Vero细胞的单细胞模拟和代谢分析
文献类型：学位论文
| 作者 | 高红亮 |
| 学位类别 | 博士 |
| 答辩日期 | 2000-09 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 欧阳藩 |
| 关键词 | Vero细胞 同步化 单细胞模型 代谢分析 |
| 其他题名 | Single cell dimulation and metabolic analysis of vero cells |
| 学位专业 | 生化工程 |
| 中文摘要 | 动物细胞大规模培养技术目前已经成为生物工程领域一个重要分支，但这一技术比较复杂，生产成本高，这在一定程度上限制了其自身的发展。目前国内外都在对动物细胞大规模培养技术进行更为广泛和深入的研究，研究方向多集中于宏观的反应和过程，而较少从细胞水平上对生长和代谢进行研究。大规模细胞培养中的宏观反应过程是以单个细胞为基础的，为了能够深入地了解细胞培养过程，更好地控制和优化这一过程，本文对单细胞的生长、代谢和模拟进行了研究，为动物细胞大规模培养技术的发展提供一些理论基础和实验数据。Vero细胞是由世界卫生组织推荐的用于生产人用免疫产品的连续细胞系之一，已经用于生产脊髓灰质炎、狂犬病疫苗和几种重组蛋白药物，具有广阔的应用前景，因此本文以Vero细胞为研究对象，全文主要分为以下几个部分：1.单个细胞生长和代谢的研究。由于目前无法直接测定单个细胞的生长和代谢，本文采用了同步化的方法。细胞同步生长后再测量得到单个细胞生长和代谢的数据。研究表明：用胸腺嘧啶核苷(TdR)双阻断法可以很好地实现Vero细胞的同步生长。处于指数生长早期的Vero细胞，经两次2mmol/LTdR阻断后，在方瓶中贴壁生长和在旋转瓶中微载体悬浮培养的细胞同步化指数（SI）分别为62%和59.8%。在Vero细胞的一个分裂周期中，细胞对底物的消耗和产物的生成有一定的周期行为。主要表现为细胞在G2期对底物的消耗速率和产物的生成速率增加，在M期对底物的消耗速率和产物的生成速率迅速降低。葡萄糖和乳酸的这种趋势更为明显。培养条件最适时Vero细胞的世代时间为18.5h，各细胞时相的时间分别为：G1期13h，S期4h, G2和M期共1.5h。2.建立了Vero细胞的单细胞模型。根据所得到的生长和代谢数据，结合大量的文献资料，首次建立了包括细胞周期调控在内的Vero细胞的单细胞模型。该模型包括了主要底物葡萄和谷氨酰胺的代谢途径；主要代谢产物乳酸和氨的产生途径；胞内大分子物质蛋白质、糖类、脂类、DNA和RNA的生成途径；能量代谢与调节；底物和产物的跨膜运输；胞内主要代谢调节；细胞周期调节和氨对细胞生长的抑制等过程。该模型的主要特点是：（1）模型把细胞作为一个能随外界条件改变而变化的反应器；（2）模型可以预测细胞大小和形状的变化，包括在不同细胞时期和不同外界条件时的变化；（3）在模型中细胞被分为蛋白质、DNA、RNA、糖类和脂类几个组分；（4）模型把细胞周期分为G1、S、G2和M期，利用细胞周期蛋白和依赖于周期蛋白的激酶对细胞的控制，可以预测细胞随外界条件的周期变化；（5）模型详细考虑了代谢底物葡萄糖、谷氨酰胺和副产物乳酸、氨对细胞生长的影响；（6）模型可以预测细胞对底物的消耗和产物的生成：（7）模型中包括了细胞普遍存在的应急反应。当外界营养缺乏时细胞会生成应急因子（p）ppGpp来调节细胞内的一系列反应，使细胞在营养缺乏时能够存活。模型模拟的细胞周期时间与实验值相相很少，对同步生长的Vero细胞的底物消耗和产物生成的模拟与实验值可以很好地吻合。模型详细计算了Vero细胞在一个分裂周期中形态和大小的变化，特别是在M期时细胞的形态变化。模拟结果表明蛋白质、糖类和脂类在一个细胞周期的积累和指数型。模型描述了主要的细胞周期蛋白(cyclins)和依赖于细胞周期蛋白激酶（CDK）对细胞周期的调控：细胞进入S期和M期时的调控，使得单细胞模型具有明显的周期特征。3.研究了Vero细胞的各种氨基酸的代谢，并详细计算了单个Vero细胞代谢流量分布。Vero细胞在代谢过程中大量积累Glu、Asp、Ala和Pro等4种氨基酸。Vero细胞对Gln、Arg、His、Leu、Thr、lle和Lys等氨基酸消耗比较快，而对Ser、Tyr、Phe、Val和Met等消耗较慢。谷氨酰胺对Vero细胞的氨基酸代谢影响比较大，而葡萄糖则影响较小。对半连续培养的Vero细胞代谢流量分布计算表明，Vero细胞的主要代谢特点是：（1）葡萄糖代谢主要有3个代谢终产物：乳酸、脂类和丙氨酸；（2）谷氨酰胺氮源主要流向氨和非必需氨基酸（丙氨酸、天冬氨酸、谷氨酸和脯氨酸）；（3）谷氨酰胺碳骨架的主要流向为完全氧化为CO_2、乳酸和非必需氨基酸（天冬氨酸、谷氨酸和脯氨酸）；（4）丙氨酸的碳骨架大部分来自于葡萄糖，只有少部分来自谷氨酰胺；（5）乳酸不仅是葡萄糖的代谢终产物，也是谷氨酰胺的代谢终产物。4.通过环境扫描电镜清楚地观察了Vero细胞在微载体上的贴壁、伸展、迁移和生长过程，得到了Vero细胞在微载体Cytodex-3上自然形态的照片。细胞接种后附和伸展速度很快，4h后绝大多数细胞完成了贴壁过程。微载体上生长的细胞形态差异很大，呈椭圆形、不规则圆形、长条形和扁平形等。Vero细胞在微载体Cytodex-3生长过程中会发生迁移现象，但迁移较少。Vero细胞同步化后S期到M期细胞很大，G1期后大细胞迅速减少。微载体上生长的细胞与方瓶中生长的细胞形态完全不同。微载体上生长的细胞为三维立体结构，而在方瓶中细胞完全铺展，失去三维立体结构。形态上可见的细胞分裂从开始到结束仅为6min，这期间细胞形态变化很大。 |
| 英文摘要 | Large scale culture of animal cells is an important field of the biotechnology industry due to the production of complex therapeutic proteins. However, growth of animal cells is restricted to a narrow rage of environmental conditions and needs expensive medium and apparatus. The complexity and high culture cost of cell culture technology also restrict its development seriously. Because of the high value products from animal cell culture processes, intensive efforts have being devoted to the research and development of large scale industrial cell culture. Much research has focussed on the following fields, including novel bioreactor design, culture process design, process monitoring and control, and so