三种方法体外同步分离培养大鼠肝枯否细胞和星状细胞的比较  

Comparison of three methods of isolating and cultivating synchronously rat Kupffer cell and hepatic stellate cell in vitro

作　　者：成家茂[1] 潘志恒[2] 谢瑶[3] 何宏文[3] 何义华[2] 

机构地区：[1]大理医学院人体解剖学教研室,1671000 [2]中山大学附属第三医院中医科,510630 [3]中山大学人体解剖学教研室,510080

出　　处：《现代消化及介入诊疗》2007年第4期220-224,共5页Modern Interventional Diagnosis and Treatment in Gastroenterology

基　　金：国家中医药管理局基金(No:04-05JP51)

摘　　要：目的比较淋巴细胞分离液(LS)、Percoll液(PS)和Nycodenz液(NS)同步分离培养大鼠肝枯否细胞(KC)和星状细胞(HSC)的优缺点。方法①采用链酶蛋白酶E、胶原酶Ⅳ联合原位灌流消化,分别以LS、PS和NS的非连续密度梯度离心法同步分离培养大鼠KC和HSC。②碳素墨汁吞噬试验鉴定KC;α-SMA免疫细胞化学染色法鉴定HSC。③倒置相差显微镜下观测细胞形态,台盼蓝染色显示细胞活性,计数后计算细胞的得率、存活率和纯度。结果体外成功地同步分离培养了KC和HSC;培养的KC吞噬功能明显,而传代HSCα-SMA免疫细胞化学染色阳性者几乎达100%;采用PS或NS体外同步分离KC的细胞得率和纯度均显著高于采用LS分离细胞所得(P<0.01),而采用NS体外同步分离肝KC的细胞存活率显著高于PS或LS分离细胞的存活率(P<0.05);分别采用LS、PS和NS同步分离HSC,三者的细胞存活率逐次升高(P<0.05或P<0.01),而使用LS同步分离的细胞虽然细胞得率比较高(P<0.05),但其细胞纯度较使用PS和NS明显降低(P<0.05),PS和NS分离获得的HSC得率和纯度均无明显统计学差异。结论采用Percoll液和Nycodenz液体外同步分离培养大鼠肝KC和HSC效果好,且后者的细胞存活率最高。Objective To compare the methods of synchronously isolating and cultivating rat Kupffer cell （KC） and hepatic stellate cell （HSC） in vitro by lymphocyte isolation medium （LS）, Percoll gradients （PS） and Nycodenz gradients （NS）. Methods Rat primary KCs and HSCs were isolated and cultured by in situ perfusion with collagenase Ⅳ and pronase E, discontinuous density gradient centrifugation with LS, PS and NS. KCs were identificated by prepared Chinese ink phagocytosis test, and HSCs were identified by Immunocytochemical staining for α-smooth muscle actin （α- SMA）. The morphological changes of KCs and HSCs were monitored under inverted phase contrast microscope. Trypan blue staining was used to display the cell activity as the cell yield, survival rate and purity were evaluated after counting number. Results KCs and HSCs were isolated synchronously and cultured successfully in vitro. The living KCs had strong phagocytosis activity, and the positive α-SMA-staining cells in passage HSCs almost reached to a hundred percent. The yield and purity of KCs by PS or NS were higher than those by LS （P 〈 0.01）, and the survival rate by NS was higher than that by PS or LS （P 〈 0.05）. The survival rate of HSCs was increased gradually by LS, PS and NS （P 〈 0.05 or P 〈 0.01）. Though the yield of HSCs by LS was higher than that by PS or LS （P 〈 0.05）, the purity of HSCs by LS was significantly lower than that by PS or NS （P 〈 0.05）. There was no statistically different regarding the yield and purity of HSCs between by PS and by NS. Conclusions KCs and HSCs was isolated and cultured efficiently by Percoll or Nycodenz gradients, the highest cell survival rate was obtained by Nycodenz gradients.

关 键 词：细胞分离 枯否细胞 肝星状细胞 细胞培养 大鼠 

分 类 号：R285.5[医药卫生—中药学] R329-33[医药卫生—中医学]