一种简易高效的大鼠原代肝细胞分离方法
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摘要
目的:寻找一种简易高效的大鼠原代肝细胞分离方法。方法:在Seglen两步胶原酶灌注法的基础上加以改进,按门静脉灌注法分离培养大鼠肝细胞,重复10次分离肝细胞实验,观察各项指标结果。采用胰酶、Ⅳ型胶原酶经门静脉灌注,大鼠肝脏肝门部结构、肝上及肝下腔静脉封闭保留胶原酶消化分离获取肝细胞,经80目和200目的筛网依次过滤细胞后,细胞悬液分别以1 000、500、300 r/min离心各5 min以纯化肝细胞,以台盼蓝排染法测定细胞活性,HE染色鉴定肝细胞纯度。结果:平均每只成年Wistar雄性大鼠可以获得(1.53±0.31)×10~8个肝细胞,平均获得肝细胞活率可达到94.63%,平均获得肝细胞纯度为96.7%。结论:本实验介绍的方法简便易行,且肝细胞产量较高,活力好,纯度高,适宜在一般条件的实验室推广和应用。
Objective:To search a facility and high efficicenty separation method of rat primary hepatocytes.Methods:Rat hepatocytes were isolated and purified by using the modified Seglen' s two-step method,portal vein perfusion.Hepatocyte tests were repeated about ten times to observe.Rats served as hepatocyte donors using Ⅳ type collagenase and pancreatin via the portal vein.Liver hepatic portal structures,the upper and inferior vena cava of the donors were closed to retain collagenase digestion to obtain hepatocytes.Following filtration through 80-mesh and 200-mesh sieves,the suspension was transferred to a centrifuge tube,and then hepatocytes were purified at 1 000,500,300 r/min centrifugation about respectively(each 5 minutes).The cell viability and purity were identified by trypan blue and HE stain.Results:The average yield of hepatocytes were(1.53±0.31)×10~8 cells per rat with an average viability of 94.63%,and the purity was 96.7%.The hepatocytes showed as oval,roundness and polygon with HE stain,and there was one or two circular nuclei in each cell.Conclusion:The separation method of hepatocytes was simple and easy,with advantages of high yield good viability and high purity,so it would be applied widely in ordinary laboratory.
Objective:To search a facility and high efficicenty separation method of rat primary hepatocytes.Methods:Rat hepatocytes were isolated and purified by using the modified Seglen' s two-step method,portal vein perfusion.Hepatocyte tests were repeated about ten times to observe.Rats served as hepatocyte donors using Ⅳ type collagenase and pancreatin via the portal vein.Liver hepatic portal structures,the upper and inferior vena cava of the donors were closed to retain collagenase digestion to obtain hepatocytes.Following filtration through 80-mesh and 200-mesh sieves,the suspension was transferred to a centrifuge tube,and then hepatocytes were purified at 1 000,500,300 r/min centrifugation about respectively(each 5 minutes).The cell viability and purity were identified by trypan blue and HE stain.Results:The average yield of hepatocytes were(1.53±0.31)×10~8 cells per rat with an average viability of 94.63%,and the purity was 96.7%.The hepatocytes showed as oval,roundness and polygon with HE stain,and there was one or two circular nuclei in each cell.Conclusion:The separation method of hepatocytes was simple and easy,with advantages of high yield good viability and high purity,so it would be applied widely in ordinary laboratory.
引文
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[2] Guo XG,Miao JY,Wang ZY.Protective effect of L-NAME to chronic liver induced by CC14 in rats[J].Journal of the Fourth Military Medical University,2006,22(22):2061-2063.
[3] Han JQ.New progress in vitro hepatocyte culture[J].Journal of Hebei Medical University,2002,23(3):184-186.[韩聚强.体外肝细胞培养技术新进展[J].河北医科大学学报,2002,23(3):184-186.]
[4] Luo MZ,Wang SR,Qi H.A review in primary culture of hepatocytes[J].Journal of Shaanxi Normal University(Natural Science),2004,32(1):72-75.[罗明制,王淑瑞,齐浩.肝细胞原代培养研究综述[J].陕西师范大学学报(自然科学版),2004,32(1):72-75.]
[5] Seglen PO.Preparation of isolated rat live cells[J].Methods Cell Briol,1976,13:29-83.
[6] Liu ML,Mars WM,Zarnegar R,et al.Collagenase pretreatment and the mitogenic effects of hepatocytes growth factor and transforming growth factor in adult rat liver[J].Hepatology,1994,19(6):152-159.
