小鼠肝脏窦状内皮细胞分离及培养的一种新方法

第30卷第1期2010年1月

国际病理科学与临床杂志 htt p://www .gjbl .net

I nternati onal Journal of Pathol ogy and ClinicalMedicine

Vol .30　No .1

Jan .　2010

收稿日期:2009-10-09 修回日期:2010-01-23作者简介:赵秀华,硕士研究生,主要从事成体干细胞的研究。通讯作者:程腊梅,E 2mail:chenglamei2000@yahoo .com

基金项目:国家重点基础研究发展计划项目(2007CB947900);湖南省自然科学基金重点项目(08JJ3075)。The work was supported by

Nati onal Key Basic Research and Devel opment Pr ojects (2007CB947900)and Key Pr ojects of Natural Science Foundati on of Hunan Pr ovince,P .R.China (08JJ3075).

・Arti cles ・

・论　著・

小鼠肝脏窦状内皮细胞分离和培养的一种新方法

赵秀华,罗彬,罗盘,程腊梅

(中南大学生殖与干细胞工程研究所,长沙410078)

[摘要]　目的:建立小鼠肝窦状内皮细胞(liver sinus oidal endothelial cells,LSEC )的分离培养方法,研究其生

物学特性。方法:中性蛋白酶消化小鼠肝脏组织,Percoll 密度梯度离心分离消化后的细胞悬液,用内皮细胞筛选培养基及酶差异消化法纯化LSEC;免疫细胞化学染色检测LSEC 的表面标志,分析LSEC 的超微结构,观察D iI 荧光标记的乙酰低密度脂蛋白(acetylated 2l ow density li pop r otein,D iI 2Ac 2LDL )摄取能力,以体外成血管能力分析LSEC 的功能。结果:分离纯化后的细胞呈典型鹅卵石样内皮细胞形态,这些细胞表达V III 因子,不表达CD31,CD90和

CK19,具有窦状内皮细胞特征性的窗孔结构;能摄取D iI 2Ac 2LDL,并有一定的成血管能力。结论:建立了一种分离、

培养LSEC 的方法,为研究LSEC 的功能奠定了基础。

[关键词]　肝窦状内皮细胞;　条件培养基;　窗孔;　成血管结构;　小鼠doi:10.3969/j .issn .167322588.2010.01.001

A new m ethod for the isol a ti on and culture of

li ver si n uso i da l endotheli a l cells

Z HAO Xiuhua,LUO B ing,LUO Pan,CHENG La mei

(Institute of Reproductive and S te m Cell Engineering,Central South U niversity,Changsha 410078,China )

[Abstract]　O bjecti ve T o establish a method f or the is olati on and culture of mouse liver sinu 2s oidal endothelial cells (LSEC ),and t o study their bi ol ogical characteristics .M ethods L iver tissue fr om mouse was treated with a s oluti on containing 2U /mL dis pase,and then the cell sus pensi on was separated by Percoll density gradient centrifugati on .LSEC was purified by culturing in an endothelial cell selective mediu m and passaged with enzy me discrepancy digesti on .LSEC i m munophenoty p ic characteristicswere an 2alyzed by using cyt o 2i m munostaining .The ultrastructures of LSEC,the ability of up taking the acetylated l ow 2density li pop r otein (acetylated LDL ),and the vascular structures in vitr o were detected .Results The is olated cells showed the typ ical cobblest one mor phol ogy characteristic of endothelial cells .These cells ex 2p ressed fact or Ⅷbut not CD31,C D90,and CK19.They were able t o up take the acetylated l ow 2density li p 2op r otein and t o f or m vascular structure .The results of electr on m icr oscope sho wed that these cells possessed typ ical transcellular fenestrati ons with a sinus oidal origin .Conclusi on W e devel op a method f or the

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