体外构建三维肝肿瘤模型及药物筛选

doi:10.3969/j.issn.2095-4344.2014.27.020

摘要/Abstract

摘要：

背景：体外构建三维肿瘤模型替代现有二维平面肿瘤细胞模型用于药物筛选是肿瘤药筛技术发展的必然趋势。
目的：体外构建三维肝肿瘤模型体系，并用于抗肿瘤药物的敏感性研究。
方法：以人肝癌细胞HepG2作为模型细胞，以壳聚糖/胶原混合材料制备水凝胶支架，体外构建肝(肿瘤)细胞的三维培养体系，表征三维肝(肿瘤)细胞聚集体的形态、生长、细胞骨架分布等，并以二维平面培养的肝肿瘤细胞为对照，研究三维肝肿瘤模型对临床常用的化疗药物的敏感性。
结果与结论： ①肝细胞在壳聚糖/胶原水凝胶支架中培养10 d后形成三维的聚集细胞团。②肝细胞在水凝胶支架中生长速度略慢于二维平面培养，但在三维体系下肝细胞能长时间保持细胞活性。③在水凝胶支架中肝细胞三维生长后，纤维蛋白骨架发生重排，结构与在体肝组织更接近。④在水凝胶支架中的三维肝肿瘤细胞模型对化疗药物的敏感性降低。由此可见，在壳聚糖/胶原水凝胶支架中形成的三维肝(肿瘤)模型，其细胞骨架结构更接近体内肝组织，因此可用于体外药筛模型研究。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

关键词: 实验动物, 组织构建, 细胞模型, 壳聚糖, 胶原, 水凝胶, 抗肿瘤, 三维, 药物筛选

Abstract:

BACKGROUND: In vitro construction of three-dimensional (3D) tumor model has been growing to substitute two-dimensional (2D) tumor model for drug screening.
OBJECTIVE: To develop 3D hepatocarcinoma model for the sensitive study of antitumor drugs.
METHODS: Taking HepG2 as cell model, hydrogel scaffold was fabricated with chitosan/collagen to construct in vitro 3D hepatocarcinoma model. The 3D hepatocarcinoma aggregates were characterized regarding to the morphology, growth and cytoskeleton distribution and so on. Finally, the sensitive assay of in vitro 3D hepatocarcinoma model to the clinical antitumor drugs was studied with 2D hepatocarcinoma model as control.
RESULTS AND CONCLUSION: (1) HepG2 cells in chitosan/collagen scaffold grew to form 3D cell aggregates after 10-day culture. (2) Although the growth rate of HepG2 cells in chitosan/collagen scaffold was slightly slower than that of cells in 2D culture, the HepG2 cell viability of 3D culture could be maintained longer. (3) It was found that the fibrin skeleton of HepG2 cells in chitosan/collagen scaffold rearranged and displayed structural similarity to in vivo hepatic tissue. (4) The sensitivity of in vitro 3D hepatocarcinoma model to the clinical antitumor drugs was significantly lower than that of 2D cells. In conclusion, the in vitro 3D hepatocarcinoma model developed in chitosan/collagen scaffold provided cytoskeleton structure closer to in vivo hepatic tissue, which is potential system for in vitro drug screening.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: collagen, chitosan, hepatocytes, antineoplastic combined chemotherapy protocols

中图分类号:

R318

刘劲松. 体外构建三维肝肿瘤模型及药物筛选[J]. 中国组织工程研究, 2014, 18(27): 4389-4394.

Liu Jin-song. In vitro constructing a of three-dimensional hepatocarcinoma model for drug screening[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(27): 4389-4394.

