小鼠胰星状细胞的分离培养及鉴定

Isolation, culture and identification of mouse pancreatic satellite cells

目的:建立简单易行的小鼠胰星状细胞(PSCs)原代培养方法,为胰腺纤维化研究提供可靠的体外细胞模型.方法:采用植块法结合酶消化法进行培养,即在无菌条件下取小鼠正常胰腺组织,经过剪碎、胰蛋白酶消化后植入培养瓶中贴壁培养,并予限制性条件培养基进行纯化,通过倒置生物显微镜对传代前后所培养细胞进行形态学、自发荧光及油红染色脂滴的观察,并结合细胞免疫化学和免疫荧光等方法来鉴定小鼠PSCs.结果:小鼠原代PSCs培养3-4d后油红染色阳性,并能观察到自发荧光现象;传代后的细胞形态主要表现为体积较大,伪足发达,呈"星芒状"或"乌贼样";细胞免疫荧光染色和细胞免疫化学染色显示α-平滑肌肌动蛋白(α-SMA)表达阳性.结论:植块与酶消化结合法分离小鼠胰腺星状细胞,简单实用、成功率高,能够满足体外实验的要求.

AIM： To establish a simple method for culture of primary mouse pancreatic stellate cells （PSCs） and provides a reliable in vitro cell model of pancreatic fibrosis. METHODS： Under sterile conditions, mouse pancreas tissue was collected, minced with scissors, digested with trypsin, and cultured in culture flasks. Cells were then purified in conditioned medium. To identify mouse PSCs, morphological observation, autofluorescence detection, oil red O staining, immunocytochemical staining and immunocytofluorescent staining were performed.RESULTS： Autofluorescence and oil red O-stained lipid droplets were observed in primary PSCs cultured for 3 to 4 days. Passaged PSCs were large and had well-developed ＂astral-like＂ or ＂squid-like＂ pseudopodia. Immunocytochemical staining and immunocytofluorescent staining showed that PSCs expressed α-smooth muscle actin （α-SMA）. CONCLUSION： Enzymatic digestion in combination with tissue culture is a simple method for separation of mouse PSCs that has a high success rate and can meet the requirements of in vitro experiment.