on, but little attention has been paid on the research of a single cell system although the single cell is the base of the bioreaction process. In order to understand intensively and control reasonably the process of large scale cell culture, the single cell system should be studied. Vero cell line is a continuous cell line recommended by the World Health Organization as a substrate for the production of immunobiologicals for human use and has been employed in the preparation of human vaccines and recombined proteins. This work attempts to elucidate and simulate the growth and metabolism of a single Vero cell. The whole work was divided into 4 parts: 1. The growth and metabolism of a single Vero cell. Since it is impossible to study directly the growth and metabolism of a single Vero cell, this work adopted the synchronous culture methods to get the relative data approximately. Results showed that Vero cells grew synchronously by treating the double thymidine (TdR) block method. The proper operation procedure is as follows: exponentially growing Vero cells were arrested with 2mmol/L TdR for 11 hours, cultured in normal medium for 14 hours and arrested again with TdR for another 11 hours. By this way, the Synchronous index (Si) reached 62.0% for the Vero cells culture in flask and 59.8% for Vero cells growing on microcarriers Cytodex-3 in spinner flask. The uptake of substrate and production of byproduct showed the periodical behaviors with the Vero cell division cycle. The substrate uptake rates and byproduct production rates of Vero cell increased at the G2 phase and decreased at the M phase. The generation time of Vero cell is 18.5h under the optimal culture conditions, and the lengths of G1, S, G2 and M phase are13, 4 and 1.5h respectively. 2. Establishment the single Vero cell model. The single cell model of Vero cell was developed based on experimental results of the synchronous Vero cell culture and literature data. This model describes metabolic behaviors of substrates, products and cell regulation, including metabolic pathways of the main substrates glucose and glutamine, production pathways of the byproducts ammonium and lactate, biosynthesis of intracellular protein, carbohydrate, lipid, DNA and RNA, metabolism and regulation of energy, trans-membrane transport of glucose, glutamine, ammonium and lactate, in ViVo metabolic regulation and cell cycle regulation. The main characteristics of the single cell model are as follows: ① the model takes the single cell as a bioreactor that can response to the change of environments; ② The model can predict cell shape and size as function of culture condition and cell cycle; ③ the whole cellular component was divided into five parts, i.e. protein, carbohydrate, lipid, RNA and DNA; ④ The model can predict the cell division cycle: G1 S, G2 and M phase, with the cyclin and CDKs regulation; ⑤ The model can predict the effects of the substrates (glucose and glutamine) and byproducts (ammonium and lactate) on the cell growth; ⑥ The model can predict the rates of substrate uptake, byproduct production; ⑦ the model can