微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

微囊化干细胞及其应用研究进展

干细胞极强的自我更新能力和多向分化潜能使其可以成为绝佳的种子细胞来源,用于各种疑难疾病的治疗。微胶囊不仅可以为细胞提供三维生长微环境,而且具有良好的免疫隔离性能和生物相容性。微囊化干细胞技术为干细胞大规模、高活性体外培养及长期保存提供了新的技术支持,为细胞移植疗法开辟了新途径。以下首先简述了微囊化技术的发展情况,然后介绍了目前用于微囊化干细胞的材料、制备方法及其免疫隔离作用,重点阐述了近年来微囊化各种不同类型干细胞的研究和应用进展。最后,提出目前微胶囊化干细胞的问题所在并对此技术进行展望。     

微囊化重组CHO细胞制备和培养条件的优化

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,然而由于目前微囊化细胞规模化制备和培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。以重组CHO细胞为模型,考察了不同的微囊制备和培养条件对微囊化细胞生长和内皮抑素表达的影响。实验表明,种子细胞所处的生长阶段和细胞接种密度对微囊化细胞生长和内皮抑素表达的影响较大,对数生长期的细胞进行包囊并且细胞接种密度为1×106~2×106cells/mL微囊时微囊内细胞生长良好、内皮抑素表达量高。微囊制备时间对细胞活性和内皮抑素表达也有较大的影响,制备时间延长对细胞的损伤增大,因此制备时间应控制在5h以内。生物微胶囊在制备过程中会造成细胞损伤,而体外培养是恢复细胞活性的良好方法,在培养过程中微囊接种量为5%时对细胞生长和内皮抑素表达有利。     

不同生境下木麻黄脯氨酸含量和质膜ATPase活性的研究

采集生长于恶劣环境和中生环境的普通木麻黄(Casuarina)小枝,超速离心提取粗质膜制剂后,用两相系统法纯化得到质膜微囊,研究不同生境下木麻黄的质膜ATPase活性,并测定木麻黄小枝的游离脯氨酸含量。实验结果表明:同一生境中的木麻黄ATPase活性相对一致,而同一树种木麻黄不同生境下质膜微囊H~+-ATPase、Ca~(2+)-ATPase、K~+-ATPase活性有显著差异,表现出以渗透胁迫为主的恶劣环境下的木麻黄质膜微囊ATPase活性和木麻黄细胞内游离脯氨酸明显高于中生环境下生长的木麻黄。说明普通木麻黄在干旱和盐胁迫下能调整生理生化过程来提高其质膜ATPase活性和增加细胞内脯氨酸含量提高渗透调节能力以保证其在恶劣环境的正常生长。     

用壳聚糖/海藻酸钠制备干扰素-τ缓释微囊

目的:用壳聚糖和海藻酸钠为原料,制备干扰素-τ微囊,希望发展一种口服干扰素制剂。方法:使用注射器手工滴制的方法,在滴加过程中,速度和距离是影响囊形的主要因素。结果:壳聚糖/海藻酸钠微囊法应用于干扰素-τ药物的包封,其制备简单快速,干扰素-τ包封率很高,并且具有肠溶缓释作用。结论:壳聚糖/海藻酸钠微囊有望用于制备干扰素-τ或其他肽类药物的口服制剂。     

海藻酸钠漂浮微囊的制备以及载药应用研究

目的:本文研究了一种海藻酸钠漂浮微囊的制备方法用以实现胃部持续给药。方法:采用微胶囊发生器制备海藻酸钠漂浮微囊,壁材为海藻酸钠,芯材为食用油的漂浮微囊,衡量不同的制备参数对微囊的理化特性影响;采用克拉霉素作为模型脂溶性药物,测量漂浮药物递送系统的控制释放性质、以及微囊载药特性和小鼠体内漂浮验证。结果:成功制备出了具有漂浮特性的海藻酸钠微囊,其中泵送速度对微囊性质的影响最大。制备出的微囊具有低细胞毒性,可以实现90%的药物包埋率。此外,微囊可以在小鼠的胃中保存超过6小时,具有良好的漂浮特性。结论:海藻酸钠漂浮微囊是一种有效的胃部药物递送系统,可明显延长药物在胃部的滞留时间。     

微囊蛋白基因及其与疾病关系研究进展

微囊蛋白(caveolin)基因家族已鉴定出3个成员:微囊蛋白-1、微囊蛋白-2、微囊蛋白-3,并被定位于抑癌基因位点区域.微囊蛋白-1是细胞质膜微囊的标记蛋白,其中间疏水区域在细胞膜内形成发夹结构,并使其N端区域与C端区域在细胞膜内表面聚合形成支架结构.微囊蛋白-1与微囊蛋白-2以组成异源寡聚体的形式存在,在脂肪细胞、内皮细胞和成纤维细胞中表达最丰富,微囊蛋白-3则特异表达于肌肉.离体与活体研究结果均表明微囊蛋白-1可能具有抑癌功能,并可能在细胞信号传导中起刹车作用.微囊蛋白-1基因敲除小鼠心血管NO与Ca²⁺信号途径受损、功能异常,肺泡上皮细胞出现异常扩增,脂质代谢失衡,身体消瘦.微囊蛋白-2基因敲除小鼠肺功能与耐力均严重受损,与微囊蛋白-1基因敲除小鼠的表型非常相似.微囊蛋白-3为维持心脏正常功能所必需,可能还与一些肌肉营养不良症有关.     

