• 首都醫(yī)科大學(xué)北京宣武醫(yī)院普外科（北京100053）;

目的　 建立簡單、有效而穩(wěn)定的大鼠肝臟Kupffer細(xì)胞分離、純化的實(shí)驗(yàn)方法。
方法 　①大鼠肝臟的膠原酶離體灌注； ②Percoll液不連續(xù)梯度離心分離Kupffer細(xì)胞； ③選擇性貼壁純化Kupffer細(xì)胞； ④吞噬墨汁實(shí)驗(yàn)、DAB染色，光鏡、電鏡下觀察鑒定Kupffer細(xì)胞。
結(jié)果 　最終獲得的大鼠肝臟Kupffer細(xì)胞活性 gt;90%，純度為95%，光鏡下Kupffer細(xì)胞具有較強(qiáng)的貼壁及吞噬碳粒能力，DAB染色呈黃色煎蛋樣結(jié)構(gòu)； 新分離的Kupffer細(xì)胞呈圓形，形態(tài)大小一致； 2 d后，細(xì)胞發(fā)生伸展，呈多角形或星形。電鏡下觀察可見典型特點(diǎn)： 有偽足及杯口狀改變，有豐富的溶酶體和吞噬體。
結(jié)論 　本實(shí)驗(yàn)建立的大鼠肝臟Kupffer細(xì)胞的分離培養(yǎng)方法效果穩(wěn)定，同時(shí)培養(yǎng)的細(xì)胞具有良好的生物學(xué)性狀。

引用本文： 蔡偉,李非. 大鼠肝臟Kupffer細(xì)胞的分離、純化. 中國普外基礎(chǔ)與臨床雜志, 2007, 14(3): 292-295. doi: 復(fù)制

版權(quán)信息： ?四川大學(xué)華西醫(yī)院華西期刊社《中國普外基礎(chǔ)與臨床雜志》版權(quán)所有，未經(jīng)授權(quán)不得轉(zhuǎn)載、改編

1. Burt AD, Le Bail B, Balabaud C, et al. Morphologic investigation of sinusoidal cells [J]. Semin Liver Dis, 1993; 13(1)∶21.
2. Seglen PO. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion [J]. Exp Cell Res, 1973; 82(2)∶391.
3. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and re-ticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
4. 韓偉， 成令忠， 顧云娣. 大鼠肝枯否細(xì)胞分離培養(yǎng)與免疫活性的研究 [J]. 解剖學(xué)雜志, 1988; 11（2）∶71.
5. Heuff G, Steenbergen JJ, Van de Loosdrecht AA, et al. Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study [J]. J Immunol Methods, 1993; 159(1-2)∶115.
6. Knook DL, Seffelaar AM, de Leeuw AM. Fat-storing cells of the rat liver. Their isolation and purification [J]. Exp Cell Res, 1982; 139(2)∶468.
7. Ikejima K, Enomoto N, Seabra V, et al. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells [J]. Am J Physiol, 1999; 276(3 Pt 1)∶G591.
8. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
9. 高春芳， 孔憲濤， 范列英. 大鼠肝貯脂細(xì)胞、 Kupffer細(xì)胞的分離、 培養(yǎng)和鑒定 [J]. 中華消化雜志， 1995; 15(3)∶142.
10. Nagelkerke JF, Barto KP, van Berkel TJ. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells [J]. J Biol Chem, 1983; 258(20)∶12221.
11. Valatas V, Xidakis C, Roumpaki H, et al. Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures [J]. Cell Biol Int, 2003; 27(1)∶67.
12. 成令忠主編. 組織學(xué) [M]. 第2版. 北京: 人民衛(wèi)生出版社, 1993∶1200~1209.
  1. 1. Burt AD, Le Bail B, Balabaud C, et al. Morphologic investigation of sinusoidal cells [J]. Semin Liver Dis, 1993; 13(1)∶21.
  2. 2. Seglen PO. Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion [J]. Exp Cell Res, 1973; 82(2)∶391.
  3. 3. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and re-ticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
  4. 4. 韓偉， 成令忠， 顧云娣. 大鼠肝枯否細(xì)胞分離培養(yǎng)與免疫活性的研究 [J]. 解剖學(xué)雜志, 1988; 11（2）∶71.
  5. 5. Heuff G, Steenbergen JJ, Van de Loosdrecht AA, et al. Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study [J]. J Immunol Methods, 1993; 159(1-2)∶115.
  6. 6. Knook DL, Seffelaar AM, de Leeuw AM. Fat-storing cells of the rat liver. Their isolation and purification [J]. Exp Cell Res, 1982; 139(2)∶468.
  7. 7. Ikejima K, Enomoto N, Seabra V, et al. Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells [J]. Am J Physiol, 1999; 276(3 Pt 1)∶G591.
  8. 8. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of Percoll centrifugation and selective adherence [J]. J Leukoc Biol, 1985; 38(2)∶213.
  9. 9. 高春芳， 孔憲濤， 范列英. 大鼠肝貯脂細(xì)胞、 Kupffer細(xì)胞的分離、 培養(yǎng)和鑒定 [J]. 中華消化雜志， 1995; 15(3)∶142.
  10. 10. Nagelkerke JF, Barto KP, van Berkel TJ. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells [J]. J Biol Chem, 1983; 258(20)∶12221.
  11. 11. Valatas V, Xidakis C, Roumpaki H, et al. Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures [J]. Cell Biol Int, 2003; 27(1)∶67.
  12. 12. 成令忠主編. 組織學(xué) [M]. 第2版. 北京: 人民衛(wèi)生出版社, 1993∶1200~1209.