摘要
试验旨在探讨胶原酶消化法分离并培养肉鸡肺动脉平滑肌细胞(pul monary artery smooth muscle cell,PASMC)的方法。将1～7日龄雏鸡的肺动脉中膜剪成小于1 mm2的组织块,0.1%Ⅱ型胶原酶消化分离PASMC并进行原代培养和传代培养。倒置显微镜下观察培养细胞的形态,对培养细胞进行HE染色和光学显微镜观察,并采用抗α-肌动蛋白单克隆抗体(α-Smooth muscle actin)免疫组化染色法检测细胞胞浆内α-肌动蛋白的表达,用以鉴定所培养的细胞。结果表明,倒置显微镜下细胞为长梭形,呈典型的"峰—谷"状,未见明显异型细胞。α-actin胞浆染色阳性细胞数占总细胞数的97%以上。因此,胶原酶消化法可用于分离培养肉鸡PASMC,且有方法简单,成活细胞数多、传代周期短的特点,可用于肉鸡心血管疾病如肺动脉高压和肺血管重构的研究工作。
Experiment was designed to culture pulmonary artery smooth muscle cells of broiler with the enzymaic digestion method.Pulmonary artery media layer from 1 to 7 day old broilers was cut into less than 1 mm×1 mm pieces,and was digested for pulmonary artery smooth muscle cells by 0.1% collagenase type Ⅱ.The obtained cells were then cultured and subcultured.Identifications of the cells were performed with the morphological feature and immunohistochemical stain using monoclonal antibody against smooth muscle α-action.The results showed that live cells and HE stained cells were typical spindle-shaped and overlapping layer form of vascular smooth muscle cells under phase contract and light microscope.α-actin positive stained cells were more than 97%.Therefore,the results showed that 0.1% collagenase type Ⅱcould digest and isolate broiler PASMC.This method for culture of PASMC has advantages of easy performance,more living cells and short period for passaging.This method can be used for study in cadiovascular disease such as pulmonary hypentension and pulmonary vascular structural remodelling.
出处
《中国畜牧兽医》
CAS
北大核心
2010年第2期55-58,共4页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(30600440)
关键词
肺动脉平滑肌细胞
细胞培养
Ⅱ型胶原酶
pulmonary artery smooth muscle cell
cell culture
collagenase type Ⅱ
