基于人和小鼠细胞共培养体系研究隧道纳米管
Study of Tunneling Nanotubes Based on Co-Culture System of Human and Mouse Cells

作者: 姚伟城 * , 王永强 * , 邓伟溪 , 彭江云 :中山大学孙逸仙纪念医院基础与转化医学研究中心、广东省恶性肿瘤表观遗传与基因调控重点实验室,广东 广州;

关键词: 细胞共培养隧道纳米管通讯连接 Cell Co-Culture Tunneling Nanotubes Communicating Junctions

摘要:
隧道纳米管(tunneling nanotubes, TNT)是一种新型的细胞间通讯连接方式。越来越多的证据显示,TNT参与小分子、病原体和部分细胞器等的细胞间传递,在细胞生理病理过程中发挥重要作用。本文将人和小鼠细胞共培养,利用激光显微成像技术对细胞间TNT的产生过程以及特征进行观察和分析。结果显示,在人和小鼠乳腺肿瘤细胞共培养体系中,同类细胞间可以形成TNT通讯结构,但是在人和小鼠细胞之间却没有。另外,在时间序列拍摄过程中发现,细胞间TNT倾向于细胞间指向性接触后形成,同时在人和小鼠活细胞共培养体系中会出现同类型细胞“汇聚现象。因此细胞间TNT的形成可能是基于特异胞间信号诱导接触,并且只能在特定细胞间建立TNT通道连接。

Abstract: Tunneling nanotubes (TNT) is a new type of intercellular communication connection. More and more evidence shows that TNT is involved in the intercellular transmission of small molecules, pathogens, and some organelles, and plays an important role in cell physiological and pathological processes. In this study, we observed and analyzed the production and characteristics of TNT between human and mouse cells in co-cultured by laser microscopic imaging technique. The results showed that the TNT structure could be formed in the same type cells, but not in the different type cells in the co-culture system of human and mouse tumor cells. In addition, it was found that the establishment of TNT between cancer cells tended to be formed after the directional contact between cells in the process of time series shooting, and the phenomenon of convergence of the same type cells were also found in the co-culture system of human and mouse living cells. Therefore, the formation of TNT between cells maybe induced based on specific intercellular signal and TNT can only build between specific cells.

1. 引言

细胞间的通讯方式有间隙连接、外泌体以及最近发现的隧道纳米管 [1]。隧道纳米管是基于细胞之间的直接膜通道连接,比间隙连接的联系距离明显要远,同时因为是细胞间直接的封闭膜管道,所以相较于外泌体膜泡不容易受到细胞外环境因子的影响,是一种有针对性的细胞间通讯方式。最近的诸多研究都描述了纳米隧道管广泛的生物学作用,且该结构几乎在所有细胞中都可以形成 [1] - [12],也有不少报道指出在不同类型细胞间也会建立TNT联系 [13] [14] [15]。在这里,我们将分别源于人和小鼠的乳腺癌细胞进行共培养,且在该体系中对TNT进行观察和分析。

TNT是Rustom等人首先在大鼠细胞(PC12细胞)和人细胞系中进行描述 [1],后来的研究者们发现在大多数的细胞中都可以建立TNT。除此之外,在3D培养细胞群以及组织中 [16] [17] 也有发现,说明TNT不仅仅存在于实验室条件下,在生物体内也会自然存在。TNT的平均长度大概为几微米到上百微米不等,在培养细胞状态下总是以最短距离连接两个细胞,在组织中由于细胞密度高挤压而呈现弯折形态。在培养皿中,连接两个细胞的TNT通常起始端和终端位于细胞下底部,而中间部分悬浮于培养基基质中,而非贴壁。已有的研究发现TNT对膜蛋白酶-EDTA消化液的处理并不敏感,但是会因细胞松弛素D的处理而消除 [18] [19],说明TNT可能是细胞间内部骨架的直接连接。

已有的报道显示,TNT有助于小分子、RNA、自噬体、溶酶体、线粒体、病毒等的细胞间运输 [8] [13] [14] [15] [20] [21] [22] [23]。除此之外,也会参与病理条件下的细胞间通讯,例如在胱氨酸病中,巨噬细胞中的溶酶体经TNT转移到成纤维细胞;急性呼吸窘迫症中线粒体经由间充质基质细胞转移到巨噬细胞中 [15] [21]。但是作为一种新型的胞间物质传递结构,其研究依然相对比较少。虽然在分子水平上对TNT已有描述 [24] [25] [26],但是其分子促成机制、调控机制以及有无相关标志物和可否用于特异性治疗等都还在研究讨论阶段。本文以人鼠细胞共培养体系为基础,通过实验观察和分析发现TNT建立可能需要特殊条件下才可以进行,并且对TNT形成过程做了一定的实验观察和分析。

