Wistar大鼠枯否细胞的分离、鉴定与培养

Isolation,Identification and Training of Wistar Rat Kupffer Cells

目的改良、完善枯否细胞(Kupffer cell,KC)的分离培养方法。方法选用健康SPF级Wistar雄性大鼠,采用原位灌注后再采用0.05%Ⅳ型胶原酶离体灌注的方法,经过25%和50%percoll密度梯度离心提取KC。光学显微镜观察细胞形态,台盼蓝鉴定细胞活力,墨汁吞噬实验及免疫细胞化学染色法鉴定细胞纯度。结果大鼠KC细胞数为3.15×107个。经过台盼蓝染色后,显示细胞活力为95%。细胞纯度为96%。结论该研究方法简单,可行。尽量减少分离过程中对肝脏KC的损害和丢失,缩短操作时间,保证细胞数量、纯度及活力,最终形成一套较经济实惠、方便简洁、效果较佳的实验方法。

Objective To improve the isolation and cultivation method of Kupffer cell （KC）. Methods Healthy SPF male Wistar rats were chosen and in situ perfused. 0. 05 %Ⅳ collagenase were used for perfusion in vitro, 25 G and 50% percoll density gradient eentrifugation were used to extract the KC.Optical microscope were utlized to observe cellular morphology.The cell viability were identified evaluated by trypan blue. Ink-phagoeytosing and immunocytochemistry staining were used to identify the purity of KC. Results The quantity of KC was 3.15 × 10^7. The viability of KC was 95 %, the purity of KC was 96%. Conclusion The method is simple and feasible.It is as far as possible to reduce the damage of KC in the process of separation, shorten the operation time, ensure the cell quantity, purity and viability, and eventually form a much more economical, convenient, Dratical experimental method_