不同生境下木麻黄脯氨酸含量和质膜ATPase活性的研究

采集生长于恶劣环境和中生环境的普通木麻黄(Casuarina)小枝,超速离心提取粗质膜制剂后,用两相系统法纯化得到质膜微囊,研究不同生境下木麻黄的质膜ATPase活性,并测定木麻黄小枝的游离脯氨酸含量。实验结果表明：同一生境中的木麻黄ATPase活性相对一致,而同一树种木麻黄不同生境下质膜微囊H^ -ATPase、Ca^2 ATPase、K^ -ATPase活性有显著差异,表现出以渗透胁迫为主的恶劣环境下的木麻黄质膜微囊ATPase活性和木麻黄细胞内游离脯氨酸明显高于中生环境下生长的木麻黄。说明普通木麻黄在干旱和盐胁迫下能调整生理生化过程来提高其质膜ATPase活性和增加细胞内脯氨酸含量提高渗透调节能力以保证其在恶劣环境的正常生长。     

微囊化胰岛B细胞系体外生长和分泌功能的研究

目的：研究海藻酸钠－多聚赖氨酸－海澡酸钠（APA）微囊化胰岛B细胞系BTC6－F7的生长和分泌规律,探索其作为生物人工胰岛的可能性。方法：以微囊静电液滴发生器制作APA微囊化BTC6－F7细胞,体外培养并定期测定微囊化细胞的生长和胰岛素分泌。结果：在实验观察的90d内,BTC6－F7细胞可在微囊内以细胞团的形式生长、存活。囊内细胞总数随培养时间的延长而增加,但细胞活率呈下降趋势,胰岛素分泌与囊内活细胞数的变化规律一致,最初呈上升趋势,然后较长时间维持在相对恒定的水平。结论：本研究所制备的APA微囊化胰岛B细胞可在较长时间内保持生长、存活和分泌功能,为进一步发展生物型人工胰岛奠定了基础,并可用于糖尿病的发病机理和治疗研究。     

脉络丛细胞微囊脑内移植前后的物理及生化性能研究

通过测定脉络丛细胞海藻酸盐微囊在大鼠脑内移植前及移植后的物理及生化性能变化,以探讨其应用于移植治疗神经系统疾病的可行性.用海藻酸盐多聚鸟氨酸微囊包裹猪脉络膜细胞,移植至大鼠黑质-纹状体通道,移植前、移植后4个月及6个月分别测定微囊的大小、形态及细胞的活力、分泌蛋白质及神经营养因子的能力、蛋白质组学的变化.脉络膜细胞微囊在移植前、后大小、细胞活力、蛋白质组学分析、分泌蛋白质及神经营养因子的能力无显著变化.海藻酸盐-多聚鸟氨酸CP微囊能有效地防止脉络膜细胞被受体免疫系统所攻击,使得它们能在大鼠的大脑存活6个月以上并不引起不良作用.     

Metabolic response of different osmo-sensitive Sacchromyces cerevisiae to ACA microcapsule

Microencapsulation technology is a convenient method to alter and regulate cell product formation. In order to probe the metabolic response of different osmo-sensitive Sacchromyces cerevisiae to ACA microcapsule, the hyper-osmo-sensitive type S. cerevisiae (Y02724) and wild type S. cerevisiae (BY4741) were encapsulated into liquid core ACA microcapsules. The behavior of cell growth, glucose consumption, ethanol production and the yields of glycerol and organic acids were determined. Free cell culture was used as control. The enzyme activities of NADP⁺-glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) on microencapsulation cells and free cultured cells were measured too. The results demonstrated that the growth of Y02724 in both aerobic and anaerobic conditions was seriously inhibited by ACA microcapsule, while the ethanol and acetatic acid yield of microencapsulation Y02724 in anaerobic condition were significantly higher than that of suspended cultivation. For Y02724, the microencapsulation cultivation significantly increased the GS and GOGAT activities and decreased the GDH activity in comparison with control group. ACA microcapsules did not significantly change the growth behavior and metabolic performance of BY4741, but decreased the GS activity. In conclusion, microcapsules microenvironment significantly changes the metabolism behavior of hyper-osmo-sensitive type S. cerevisiae (Y02724), but nearly had no effect on BY4741.     

