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Quasi Vivo流动培养提高人肝脏细胞原代培养时的基因表达

——让体外贴壁细胞培养更接近生理环境

 

鉴于人肝细胞原代培养在药物代谢、毒性、副作用的研究中被视为核心检验标准,而原代培养的肝细胞经常面临诸如解毒基因表达量下调的问题(如膜转运蛋白、结合酶、细胞色素P45),在Vinci B等人的肝细胞研究中(Vinci B, et al. Modular bioreactor for primary human hepatocyte culture: medium flow stimulates expression and activity of detoxification genes. Biotechnol J. 2011; 6: 554-64),认为正是传统静置培养无法模拟的某些生理刺激,引发了基因表达的下调。而Vinci B通过Quasi Vivo流动培养人原代肝脏细胞后,不论基因表达量、酶活还是生物参数,均证明流动培养可特异性地上调多种解毒酶基因的表达,而且流动培养的人贴壁肝细胞,其解毒酶基因家族的表达水平接近或高于新鲜分离的肝脏细胞。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

实验流程

1、将原代肝细胞进行静态培养,在第七天分组,一组保持静态培养,一组改为Quasi Vivo流动培养,继续培养至二十一天,进行检测。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

2、分别进行流动培养、静态培养、标准培养十四天后的肝脏细胞(FT297),形态无差异  

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

3、流动培养下的肝细胞CYP1A1、CYP1A2、CYP2B6、CYP3A4、GSTa等解毒酶基因表达更高,到达峰值时长有差异。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

4、多种解毒基因上调,CYP1A1等基因表达甚至高于新鲜分离的肝脏细胞(FIH)

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

5、流动培养提高了肝脏细胞CYP3A4、UGT2B4/7、CYP2C9的活性,化合物代谢速率显著提高。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

文章小结:

1、人原代肝脏细胞分别进行7-21天的流动培养、静态培养;

2、检测肝脏细胞32个基因的表达量、酶活和生物参数;

3、使用流动培养可提高人贴壁肝脏细胞解毒基因表达:

①UGT(尿苷二磷酸葡萄糖醛酸转移酶)基因家族表达上调:UGT1A1/UGT2B4/UGT2B7;

②多药耐药相关蛋白1 (MDR1)和MRP2基因的mRNA的表达上调;

③外源物质/药物代谢和运输基因表达上调,并激活某些酶活性:CYP1A1/1A2/2B6/2C9/3A4;

④GST(谷胱甘肽S-转移酶)基因表达上调。

 

用于(贴壁)细胞培养的Quasi Vivo流动培养系统,利用培养基循环流动,模拟血流剪切应力环境,更贴近人肝脏细胞的体内环境,能促进原代肝脏细胞增殖、贴壁肝细胞的基因表达等。作为英国Kirkstall公司核心专利产品,Quasi Vivo流动培养系统创新地模拟了体内动态环境,并可同时结合3D培养构建细胞模型。流动培养系统主要由高精度蠕动泵(用于培养基循环流动)、特殊设计的培养腔室(低通量培养用、中-高通量培养用)耗材组成。Quasi Viv设备可自行拼装,操作方便,按产品说明书清洗和灭菌后,所有耗材可长期、重复使用,大大降低了Quasi Vivo的使用成本。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

目前,Quasi Vivo流动培养系统已在全球超过70个专业研究机构获得应用,已成功构建包括呼吸系统(肺成纤维细胞、支气管上皮细胞等)、肝脏、肾脏、心血管、脑组织、糖尿病等研究模型(原代细胞模型)。

英国Quasi Vivo流动培养系统 原代肝细胞基因表达上调 Quasi Vivo中国独家代理商北京泽平

 

Quasi Vivo流动培养系统,贴壁细胞培养实验指导

 

 

Quasi Vivo参考文献

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28.Vinci, B. et al., 2011. Modular bioreactor for primary human hepatocyte culture: Medium flow stimulates expression and activity of detoxification genes. Biotechnology Journal, 6, pp.554–564. 
29.Tommaso S. et al., 2011. Engineering Quasi-Vivo in vitro organ models. Advances in Experimental Medicine and Biology Volume: 745, pp 138-153.
30.Ahluwalia A. et al., 2011. Hepatotoxicity of diclofenac in a Quasi-Vivo™ multicompartment bioreactor. oxicology Letters Volume: 205. 

 

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除人源肝脏细胞外,Quasi Vivo流动培养还可用于小鼠、大鼠、比格犬、食蟹猴原代肝脏细胞培养等各类常规实验动物的贴壁肝脏细胞培养。

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