Open Journal of Nature Science
Vol.04 No.02(2016), Article ID:17506,5 pages
10.12677/OJNS.2016.42016

Study of Cancer Cell Malignancy Based on Three-Dimensional Cell Culture Technology

Zhenchao Guo1,2, Tianzhu Zhang1*

1School of Biological Science and Medical Engineering, Southeast University, Nanjing Jiangsu

2Department of Biological Science and Food Engineering, Bengbu University, Bengbu Anhui

Received: Apr. 19th, 2016; accepted: May 3rd, 2016; published: May 9th, 2016

Copyright © 2016 by authors and Hans Publishers Inc.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

ABSTRACT

In order to understand the law of the invasion and metastasis of cancer cells and decrease the malignancy of tumor, three-dimensional culture techniques have been wildly used in tumor cells scientific research. The technology of three-dimensional cell culture is a more convenient and useful technology than traditional two-dimensional culture; it can simulate closer to the cell survival in the real environment. Through three-dimensional culture by simulating tumor microenvironment, the study of malignant tumor cell differentiation, invasion and metastasis, chemical targeted therapy and drug resistance evaluation in vitro has become a hot research topic in the field of biomedicine today.

Keywords:Three-Dimensional Cell Culture, Tumor Microenvironment, Malignancy of Cancer Cells, Drug Resistance Evaluation

基于三维培养下的肿瘤细胞恶性研究

郭振超1,2,张天柱1*

1东南大学生物科学与医学工程学院,江苏 南京

2蚌埠学院生物科学与食品工程系,安徽 蚌埠

收稿日期:2016年4月19日;录用日期:2016年5月3日;发布日期:2016年5月9日

摘 要

为了认识肿瘤侵袭转移的规律,干预治疗改善肿瘤的恶性,肿瘤细胞的科学研究已竞相采用三维培养技术。三维细胞培养与传统的二维培养方法相比,更能模拟接近机体内细胞生存的真实环境。作为一种更为方便、有用的技术,目前在三维培养条件下模拟体内肿瘤细胞微环境研究肿瘤细胞的恶性分化、侵袭转移、化学靶向治疗和耐药性评价已经成为生物医药领域的研究热点。

关键词 :三维细胞培养,肿瘤微环境,肿瘤细胞恶性,耐药性评价

1. 引言

目前的组织工程领域发展迅速,在某些领域已可重现天然组织的形貌、结构或化学组成,并已实现基本修复组织、可替代天然组织的目标。反观肿瘤领域,大量研究都未重视肿瘤所处的三维微环境,还只是处于较滞后的状态。众所周知,体内肿瘤微环境非常复杂,细胞与细胞外基质(ECM)之间处于动态的相互作用,同时还有信号分子及细胞因子等参与。和体内的肿瘤细胞相比,传统的体外二维培养细胞,呈现出的恶性程度明显下降,细胞相关的性质如分化、极性、细胞间通讯及与细胞外基质接触等均发生了变化,因而并不能代表体内的状况,因此其结果总是不能很好地反应体内体系,这就造成了模型与真实肿瘤病灶之间的差距 [1] 。

以模拟天然细胞的生活环境为目的的三维细胞培养技术,可以获得更多有意义的生物医学数据 [2] ,因此较二位培养有更多的优势 [3] ,目前已成为全世界竞相采用的科学研究手段 [4] 。对细胞的深入研究也会使人们对生命奥秘和细胞的认识更加全面,是征服当前关系人类健康的癌症甚至其它重大疾病的关键。肿瘤是机体在致癌因子的作用下,组织中的细胞失去对其生长的正常调控,导致其克隆性异常增生而形成的产物。目前对于各种癌症还没有特效药物来治疗,肿瘤研究的首要任务是明确致癌机制。三维细胞培养技术使研究人员更明晰正常细胞、癌前病变细胞、生命有限的肿瘤细胞以及完全转化或永生化的肿瘤细胞的生物学特征,这些逐级演化的细胞是体外研究多阶段致癌机制的基础 [5] 。