图/表（结果） 4

2.1 肝细胞三维培养后的形态变化人肝癌细胞在水凝胶支架内呈三维立体方式生长，随培养时间的延长聚集成细胞团。将培养14 d的细胞团通过石蜡切片，苏木精-伊红染色结果显示，细胞团内细胞核蓝染，细胞排列紧密，有规则(图1A)。进一步通过扫描电镜观察细胞团的超微结构显示，在水凝胶支架中的细胞呈圆形，大小均一，多层排列，细胞间彼此接触，聚集呈不规则的多细胞聚集体 (图1B)。而二维培养细胞呈扁平多边形(图1C)，细胞间不具有三维空间关系。

2.2 肝细胞三维培养体系的生长特性从细胞生长曲线可以看出，与游离的二维培养方式相比，在水凝胶支架中，肝细胞生长曲线的延滞期略长于二维平面培养组。但二维培养的细胞在迅速进入对数生长期后，平台期时间维持很短，很快即从培养瓶中脱落，死亡。相比之下，在水凝胶支架中，细胞在平台期活性维持更持久，更利于细胞活性的长期保持(图2)。

2.3 肝细胞三维培养体系的细胞骨架细胞骨架是细胞内蛋白质组成的网络系统^[42]。与贴壁细胞相比，在水凝胶支架中聚集生长后，细胞形态发生很大变化，由伸展的多边形变成球形。因此，通过标记细胞骨架蛋白，进一步研究在三维培养体系中的细胞骨架排布。借助FITC-鬼笔环肽标记细胞的纤维肌动蛋白，利用激光共聚焦扫描显微镜，观察纤维肌动蛋白在细胞上的分布。结果显示纤维肌动蛋白在水凝胶支架中三维细胞团内，呈现出完全不同于二维贴壁细胞的结构，大量的纤维肌动蛋白趋向于胞周分布，形成纤维肌动蛋白环，极性增强，与小鼠肝组织内的结构更接近(图3)。由此可见，肝细胞在水凝胶支架中聚集生长后，引起细胞骨架的重排和细胞形态的改变，形成更接近在体的结构。

2.4 肝细胞三维培养体系对化疗药物的敏感性以化疗药物5-氟尿嘧啶、顺铂、阿霉素、多烯紫杉醇为模型，研究上述制备的肝癌细胞三维体系对化疗药物的敏感性。结果显示4种药物对二维平面培养和三维立体培养的细胞均有抑制作用。但在传统的二维平面培养组中所有化疗药物对肝癌细胞抑制作用更显著，而在三维培养体系中，达到半数致死量的药物浓度要较二维平面培养组提高10倍左右。在4种化疗药物中，无论二维培养组、三维培养组，顺铂对肝癌细胞的抑制作用都优于其他药物。但5-氟尿嘧啶在二维培养体系中抑制效果要优于多烯紫杉醇，但在三维培养组中药物的抑制效果相反，5-氟尿嘧啶的化疗效果最差(图4)。

参考文献

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引言

目前，常规药物筛选研究中，在进入临床试验前，还是借鉴二维细胞试验筛选后再开展动物实验。但随着体外三维培养技术的发展，研究者越来越认识到，由于细胞在体内与细胞外基质共同构成三维环境，因而细胞在体外二维平面培养所表现的生物学特性与在体存在着较大差异^[1-2]，甚至出现相悖的结果。因此，在体外发展三维肿瘤细胞模型^[3-5]，将替代二维肿瘤细胞模型，成为抗肿瘤药物筛选技术发展的必然趋势^[6-8]。细胞三维培养的概念早在20世纪50年代即被提出。Moscona实验室首先用细胞的球形聚集培养方法研究了胚胎细胞和肿瘤细胞的形态发生，开辟了三维培养技术的先河^[9-15]。随着三维培养技术的不断发展，研究者都试图在体外提供与组织来源相似的细胞生长微环境，利于细胞的定向诱导分化^[16-21]，及细胞表型的有效维持^[22-25]。因此，开展细胞三维培养技术的目的就是在体外构建出与体内相同的三维微环境体系。

具有良好生物相容性与加工成型性能的支架材料^[26-27]，是有效模拟细胞外基质、提供细胞体外构建三维组织模型的关键因素之一^[28-30]。作者拟采用壳聚糖与胶原共混制备成水凝胶支架材料，通过壳聚糖的引入适当增加体系中的正电荷利于细胞有效黏附^[31-34]，而胶原是细胞外基质成分利于细胞功能表达^[35-37]，水凝胶支架则能更真实的模拟体内微环境体系^[38-41]。实验以肝(肿瘤)细胞为模型，通过体外构建肝细胞的三维培养体系，观察三维支架对肝细胞增殖形成细胞团的形态、活性、细胞骨架排布，并在此基础上，以二维生长为对照，研究此三维模型对抗肿瘤药物的敏感性。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程
全文链接：

