大鼠原代肝细胞分离培养的实验研究

Experimental study on isolation and cultivation of primary rat hepatocytes

目的:探讨胶原酶经肝门静脉灌流分离肝细胞及培养的可行性。方法:取10只SD大鼠,按照改良Seglen原位两步胶原酶灌流法分离培养原代肝细胞。以SD大鼠作为肝细胞供体,采用Ⅳ型胶原酶经门静脉灌注,下腔静脉封闭保留胶原酶消化分离肝细胞,经200目筛过滤,滤液以200 r/min离心3～5 min,重复3次,纯化肝细胞。台盼蓝拒染实验检测分离细胞活性。将10只SD大鼠分离得到的肝细胞混合,稀释50倍后接种于包被有鼠尾胶原的培养皿或培养板中,倒置显微镜下观察分离肝细胞的形态特征,糖原染色法(PAS法)鉴定分离肝细胞,MTT法测定肝细胞在培养不同时期的增殖情况。结果:平均每只大鼠肝脏分离获得(1.8±0.14)×108/mL肝细胞,细胞活率>85%。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。MTT实验结果表明在培养的第八天肝细胞增殖达到峰值,培养的第九天细胞活力开始下降。结论:采用胶原酶经肝门静脉灌流分离肝细胞是一种简单、高效的获得肝细胞的方法。

Objective：To investigate the feasibility of isolation and cultivation of primary rat hepatocytes by collagenase perfusion through hepatic portal vein.Methods：10 Sprague-Dawley rats as liver cell donor were used to obtain hepatocytes by Seglen in situ two-step collagenase perfusion through hepatic portal vein.Following filtration through 200-mesh sieves,the suspension was transferred to a centrifuge tube,and then purified hepatocytes at 200 r/min centrifugation for three times（3~5 min each time）.Trypan blue staining was utilized to measure cell viability.Hepatocytes were cultured on the plates covered with rat tail collagen.The morphology of hepatocytes was observed under the inverted microscope.The isolated liver cells were identified by Periodic acid-Schiff reaction（PAS）.The proliferation of the hepatocytes was detected by MTT.Results：The yield of the hepatocytes was（1.8±0.14）×108/mL from each rat.The viability of harvested primary hepatocytes were above 85%.Numerous glycogenosomes were found in cultured hepatocytes and revealed redness by PAS staining.The MTT results showed that the proliferation ability of the cultured hepatocytes reached peak on 8 d cultivation,but the viability of the hepatocytes began to decline on 9 d.Conclusion：Obtaining liver cells from the rat liver by collagen perfusion through hepatic portal vein is a simple and effective method.