSC recovery, viability and functionality can be obtained using CryoStor with 10% DMSO and that similar if not better numbers can be obtained with 5% DMSO compared with dextran-40 with 10% DMSO.     

Short- and long-term effects of solar ultraviolet radiation on the red algae Porphyridium cruentum (S. F. Gray) N?geli.

During spring 2002 and fall 2003 we carried out experiment in tropical southern China to determine the short- and long-term effects of solar ultraviolet radiation (UVR, 280-400 nm) on photosynthesis and growth in the unicellular red alga Porphyridium cruentum. During the experimentation, cells of P. cruentum were exposed to three radiation treatments: (a) samples exposed to PAR (400-700 nm) + UV-A (315-400 nm) + UV-B (280-315 nm)(PAB treatment); (b) samples exposed to PAR + UV-A (PA treatment) and, (c) samples exposed only to PAR (P treatment). To assess the short-term impact of UVR as a function of irradiance, we determined photosynthesis versus irradiance (Pvs.E) curves. From these curves the maximum carbon uptake rate (P(max)) and the light saturation parameter (E(k)) were obtained, with values of approximately 12.8-14.4 microg C (microg chl a)(-1) h(-1), and approximately 250 micromol m(-2) s(-1), respectively. A significant UVR effect on assimilation numbers was observed when samples were exposed at irradiances higher than E(k), with samples exposed to full solar radiation having significant less carbon fixation than those exposed only to PAR. Biological weighting functions of P. cruentum were used to evaluate the UVR impact per unit energy received by the cells; the data indicate that the species is as sensitive as natural phytoplankton from the southern China Sea; however, it is much more resistant than Antarctic assemblages. When evaluating the combined effects of mixing speed and UVR, it was seen that samples rotating fast within the upper mixed layer were less inhibited by UVR as compared to those under slow mixing or in fixed samples. Growth of P. cruentum over a week-long experiment was not affected by neither UVR nor UV-A; additionally, low photoinhibition was found at the end as compared to that at the beginning of this experiment. Our results thus indicate that, although on short-term basis P. cruentum is affected by solar UVR, it can acclimate to minimize UVR-induced effects when given enough time.     

Impact of Procedural Steps and Cryopreservation Agents in the Cryopreservation of Chlorophyte Microalgae

The maintenance of traditional microalgae collections based on liquid and solid media is labour intensive, costly and subject to contamination and genetic drift. Cryopreservation is therefore the method of choice for the maintenance of microalgae culture collections, but success is limited for many species. Although the mechanisms underlying cryopreservation are understood in general, many technical variations are present in the literature and the impact of these are not always elaborated. This study describes two-step cryopreservation processes in which 3 microalgae strains representing different cell sizes were subjected to various experimental approaches to cryopreservation, the aim being to investigate mechanistic factors affecting cell viability. Sucrose and dimethyl sulfoxide (DMSO) were used as cryoprotectants. They were found to have a synergistic effect in the recovery of cryopreserved samples of many algal strains, with 6.5% being the optimum DMSO concentration. The effect of sucrose was shown to be due to improved cell survival and recovery after thawing by comparing the effect of sucrose on cell viability before or after cryopreservation. Additional factors with a beneficial effect on recovery were the elimination of centrifugation steps (minimizing cell damage), the reduction of cell concentration (which is proposed to reduce the generation of toxic cell wall components) and the use of low light levels during the recovery phase (proposed to reduce photooxidative damage). The use of the best conditions for each of these variables yielded an improved protocol which allowed the recovery and subsequent improved culture viability of a further 16 randomly chosen microalgae strains. These isolates included species from Chlorellaceae, Palmellaceae, Tetrasporaceae, Palmellopsis, Scenedesmaceae and Chlamydomonadaceae that differed greatly in cell diameter (3–50 µm), a variable that can affect cryopreservation success. The collective improvement of each of these parameters yielded a cryopreservation protocol that can be applied to a broad range of microalgae.     