describe an important cell regulation: stringent response. The model's simulation shows that the model can predict the length of cell cycle phase of Vero cell exactly. The predicted values of substrate uptake and byproduct production were in good accordance with experimental data of synchronous culture of Vero cells. The model described the shape and size of single Vero cell during a cell division cycle. The model prediction results indicated that the accumulations of intracellular protein, carbohydrate and lipid were in exponential modes. 3. The metabolic behaviors of Vero cell. The metabolism of amino acids of Vero cell in batch culture and semi-continuous culture was studied intensively, and the metabolic network distributions of a Vero cell was calculated by metabolic flux balance model. The results showed that 12 amino acids (Gln, Arg, His, Leu, Thr, lle, Lys, Ser, Tyr, Phe, Val and Met) were consumed, and 4 (Glu, Asp, Ala and Pro) produced during the growth of Vero cells. 6 amino acids (Gln, Arg, His, Leu, Thr, lle and Lys) were consumed quickly and 5 (Ser, Tyr, Phe, Val and Met) slowly. The amino acid metabolism was influenced mainly by glutamine and to a lesser extent by glucose. Metabolic flux distribution of animal cells using metabolic flux balance model was analyzed quantitatively. Results showed that glucose has three main end products: lactate, alanine and lipid. The glutamine nitrogen was mainly flow to ammonium and nonessential amino acid: alanine, aspartic acid, glutamate and proline, the carbon skeleton of glutamine was mainly used for CO_2, lactate and nonessential amino acid: aspartic acid, glutamate and proline. The main carbon skeleton of alanine was from glucose, and a little from glutamine. Lactate came from both glucose and glutamine. 4. Study on the growth morphology of Vero cell on Cytodex-3 by the environmental scanning electron microscopy (ESEM). A series of striking changes took pace in morphology of Vero cell at the beginning of cultivation. To understand the changes of morphology of cells deeply, adhesion, spreading and migration of Vero cells were observed and imaged by ESEM. It was found that Vero cells attached the microcarrier immediately after the suspended cells were inoculated, and the cells began to flatten and extend themselves out of the surface of microcarrier. The spherical cells became oval, longer, flatten, and other irregular shapes in after 4 hours incubation. Migration of cells along the surface of microcarrier was observed during the cell adhesion and spreading. The synchronous cells get very large in volume from S to M phase, and became smaller at G1 phase. The morphology of cells growing on Cytodex-3 and in flask was quite different. The cell became almost plane with two-dimensions on flask and has three-dimensions on Cytodex-3. |
| 语种 | 中文 |
| 公开日期 | 2013-09-26 |
| 页码 | 212 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1923]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 高红亮. Vero细胞的单细胞模拟和代谢分析[D]. 中国科学院研究生院. 2000. |
入库方式： OAI收割
来源：过程工程研究所
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