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[8] Pfeiffer E,Kegel V,Zeilinger K,et al.Featured article:Isolation,characterization,and cultivation of human hepatocytes and nonparenchymal liver cells[J].Exp Biol Med (Maywood),2015,240(5):645-656.
[9] Hasebe T,Tanaka H,Sawada K,et al.Bone morphogenetic proteinbinding endothelial regulator of liver sinusoidal endothelial cells induces iron overload in a fatty liver mouse model[J].J Gastroenterol,2017,52(3):341-351.
[10] Lacotte S,Slits F,Orci LA,et al.Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma[J].Oncoimmunology,2016,5(11):e1234565.
[11] Yao YQ,Zhang DF,Huang AL,et al.Low concentration of collagenase in situ circulatory irrigation was used to isolate primary rat hepatocytes[J].Modern Medicine & Health,2001,17(2):84-85.[姚云清,张定风,黄爱龙,等.低浓度胶原酶原位循环灌流法用于原代大鼠肝细胞分离[J].现代医药卫生,2001,17(2):84-85.]
[12] Chen HM,Liao H,Gao J.Advances in hepatocyte culture methods[J].Journal of Cell Biology,2002,24(3):163-166.[陈慧梅,廖红,高静.肝细胞培养方法研究进展[J].细胞生物学杂志,2002,24(3):163-166.]
[13] Zhang LF,Wang P,Hu X.Comparison of three primary hepatocyte separation methods[J].Journal of Jiangxi Medical College,2009,49(5):40-43.[张丽芳,王萍,胡晓.三种原代大鼠肝细胞分离方法的比较[J].江西医学院学报,2009,49(5):40-43.]
[2] Guo XG,Miao JY,Wang ZY.Protective effect of L-NAME to chronic liver induced by CC14 in rats[J].Journal of the Fourth Military Medical University,2006,22(22):2061-2063.
[3] Han JQ.New progress in vitro hepatocyte culture[J].Journal of Hebei Medical University,2002,23(3):184-186.[韩聚强.体外肝细胞培养技术新进展[J].河北医科大学学报,2002,23(3):184-186.]
[4] Luo MZ,Wang SR,Qi H.A review in primary culture of hepatocytes[J].Journal of Shaanxi Normal University(Natural Science),2004,32(1):72-75.[罗明制,王淑瑞,齐浩.肝细胞原代培养研究综述[J].陕西师范大学学报(自然科学版),2004,32(1):72-75.]
[5] Seglen PO.Preparation of isolated rat live cells[J].Methods Cell Briol,1976,13:29-83.
[6] Liu ML,Mars WM,Zarnegar R,et al.Collagenase pretreatment and the mitogenic effects of hepatocytes growth factor and transforming growth factor in adult rat liver[J].Hepatology,1994,19(6):152-159.
[7] Papeleu P,Vanhaecke T,Henkens T,et al.Isolation of rat hepatocytes:Methods in molecular biology[M].Totowa:Humana Press,2005:229-238.
[8] Pfeiffer E,Kegel V,Zeilinger K,et al.Featured article:Isolation,characterization,and cultivation of human hepatocytes and nonparenchymal liver cells[J].Exp Biol Med (Maywood),2015,240(5):645-656.
[9] Hasebe T,Tanaka H,Sawada K,et al.Bone morphogenetic proteinbinding endothelial regulator of liver sinusoidal endothelial cells induces iron overload in a fatty liver mouse model[J].J Gastroenterol,2017,52(3):341-351.
[10] Lacotte S,Slits F,Orci LA,et al.Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma[J].Oncoimmunology,2016,5(11):e1234565.
[11] Yao YQ,Zhang DF,Huang AL,et al.Low concentration of collagenase in situ circulatory irrigation was used to isolate primary rat hepatocytes[J].Modern Medicine & Health,2001,17(2):84-85.[姚云清,张定风,黄爱龙,等.低浓度胶原酶原位循环灌流法用于原代大鼠肝细胞分离[J].现代医药卫生,2001,17(2):84-85.]
[12] Chen HM,Liao H,Gao J.Advances in hepatocyte culture methods[J].Journal of Cell Biology,2002,24(3):163-166.[陈慧梅,廖红,高静.肝细胞培养方法研究进展[J].细胞生物学杂志,2002,24(3):163-166.]
[13] Zhang LF,Wang P,Hu X.Comparison of three primary hepatocyte separation methods[J].Journal of Jiangxi Medical College,2009,49(5):40-43.[张丽芳,王萍,胡晓.三种原代大鼠肝细胞分离方法的比较[J].江西医学院学报,2009,49(5):40-43.]