Probing the role of microenvironment for microencapsulated Sacchromyces cerevisiae under osmotic stress

Cell encapsulation opens a new avenue to the oral delivery of genetically engineered microorganism for therapeutic purpose. Osmotic stress is one of the universal chemical stress factors in the application of microencapsulation technology. In order to understand the effect and mechanism of the encapsulated microenvironment on protecting cells from hyper-osmotic stress, yeast cells of Saccharomyces cerevisiae Y800 were encapsulated in calcium alginate micro-gel beads (MB), alginate-chitosan-alginate (ACA) solid core microcapsules (SCM), and ACA liquid core microcapsules (LCM), respectively. The stress-induced intracellular components and enzyme activity including trehalose, glycerol and super oxide dismutase (SOD) were measured. Free cell culture was used as control. The survival of encapsulated cells and the cells released from MB, SCM and LCM after osmotic shock induced by NaCl solution (1, 2 and 3M) was evaluated. An analysis method was established to probe the effect of encapsulated microenvironment on the cell tolerance to osmotic stress. The results showed that LCM gave rise to the highest level of intracellular trehalose and glycerol, and SOD activity, as well as the highest survival rate of encapsulated cells or cells released from microcapsule. It was demonstrated that LCM was able to induce the highest stress response and stress tolerance of cells, which was adapted during culture, while SCM failed. The theoretical analysis revealed that it was the liquid alginate matrix in microcapsule that played a central role in domesticating the cells to adapt to hyper-osmotic stress. This finding provides a very useful guideline to cell encapsulation.     

Differential role of microenvironment in microencapsulation for improved cell tolerance to stress

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.     

Characterization of the adaptive response and growth upon hyperosmotic shock in Saccharomyces cerevisiae

Molecular and physiological details of osmoadaptation in yeast Saccharomyces cerevisiae are well characterized. It is well known that a cell, upon osmotic shock, delays its growth, produces a compatible solute like glycerol in yeast to maintain the osmotic equilibrium. Many genes are regulated by the hyperosmolarity glycerol (HOG) singling pathway, some of which in turn control the carbon flux in the glycolytic pathway for glycerol synthesis and reduced growth. The whole process of survival of cells under hyperosmotic stress is controlled at multiple levels in signaling and metabolic pathways. To better understand the multi-level regulations in yeast to osmotic shock, a mathematical model is formulated which integrates the growth and the osmoadaptation process. The model included the HOG pathway which consists of Sho1 and Sln1 signaling branches, gene regulation, metabolism and cell growth on glucose and ethanol. Experiments were performed to characterize the effect of various concentrations of salt on the wild-type and mutant strains. The model was able to successfully predict the experimental observations for both the wild-type and mutant strains. Further, the model was used to analyze the effects of various regulatory mechanisms prevalent in the signaling and metabolic pathways which are essential in achieving optimum growth in a saline medium. The analysis demonstrated the relevance of the combined effects of regulation at several points in the signaling and metabolic pathways including activation of GPD1 and GPD2, inhibition of PYK and PDC1, closure of the Fps1 channel, volume effect on the glucose uptake rate, downregulation of ethanol synthesis and upregulation of ALD6 for acetate synthesis. The analysis demonstrated that these combined effects orchestrated the phenomena of adaptation to osmotic stress in yeast.     

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (μ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.     

酵母细胞甘油代谢与生理功能研究进展

甘油是酵母细胞生长代谢过程中常见的多元醇物质。尽管甘油的结构简单,代谢途径并不复杂,但是其在细胞内的生理功能十分重要。甘油代谢过程主要参与细胞的高渗透压生理调节和厌氧条件下的胞内氧化还原平衡调节。近年来许多学者在酵母细胞的甘油代谢及生理功能方面开展了深入的研究。在扼要介绍甘油生理代谢的基础上,重点阐述甘油代谢参与细胞高渗压甘油应答信号途径和氧化还原平衡调节的生理机制,同时就酵母细胞甘油合成的代谢工程进行归纳和评述。     

A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism

Abstract The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed. This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation. Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli . The glycerol overproducing phenotype of two isolates was confirmed by chemical analysis.     

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

光镊拉曼光谱分析酿酒活性干酵母的活化与生长

应用光镊拉曼光谱新技术(LTRS)对酿酒活性干酵母复水活化与生长进行动态观察, 探索从分子光谱角度窥视胞内糖类、核酸、蛋白等生物大分子的变化过程, 及葡萄糖消耗和乙醇生成的动态过程。结果显示, 酿酒活性干酵母复水活化后, 第6小时和9小时, 即酵母对数生长中期及乙醇产生前期, 是调控酵母细胞生理变化的2个重要的时间点。核酸类物质在细胞活化后迅速增加, RNA在第6小时达到最大值; 而蛋白质和脂类物质从第6小时开始快速增加, 在第9小时达 到最大值, 而后呈下降趋势; 胞内乙醇则是在9 h开始出现, 在9