材料方法

设计：细胞学对比观察。

时间及地点：实验于2012年8月至2014年3月在解放军463医院中心实验室完成。

材料：

细胞：HepG2 人肝癌细胞购自中科院上海细胞库。

方法：

肝细胞的二维培养：HepG2细胞以MEM/EBSS培养液(含体积分数10%胎牛血清，1×10⁵ U/L青霉素，1×10⁵ U/L链霉素，1 mmol/L丙酮酸钠)在体积分数5% CO₂细胞培养箱中进行常规二维平面培养。

肝细胞三维培养：二维平面培养的HepG2细胞，待细胞长到对数生长期，用0.25%胰酶消化，生理盐水洗涤3次，离心收集细胞，计数，悬浮于冰浴中的胶原/壳聚糖混合液(二者等体积混合)中，混合均匀后，将温度升至37 ℃，即形成水凝胶支架。加入MEM培养液，在体积分数5% CO₂细胞培养箱中进行培养。

肝细胞形态学观察：将肝细胞三维培养一段时间后，体积分数4%甲醛溶液固定后梯度乙醇脱水。一部分样品进行常规石蜡包埋后切片，切片厚度6 μm，苏木精-伊红染色后，显微镜下观察细胞形态。另一部分样品修剪成约 2 mm见方的样品切块，用1%锇酸固定，用导电胶将样品粘在扫描电镜专配的标本台上，扫描电镜观察。

肝细胞骨架结构表征：将待观察的细胞样品用体积分数4%甲醛固定后，用5 mg/L FITC-鬼笔环肽染色1 h，PBS充分漂洗，用激光共聚焦扫描显微镜观察。小鼠肝组织冰冻切片，甲醛固定后，染色方法同前。激光共聚焦扫描显微镜检测：两荧光通道同时激发(激发/发射：488/535 nm；543/630 nm)，采用激光共聚焦扫描显微镜的Z Series程序进行光学切片，xyz扫描模式对样品进行断层扫描，获得无损伤连续光学切片，再用process/3Dvisualization软件对不同层面图像做三维重建。

肝细胞活性表征：MTT法测定二维及三维培养体系下，肝细胞的生长曲线。

细胞毒性实验：CCK-8法检测药物对细胞的毒性，将药物分别设计系列浓度梯度，在96孔板中，分别接种二维培养的细胞及三维培养的细胞，每孔加入2×10⁴个细胞，加入不同剂量的5-氟尿嘧啶、顺铂、阿霉素、多烯紫杉醇，每组设置3个平行孔。加样后，于37 ℃，体积分数5% CO₂的培养箱中培养48 h后，每孔加入CCK-8，孵育2 h后， 450 nm测吸光度值，Origin Pro9软件绘制曲线，计算半数抑制浓度(IC50)。

统计学分析：各组所得计量数据采用x(_)±s表示，用SPSS 18.0软件(美国SPSS公司)处理数据，两组间均数比较t 检验，检验水准α=0.05，P < 0.05 为差异有显著性意义。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

