改良组织块法培养C57BL／6小鼠胰腺星状细胞

Modified tissue culture for C57BL/6 mouse pancreatic stellate cells

目的通过改良传统组织块法培养C57BL／6小鼠胰腺星状细胞（PSCs），建立一种简便易行、快速获得大量小鼠PSCs的培养方法。方法分离C57BL／6小鼠胰腺组织，经胎牛血清清洗后剪切成0．5—1mm’组织块，经贴壁培养4d取原代PSCs，并传代。采用油红0染色观察细胞内脂滴变化，采用细胞免疫法及蛋白质印迹法检测细胞α—SMA、Desmin表达。结果贴壁4d的原代PSCs多呈椭圆形或星形，胞质内大量脂滴，表达α-SMA及Desmin蛋白的细胞少；传代后细胞变大、伪足丰富，脂滴减少或消失，原代、传代PSCs的d—SMA蛋白相对表达量分别为0．653±0．071、2．290±0．055，传代细胞的表达显著高于原代细胞（P〈0．05），表达Desmin蛋白的细胞数量显著增加。结论改良组织块法可高产和高纯度地获得小鼠静息态和激活态两种状态的PSCs，可以满足体外研究的需要。

Objective To culture C57BL/6 mouse pancreatic stellate cells （PSCs） with a modified method, and to establish a simple cultivation method with a high yield of PSCs. Methods Pancreatic tissues in C57BL/6 mice were collected, washed by fetal bovine serum, minced into 0.5 - 1 mm3, and cultured in sterile culture flasks. After 4days, primary PSCs were collected and passed on. The changes in intracellular lipid droplets were identified by oil red O staining; α-SMA, desmin expression was detected by immunocytochemical or immunocytofluorescent staining, and Western blot. Results Outgrowth ceils in the fourth day were oval or star with abundant lipid droplets and fewer ceils expressed α-SMA and desmin protein. PSCs turned into bigger, more pseudopodial ones with decreasing lipid droplets and significantly increasing expression of α-SMA and desmin protein after the passage. Protein expression of α-SMA in primary and passage PSCs were 0. 653 ± 0.071, 2. 290 ± 0.055, respectively; and the expression in passage PSCs was significantly higher than that in primary PSCs （P 〈0.05 ）, and the number of cells expressing desmin protein was significantly increased. Conclusions Modified tissue culture is a high yield and high purity method for both quiescent and activated PSCs, which can meet the need of in-vitro studies.