摘要
目的 建立一种简便而可靠的肝星形细胞 (HSC)的分离方法。 方法 雄性 SD大鼠 ,常规门脉穿刺插管 ,蠕动泵灌注不含钙 D- Hanks液后游离肝脏 ,先后用含链霉蛋白酶 E、 型胶原酶消化液 37℃体外灌注消化和振荡消化 ,细胞悬液过滤后 ,经 1 1 % Nycodenz密度梯度离心 (2 730 r/ min× 1 5 m in)分离 HSC,锥虫蓝染色排斥法鉴定细胞活率 ,以 1× 1 0 6 m L- 1 密度混悬后接种在培养瓶。结蛋白免疫细胞化学染色鉴定 HSC纯度。 结果 成功分离 HSC,细胞得率 (1 .2～ 2 .3)× 1 0 6 g- 1 肝组织 ,活率 >90 % ,纯度 >90 % ,可用于实验研究。 结论 链霉蛋白酶E和 型胶原酶体外灌注消化及 1 1 % Nycodenz密度梯度离心是分离大鼠 HSC简便。
Objective To establish a simple and reliable method in isolation and primary culture of rat hepatic stellate cells. Methods The liver of adult male Sprague|Dawley rat was routinely perfused through a portal vein catheter with Ca+{2+}|free D|Hanks solution. Then perfused with pronase E and type Ⅳ collagenase dissolved in Ca+{2+} containing D|Hanks solution. The liver was homogenized and incubated with pronase E, type Ⅳ collagenase and DNaseⅠ dissolved in Ca+{2+} containing D|Hanks solution for 20 minutes at 37 ℃ with constant stiring. This suspension was centrifuged by 11% nycodenze density gradient centrifugation for 15 minutes at 1500 g then filtered. The cells were collected, washed, resuspended in DMEM containing 20% calf serum and plated at density of 1×10+6 mL+{-1} in tissue culture glasses. The cells viability was determined by trypan blue exclusion staining. The purity of HSC was identified by the expression of desmin by immunocytochemistry SP method. Results The method of isolation for HSC in vitro by homogenization with pronase E, type Ⅳ collagenase and density gradient centrifugation was successfully established. The yield of the HSC was (1 2~2 3)×10+6 g+{-1}liver tissue, with viability over 90% and purity above 90%. The cell viability and yield rate were reliable and available for further research. Conclusion The method of isolating and primary culturing HSC in this study is simple, economic and reliable.
出处
《福建医科大学学报》
2004年第1期71-73,F003,共4页
Journal of Fujian Medical University
基金
福建省科技计划项目 ( 2 0 0 3 D0 5 )
关键词
肝
星形细胞
细胞分离
细胞
培养的
大鼠
hepatic
stellate cells
cell isolation
cell,cultured
rat