2. 结果

2.1. 体外培养的癌细胞间TNT形成的观察

TNT作为一种重要的细胞间通讯桥梁,在近十多年被广泛的研究,围绕其功能分析以及形成分子机制的研究逐步增多。在分别将小鼠4T1细胞系和人来源MCF-7细胞系进行培养过程中,发现在两种细胞内都会可以形成TNT结构(图1白色箭头所示)。

Figure 1. Observation of TNT formation in breast cancer cells cultured in vitro. A. Microscopic observation of TNT in 4T1 cells; B. Microscopic observation of TNT in MCF-7 cells. The structure of TNT is shown by the white arrow and the scale is 50 μm

图1. 体外培养的乳腺癌细胞间TNT形成的观察。A. 4T1细胞中TNT的显微观察;B. MCF-7细胞中TNT的显微观察。白色箭头标出的为TNT结构,标尺 = 50 μm

2.2. 共培养体系中人和小鼠癌细胞间并未形成TNT

在同一细胞系的细胞间可以自发形成TNT传输通道,这有利于细胞间高保真的物质信号传输。TNT作为一种特异性的胞间传输通道,理论上其形成条件也是相对苛刻的,那么在不同种类细胞间是否也有可以形成?为了验证此想法,本文将人和小鼠来源的乳腺癌细胞进行共培养,再利用激光显微断层扫描技术进行观察分析。实验中分别将4T1细胞和MCF-7细胞进行mChery和GFP活细胞标记,然后将具备颜色差异的两种细胞进行共培养。如图2所示,在4T1-GFP细胞与MCF-7-mChery细胞共培养体系中,绿色标记细胞与红色标记细胞间TNT (G-R-TNT)的计数为零,而绿色细胞之间TNT (G-G-TNT)以及红色细胞之间TNT (R-R-TNT)的计数都是接近50% (图2(A))。我们认为造成G-R-TNT计数为零有两种可能原因:1) 人和小鼠细胞间并不形成TNT结构;2) G-R的细胞标记方法造成了假阴性。为了排除实验误差的干扰,我们将同种细胞系也分别标记mChery和GFP,并将不同颜色标记的同类细胞共培养。实验结果显示,4T1和MCF-7细胞系都会形成R-G-TNT结构,只是这种TNT的计数占比仅有10%左右(图2(B))。说明虽然G-R的标记方法对细胞间建立TNT造成影响,但是并未阻止R-G-TNT结构的形成。为此我们推断,在人和小鼠乳腺癌细胞间可能并不形成TNT。

2.3. 共培养体系中细胞的定向迁移现象与TNT发生

在4T1和MCF-7细胞培养皿中,应用激光共聚焦显微镜时间序列扫描功能进行活细胞长时程观察。实验发现培养过程中细胞间TNT的建立更倾向于细胞间接触后形成(图3(A)),而在细胞发生实际接触之前其运动是否具有指向性或者引导性尚不清楚。利用人和小鼠乳腺癌细胞间构建TNT的特殊性,在两类细胞混合培养系中可以较好的观察和验证。将4T1和MCF-7细胞共培养,用移液器将两类细胞吹打混匀。在观察初期可以看出两类细胞均匀相间分布于培养皿中,随着培养时间的加长,两类细胞分别会出现不同程度的“汇聚”现象(图3(B))。由此可以看出,将人和小鼠细胞共培养,两类细胞会体现出各自不同的属性,并且向同类细胞靠近,最终构建各自的TNT链接信息交流系统。

3. 讨论

TNT是一种建立于细胞之间的封闭连接管道,可以在胞间传输化学分子甚至病毒等物质 [8] [13] [14] [15] [20] [21] [22] [23]。自从TNT被发现以来,相关的研究都在不断的丰富,但是研究依然比较局限。本文提出一种思路,将人和小鼠细胞进行共培养,用来对TNT发生发展的特征进行观察并分析,得到一些有趣的发现。

Figure 2. Observation and counting of TNT in the co-culture system of human and mouse cancer cells. A. TNT counting and analysis were performed in the co-culture system of 4T1 cells and MCF-7 cells; B. TNT counting and analysis were performed in the mixed 4T1 cells with green or red fluorescent labels respectively. The scale is 50 μm

