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细胞转染方法及脂质体介导法

发布: 2019-10-09 966阅读 222评论

细胞转染是指将外源核酸物质（DNA或者RNA）输送至真核细胞内部的技术，以进行多种细胞生理和生化的研究，包括：代谢途径研究；基因表达调控；癌症&疾病模型相关研究以及重组蛋白研究。随着基因和蛋白功能研究的发展，细胞转染技术已经成为科研试验中最常用的技术手段之一。外源基因导入真核细胞主要有四种方法：磷酸钙法、电击法、脂质体介导法和病毒介导法。

电击法是利用高脉冲电压可逆地击穿细胞膜形成瞬时的小孔促使外源DNA分子进入细胞内，适合所有类型的细胞，但细胞致死率高，DNA和细胞用量大，且需根据不同细胞类型优化电穿孔条件。

磷酸钙法是利用细胞内吞作用将磷酸钙介导共沉淀的磷酸钙-DNA复合物纳入细胞中的方法。该方法可用于瞬时或稳定转染，但对pH、温度和缓冲盐溶液浓度的微小变化十分敏感，导致该方法可重复差。且磷酸钙法对许多类型的细胞（尤其是原代细胞）具有细胞毒性，转染效率较差。

病毒介导法通过侵染宿主细胞将外源基因整合到宿主染色体中，可用于难转染细胞、原代细胞和稳定性转染，也可用于蛋白质过表达或抑制，是临床研究中常用的方法之一。但是病毒介导法对插入的目的片段长度有一定的限制，技术难度比较高，而且某些病毒起效时间比较慢，跟不上细胞快速繁殖的速度。单纯用于简单细胞系的转染，其性价比不高。

脂质体介导法利用带正电的脂质体与核酸带负电的磷酸基团形成复合物，被表面带负电的细胞膜吸附，进而通过融合或细胞内吞作用导入外源DNA/RNA（Figure 1）。脂质体转染适合用于把DNA转染入悬浮或贴壁培养的细胞中，其转染效率高，是目前实验室最方便的转染方法之一。

图1. 脂质体介导的转染和细胞内吞（Parker et al. 2003）。

脂质体转染protocol

理想的细胞转染方法，要求转染效率高、细胞毒性小。对于很多普通细胞系，一般的瞬时转染方法多采用脂质体介导法。接下来，我们主要探讨一下脂质体转染。进行实验之前，需要先规划好实验安排，一般细胞转染需要24-72h，所有要充分了解所用细胞生长速度，计算所需脂质体和DNA的用量。

实验试剂及耗材：

DMEM培养基、血清、转染试剂、灭菌PBS

培养皿、离心管、微量移液器、涡旋振荡器、恒温水浴箱、台式离心机、观察用倒置显微镜、计数板

实验步骤：

1． 细胞传代

（1） 弃掉培养皿中的培养基，用1 ml的PBS溶液洗涤两次

（2） 加入1 ml 1×Trypsin，放入细胞培养箱中化1 min（37℃，5% CO2）。用手轻拍培养皿，观察到细胞完全从底部脱落为止。

（3） 加入3~5 ml含10%血清的培养基终止消化

（4） 用1 ml枪头多次吹打，使细胞完全分散开来

（5） 将培养液移入15 ml离心管中，700~1000 rpm离心5 min（转速和时间可视细胞状态及数量进行调整）

（6） 移去培养液，用新的含血清培养液重悬细胞，细胞计数后选择0.8*106个细胞加入一个35mm培养皿（可根据实际情况进行调整）

（7） 加入合适体积完全培养液，轻轻混匀细胞使其分布均匀

（8） 将培养皿转入CO2培养箱中培养过夜

2. 细胞转染

（1） 选择合适的混合比例（脂质体体积：DNA质量1：1 — 1：2）来转染细胞（Table 1）。在两个1.5 ml离心管管中加入合适的等体积的Opti-MEM，并分别加入合适质量的DNA、脂质体。用移液枪轻轻吸吹混匀后静置孵育5 min。随后将两管混合为一管，孵育20 min。

（2） 吸去培养皿中的培养基，用PBS或无血清培养基清洗一次

（3） 加入转染混合液，将细胞放回培养箱中培养4h

（4） 根据细胞类型决定是否移除转染混合液，之后加入适量完全培养液继续培养24~48 h

表1: 不同培养皿中DNA与脂质体转染比例

如何增强转染效率？

若是转染后发现转染效率不高（若转染的质粒带有荧光标签，可以同倒置荧光显微镜观察转染效率），可以通过两种方法增强转染效率：

a) 复转染，即转染后12~24 h在进行转染，前提是该细胞系对脂质体的耐受性较好，转染后细胞死亡数较少

b) 通过药物筛选来杀死未转入成功的细胞。前提是该质粒带有抗生素抗性基因

图 2: 细胞转染流程示意图。

RECOMMEND

安必奇专业团队对细胞转染技术进行全面优化，可方便有效地用于动物细胞基因沉默和过表达。我们拥有多个成熟的细胞遗传修饰技术平台，如基因敲除稳定转染细胞株、基因过表达稳定转染细胞株、病毒颗粒组装等，可根据您的需求提供最优质的服务。

服务内容：细胞转染试剂、稳定转染的细胞株、病毒包装定制、表型及基因功能分析服务

我们的优势：快速高效-安必奇专业团队已将细胞转染服务流程进行优化、规范，从而为客户提供更高效快速的服务。客户自定义服务-安必奇拥有先进的仪器设备及专业的研究团队，可根据客户的需求提供定制的载体构建、细胞转化以及表型和基因功能分析等一站式的服务。

参考文献：

1. Alan L. Parker. et al. Nonviral gene delivery: techniques and implications for molecular medicine. Cambridge University Press. 2003; 5:3.

2. David A. Dean and Joshua Z. Gasiorowski. Liposome-Mediated Transfection. Cold Spring Harbor

Protocols. 2011. doi:10.1101/.

3. Michael Whitt. Linda Buonocore. John K. Rose. Liposome‐Mediated Transfection. Current Protocols in Immunology banner. 1992; 3(1): 10.16.1-10.16.4.

4. Tae Kyung Kim and James H. Eberwine. Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010; 397(8):3173–3178.

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