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讨论

细胞在体内微环境中，与间质细胞、细胞外基质、细胞因子等共同构成三维立体的组织微环境。而细胞三维培养技术就是将细胞接种在具有一定空间结构的支架材料中，在模拟体内物理、化学和生物条件下进行的培养。在众多的支架材料中，水凝胶支架由于在形态、基质刚度、网络孔隙等更接近在体软组织的细胞外基质结构，因此在软组织工程中，以水凝胶支架材料的研究为主体^[43-45]。由于作者希望首先在体外构建类似载体肝组织的三维培养系统，因此选择水凝胶支架材料作为肝细胞的培养体系，使其更接近肝组织微环境。肝细胞是具有高度特异性和极性的细胞，肝细胞极性是肝脏独特生理功能的基础，在肝细胞功能表达上有极为重要的意义。而细胞连接在细胞形态形成、细胞分化以及细胞极性的建立和维持中有重要作用^[46-47]。有研究报道，离体培养的肝细胞在体外会很快丧失肝细胞极性，失去肝细胞胆小管结构及其分泌功能^[48-49]。因此，实验通过水凝胶支架体系进行肝细胞的三维培养，增加细胞间接触，创造一个有利于肝细胞极性重建的三维微环境。文章中，通过标记细胞的纤维肌动蛋白，很清晰的显示出，肝细胞在水凝胶支架中三维培养聚集成团后，细胞骨架肌动蛋白发生重排，恢复了肝细胞的部分极性，其结构分布与二维平面培养的差异显著，而更接近在体肝组织。结果说明作者构建的三维细胞培养体系能够为体外培养细胞提供与组织来源相似的微环境和细胞连接。同时，在体外构建的肝细胞聚集体也可作为药物代谢体系、毒理研究、药效筛选、肝脏生物学等研究的重要手段。实验即将构建的人肝癌细胞三维聚集体用于抗肿瘤药物的敏感性研究。

越来越多的证据表明，肿瘤的发生发展过程是肿瘤细胞与其微环境相互作用的结果。即使在肿瘤发生的最早期，肿瘤细胞的失控性存活与生长也需要其微环境相应的改变才能维持。因此，肿瘤的发生不仅仅是基因突变造成的，而是肿瘤细胞在与其微环境乃至机体之间不断的相互作用中演变形成的另类组织^[50]。而常规体外二维培养的贴壁生长的肿瘤细胞处于均一的环境中，具有同步化特征，这与其在体内生长所处的环境有很大不同；而肿瘤微环境是影响细胞增殖、分化、转移等生物学特性的重要因素^[51-52]。而肿瘤微环境中除了肿瘤细胞自身，还有细胞外基质、细胞因子等。因此，体外构建肿瘤微环境，离不开细胞外基质成分。胶原是最常见的细胞外基质成分，壳聚糖作为自然界唯一的阳离子多糖利于细胞粘附，因此二者结合形成的水凝胶结构可有效模拟细胞外基质环境。同时，在水凝胶支架中，细胞成三维生长方式，更接近在体肿瘤组织，因此体外构建有效的三维培养技术^[53-54]，可模拟在体肿瘤，用于研究肿瘤发生、信号传导、抗肿瘤药物筛选等具有重要意义^[55-56]。

随着肿瘤靶点及抗癌新药的不断发现，选择一种有效且能真实模拟体内肿瘤组织的药物筛选模型是目前抗肿瘤药物筛选面临的重要问题之一。当前的药筛模型主要包括肿瘤细胞二维平面培养的体外模型和裸鼠移植性肿瘤的体内模型。其中，裸鼠移植性肿瘤模型：由于提供了局部组织微环境，形成的肿瘤更接近在体肿瘤组织，结果更可靠，但裸鼠饲养条件苛刻，价格昂贵，实验周期较长。而肿瘤细胞二维培养模型：操作简便，快捷，灵敏度较高，实验周期短，但是二维培养的细胞并不能完全代替体内生长的肿瘤球^[57-58]，由于体内成熟肿瘤组织多具有复杂的结构，包括增殖旺盛的细胞、静止细胞和坏死组织等，具有明显的异质性^[59]。同样，在肿瘤组织内部，不同部位细胞对化疗药物的敏感性也不同。距血管近的细胞营养丰富、氧供充足、增殖活性强，对化疗药物的敏感性也强；而距血管远的细胞由于营养和氧气供给困难，多出现缺氧甚至坏死现象^[60-61]，这部分细胞对化疗药物的敏感性较低。因此，实验通过目前临床常见的几种化疗药物的敏感性研究也进一步证实体外构建的三维肝癌细胞模型，由于其组织结构更接近在体组织，对药物的敏感性与二维细胞模型有显著差异。因此，体外构建接近在体的三维肿瘤细胞模型，将成为抗肿瘤药物筛选提供重要的研究模型体系，将提供较二维平面培养更可靠、更接近在体的实验结果。