图2. 人和小鼠癌细胞共培养体系中的TNT观察与计数。A. 在4T1细胞和MCF-7细胞共培养体系中进行TNT计数与分析;B. 分别带有绿色和红色荧光标记的4T1细胞混合培养,并进行TNT计数与分析。标尺 = 50 μm

Figure 3. Analysis of the cell directional migration and the formation of TNT in vitro cell culture. A. Time series shooting and analysis of the formation process of TNT. The red cells were 4T1 with mChery, the green cells were MCF-7 with GFP, and the TNT formation process was marked by the white dotted line (scale = 20 μm). B. The aggregation trend of cells in vitro cell culture by time series shooting (scale = 50 μm). C. A possible formation model of TNT

图3. 体外培养环境下细胞定向迁移现象与TNT发生分析。A. 细胞TNT形成过程的时间序列拍摄与分析。其中红色细胞为4T1-mChery,绿色细胞为MCF-7-GFP,白色虚线圈标注了细胞TNT形成过程(标尺 = 20 μm)。B. 时间序列拍摄记录培养条件下细胞的聚集趋势(标尺 = 50 μm)。C. 一种可能的TNT建立模型

首先我们分别在独立培养的人和小鼠细胞培养皿中均发现TNT的形成,更进一步体现出TNT建立的广泛性。但是在这两类细胞之间是否可以建立这种直接沟通的桥梁则不得而知。为诠释这种想法,本文将4T1细胞和MCF-7细胞进行mChery和GFP活细胞标记,然后将具备颜色差异的两种细胞进行共培养。在TNT的实验统计中并没有发现建立于人和小鼠细胞之间的TNT。因此我们推断TNT的构建与细胞种属有关系,更倾向于亲缘性靠近的细胞间,而在异物种细胞间很难形成。

将颜色差异的两类细胞吹打均匀后进行共培养,在一段时间后同类细胞会趋于“汇聚”,并且形成TNT连接结构。这种复杂培养环境中,异物种细胞精准“绕行”,并与同类细胞形成TNT连接,提示TNT建立是有指向性的。在动态观察实验中,两个原本并无直接接触的细胞,会逐步在细胞运动后接触,最后产生胞间TNT。综合以上信息,我们推论出一种可能的胞间信息交换模型(图3(C)):在培养状态下的细胞通过外分泌或者其他途径,在其周围建立起类化学物质为基础的特定信号辐射(这些信号可能包括分裂、胁迫应急等),如果有细胞可以识别到相关信号,就会做出导向性运动反应,这很可能是细胞间建立精准TNT信息交流的基础。

4. 材料以及方法

4.1. 试剂

DMEM培养基、胎牛血清、胰酶购于美国Gibco公司;4%多聚Open Access甲醛购于Thermo Fisher Scientific公司。

4.2. 细胞培养

4T1和MCF-7荧光标记细胞由黄子贤博士(中山大学孙逸仙纪念医院口腔科)友情提供,将4T1细胞和MCF-7细胞分别计数5万个,在共聚焦皿中混合培养。4T1细胞、MCF-7细胞以及两类细胞混合体系均使用10%胎牛血清及DMEM Medium培养,培养箱温度为37℃、CO2浓度为5%,细胞按1 × 105个接种于共聚焦皿中,细胞密度50%~70%备用。

4.3. 荧光成像与分析

使用激光共聚焦显微镜(LCSM 800, Zeiss, Germany)记录图像,该显微照片配备了平面复消色差×40/1.4 NA油浸物镜。实验用激光器波长分别为488 nm和561 nm。图像处理使用ZEN (blue edition)软件。

4.4. TNT的计数方法

在细胞生成浓度在70%时进行TNT计数,TNT的识别标准参考前人文献的报道 [27],只考虑大于10微米的胞间膜桥作为TNT计数。进行细胞TNT计数的图片统一使用3D断层扫描模式进行拍摄,使用ZEN (blue edition)软件纵向正交叠加处理后分析计数。

基金项目

国家自然科学基金(81772821),广东省自然科学基金(2018A030310344)。

参考文献

文章引用: 姚伟城 , 王永强 , 邓伟溪 , 彭江云 (2020) 基于人和小鼠细胞共培养体系研究隧道纳米管。 生物过程, 10, 39-45. doi: 10.12677/BP.2020.104006

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