2. 肿瘤微环境与肿瘤细胞的恶化

肿瘤微环境是指肿瘤细胞周围区域中与其相互作用的非肿瘤细胞以及细胞外基。细胞外基质在肿瘤细胞向恶性转移分化的过程中发挥了关键的作用 [6] 。有学者发现在I型/III型胶原培养基上生长的胰腺癌细胞能够发生上皮–间质转换(EMT)改变,变得具有侵袭性 [7] 。微环境及肿瘤细胞TGF-β高表达后 [8] ,在诱导EMT发生的同时,也促进其分泌基质金属蛋白酶(MMP)、尿激酶等到细胞外基质,从而降解基底膜,促进癌细胞浸润和转移 [9] 。基质金属蛋白酶(MMPs)是一族锌肽酶超家族,具有降解细胞外基质成分的能力,是目前已知的唯一能降解胶原纤维的酶类,层黏连蛋白、纤维连接蛋白等都是它的作用底物,其活性对细胞黏附系统有破坏作用。MMP中的膜型-1-基质金属蛋白酶(MTl-MMP)可直接或间接地降解细胞外基质中的多种成分,调节黏附分子和血管生成,是MMP家族中与肿瘤侵袭关系最密切的酶。

缺氧是实质性肿瘤微环境的基本特征,也是肿瘤发生恶化甚至转移的最初驱动力 [10] 。而且缺氧可促进肿瘤细胞转移、基质浸润和基底膜的重构。Lester RD等 [11] 把乳腺癌MDA-MB-468细胞株在1.0% O2孵卵器中培养50 h后观察,细胞呈成纤维样形态,Western-blot及免疫荧光染色显示波形蛋白(Vimentin)表达增加,多数细胞膜E-Cad丢失,细胞迁移及Transwell试验显示侵袭能力增强。

3. 恶性肿瘤细胞的表型特征与分子细胞学机制

上皮来源的肿瘤细胞通过肿瘤微环境中发生EMT失去彼此间的连接,获得间充质细胞的表型,获得了侵袭和转移的能力 [12] ,因此肿瘤微环境在肿瘤的生长和演进中起着重要作用。发生EMT的恶性肿瘤细胞具备转移性或成纤维细胞表型,同时失去E-钙黏蛋白(E-cadherin)等上皮细胞标志的表达,从而获得Vimentin等间质细胞标志的表达,与肿瘤的转移、浸润和演进密切相关。

对肿瘤干细胞(CSC)的成功分离和鉴定 [13] ,大部分学者认为实体瘤内肿瘤干细胞是恶性肿瘤复发转移的根本原因 [14] 。Morel等研究发现,EMT与乳腺癌肿瘤干细胞的产生密切相关。Mani等通过对乳腺癌标本进行系列基因表达分析,发现兼具CD44High/CD24low的乳腺细胞高水平表达EMT标志物N-cadherin,Vimentin的mRNA。提示乳腺上皮干细胞能从分化的细胞群EMT转化时产生,EMT能从相对分化的肿瘤细胞中形成肿瘤干细胞。Brabletz等在对人结肠癌肝转移的研究中发现,无论是原发瘤还是转移灶,在肿瘤宿主侵袭边缘发生EMT的肿瘤细胞大多具有干细胞的特性,且其细胞核内都高表达与肿瘤高转移率和肿瘤患者低生存率密切相关的β-catenin。Wnt/β-catenin信号转导途径是肿瘤干细胞自我更新和EMT机制的共通点 [15] ,肿瘤干细胞与EMT在肿瘤转移中的作用值得研究。

生长因子在肿瘤细胞的EMT过程中起着重要的调节作用,作用于生长因子及其受体的药物则能够影响EMT过程,从而达到抑制肿瘤的侵袭转移的目的。例如,PDGF/PDGFR的抑制剂STl571能够诱导人类乳腺癌细胞的凋亡,并降低该肿瘤细胞在裸鼠中的转移能力。作用于TGF-β的激酶抑制剂,能够抑制多种肿瘤的生长及其在裸鼠中的转移。EGFR酪氨酸激酶的靶向抑制药吉非替尼(Gefltinib),能够通过阻断EGFR的信号转导途径,从而抑制肿瘤细胞的增殖和生长 [16] 。RNA干扰是肿瘤治疗中的新技术,研究表明shRNA干扰沉默snail可以诱导MET改变即逆转EMT从而抑制体内肿瘤的生长。