葡萄藻冷冻保藏的研究

为给微藻大规模培养生产生物燃料提供稳定可靠的种质资源,本研究以葡萄藻为研究对象,建立了一套葡萄藻快速高效冷冻保藏的方法.通过对不同冷冻保护剂二甲基亚砜(DMSO)、甲醇(MeOH)、乙二醇(EG)、丙二醇(PG)和甘油(Gly)的毒性测试和冷冻保藏效果的比较,结果表明在以6% MeOH作为冷冻保护剂的条件下葡萄藻的存活...     

有机物质对紫球藻生长的影响

研究了有机碳、氮源扩B族维生素对紫球藻生长的影响。葡萄糖促生长作用最佳,添加2％（W／V）葡萄糖时,藻细胞生长速度比对照组明显提高,培养10d收获的生物量增加92.6％,培养液中的溶解氧含量和藻体叶绿素a含量也有变化,有机氮源的利用率低,仅蛋白胨、酵母汁可被利用。维生素B2和B12也有促长作用。     

不同实验方法检测常用有机溶剂对细菌活性的影响及其安全使用限量

【目的】采用不同实验方法测定常用有机溶剂二甲基亚砜(DMSO)、丙酮和乙醇对细菌活性的影响,以指导抗菌类药物体外抑菌实验所用溶剂的选择和添加限量。【方法】采用常规体外抑菌实验方法(纸片扩散法、肉汤稀释法),并参照生长曲线法检测有机溶剂DMSO、丙酮和乙醇对大肠杆菌(Escherichia coli)及金黄色葡萄球菌(Staphylococcus aureus)的抑菌作用,采用扫描电子显微镜(SEM)观察溶剂作用后的细菌形态变化。【结果】3种溶剂对E.coli和S.aureus抑菌率达到20%时,在肉汤稀释法下,DMSO、丙酮、乙醇的浓度(体积比)分别为1.00%、0.25%、2.00%和1.00%、1.00%、0.50%;在生长曲线法下,溶剂浓度(体积比)分别为0.50%、1.00%、0.50%和1.00%、0.50%、0.50%;而在纸片扩散法下,32%(体积比)DMSO和32%(体积比)乙醇对E.coli产生明显抑菌圈,但3种溶剂对S.aureus均无抑菌圈出现。3种方法比较后得出:当3种溶剂的抑菌率达到20%时,溶剂浓度(体积比)均低于0.5%,对细菌整体生长活性影响较小。SEM结果表明控制溶剂使用限量可有效减少其对E.coli生长过程的影响。【结论】相对于DMSO和丙酮,乙醇对微生物生长繁殖能力的影响更加明显;采用相同浓度有机溶剂时,液态条件下(肉汤稀释法和生长曲线法)微生物受到有机溶剂的影响较大。     

海藻酸微囊替代DMSO和FBS的STO细胞冷冻保存研穷

目的：改进现有的细胞冷冻保存方法,建立一个不舍二甲基亚砜（DMSO）和血清（FBS）的高效冷冻保存方法,为细胞治疗等临床实践提供优质细胞。方法：海藻酸微囊包埋鼠胚成纤维细胞（STO细胞）后用不含DMSO和FBS的冷冻保存液进行冷冻保存。,设四个对照组：添加10％DMSO和20％FBS的组、仅添加10％DMSO的组、仅添加20％FBS、DMSO和FBS均不添加组。在冷冻前后对各实验组细胞用台盼兰染色,进行细胞计数,计算细胞存活率,同时利用溴乙锭的二聚物（EthD）、钙黄绿素-AM（Calcein—AM）进行染色观察细胞的形态,且进一步验证细胞存活率;解冻复苏后用MTT法评估细胞的增殖速度和生长活力。结果：冷冻保存30天后对各组的细胞数量、细胞存活率、细胞形态和解冻复苏后细胞的生长活力进行比较发现,海藻酸微囊包埋冷冻组的细胞数、细胞存活率、细胞形态和生长活力均与添加DMSO和FBS的组之间无显著性差异,而与其它三个对照组呈显著性差异。结论：使用海藻酸微囊替代DMSO和FBS保存STO细胞,能有效的维持细胞形态、数量、存活率,同时不影响细胞的生长活力,从而建立了一个不含DMS0和FBS的高效冷冻保存方法。