4. 恶性肿瘤细胞的侵袭转移实验模型

Kim-Vy Nguyen-Ngoc等 [17] 认为细胞的不同类型和不同胞外基质对细胞的行为分化 [18] 、转移分布有重要的影响,细胞和肿瘤微环存在相互的改造作用 [19] 。小鼠正常乳腺上皮细胞和乳腺癌细胞分别在基质和I型胶原中三维培养4天,结果分化产生了完全不同的四种形态行为的细胞。ECM控制肿瘤和正常小鼠乳腺上皮细胞的迁徙模式和传播的行为 [20] 。癌症在转移时发生肿瘤细胞突破基底膜穿透细胞外基质,转移是癌症真正致人于死命的方面。Liyu Liu等 [21] 研究发现mda-mb-231转移性乳腺癌细胞协同入侵一个三维葡萄糖梯度的胶原蛋白基质。在细胞入侵胶原蛋白基质之前,采用共焦成像和标记珠子镶嵌成像。该侵入性的定量分析模型,可以跟踪入侵葡萄糖梯度显示细胞入侵前以合作的方式更换转移领头癌细胞通过行为。75%的卵巢上皮癌患者主要是腹腔内转移,卵巢癌细胞直接扩散到邻近器官后通过体腔转移通向机体远端传播。恶性腹水作为天然流体引起的生理因素,包括重力和隔下压力增大,形成整个腹膜的转移性细胞。Imran Rizvi等 [22] 通过三维培养生长控制和连续流体板状平台(流体成分调节器),研究了卵巢癌结节的转移特性 [23] 。结果表明水流动的力量,生物标志物的调节表达和肿瘤形态学符合EMT过渡、转移性进展的疾病指标。驱动部分由一个翻译后上调表皮生长因子受体(EGFR)的表达和来增加EMT过渡,与卵巢癌的预后较差相关。微流体可以设定E-cadherin减少波形蛋白的表达,探明转移性增加的潜在风险。微流体平台充实了对肿瘤发生机制的认识和治疗策略的选择,广泛适用于致命的恶性肿瘤。

肿瘤细胞对各种化疗药物的耐受性证明了肿瘤恶性潜在侵袭转移程度的高低 [24] 。Xin Liu等 [25] 认为未能考虑药物渗透的效率会导致许多癌症致命的复发。药物渗透进入实体肿瘤,对临床化疗的有效性来说是至关重要的 [26] 。应用激光解吸电离(MALDI)成像质谱(IMS)模型评估抗癌药物伊立替康在结肠癌多细胞球状体的分布。伊立替康的时变渗透可视化和三个代谢物的定位以及药物治疗是球状体映射。细胞球自外向内,可以分为增殖区、静止区和核心区,药物和氧气在细胞球内也存在着浓度梯度的变化。该测量方法允许药物渗透和分布在三维培养模型中,拥有更多的低廉成本和时效性优势 [27] 。

5. 结语

三维细胞培养在于支持生物医学工程及其它生产科研多方面的需要。因此,要充分考虑培养基分子的浓度和分布梯度,模拟机械刺激和电刺激细胞的生长状况,利用共聚焦和Mcro-CT截图图像分析,结合分子影像技术实时监测细胞生活状态。采用脱细胞技术、细胞共培养技术等,使实验结果更加接近实际。

肿瘤微环境是肿瘤细胞发生、发展和演化的场所,也是征服治疗癌症必须全面认识不可回避的关键领域。EMT是肿瘤细胞在肿瘤微环境中获得侵袭和转移能力的重要方式,涉及一系列分子事件及调控通路。三维肿瘤微环境中相信还有许多与转移侵袭及EMT相关的因子尚未发现。根据不同的科研目标,分类建立有针对性的研究模型,对研究认识肿瘤侵袭转移的规律及EMT的发生机制,以及寻找有效的靶点进行临床干预治疗,通过改善微环境改变进展期肿瘤的恶性预后有非常深远的意义。

基金项目

感谢江苏省研究生创新工程基金项目(KYLX_0191)以及蚌埠学院自然科学重点研究项目(2015ZR03 zd)对本论文工作的支持!

文章引用

郭振超,张天柱. 基于三维培养下的肿瘤细胞恶性研究
Study of Cancer Cell Malignancy Based on Three-Dimensional Cell Culture Technology[J]. 自然科学, 2016, 04(02): 132-136. http://dx.doi.org/10.12677/OJNS.2016.42016

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