摘要
目的采用酶消化法进行人脐静脉内皮细胞分离培养,并比较不同消化酶获得细胞数量及细胞存活率的差别。方法分别用0.125%、0.25%胰蛋白酶和0.1%I型胶原酶消化获取脐静脉内皮细胞,比较三种酶液消化的结果,在倒置相差显微镜下观察细胞形态特点,同时用Ⅷ因子免疫荧光法和DiI-acLDL摄取实验对所得细胞进行鉴定。结果0.125%、0.25%胰蛋白酶和0.1%I型胶原酶的最佳消化时间分别为15min、12min、15min,倒置相差显微镜下观察所得细胞呈典型内皮细胞形态,免疫荧光法检测Ⅷ因子相关抗原阳性,且细胞具有DiI-acLDL摄取能力。结论采用酶消化法分离培养脐静脉内皮细胞简便易行,但需严格掌握消化时间以减少细胞损伤,0.1%I型胶原酶是最理想的内皮细胞消化酶。
Objective To compare the efficacy of three enzyme digestion to isolate the primary endothelial cells from human umbilical vein in vitro. Methods Trypsin （0. 125 %, 0. 25 %） or 0. 1% type Ⅰ collagenase was respectively infused into 20-centimeter human umbilical vein and remained inside for 6, 8, 10, 12, 15, 20 min, then the digestion solution and PBS washes were collected to obtain endothelial cells. After the collected endothial cells were cultured with routine method, the cells were observed under inverted phase contrast microscope, their factor Ⅷ related antigen was detected by immunofluorescence method and their ability to uptake DiⅠ-acLDL was examined. Results The optimal digestion time by 0. 125% trypsin, 0. 25% trypsin or 0. 1% type Ⅰ collagenase for primary culture of human umbilical vein endothelial cell was 15 min, 12 min and 15 min respectively. The passaged cells displayed the typical morph of endothelial cells, factor Ⅷ related antigen-positive and the ability to uptake DiⅠ-acLDL. Conclusion Enzyme digestion is simple and reliable to isolate primary endothelial cells from human umbilical vein. The digestion time should be controlled strictly to minimize the cell injury. 0. 1% type Ⅰ collagenase may be the best choice to isolate the endothelial cells.
出处
《华南国防医学杂志》
CAS
2007年第6期1-3,共3页
Military Medical Journal of South China
基金
国家自然科学基金(30470729)
关键词
人脐静脉
内皮细胞
细胞培养
Human umbilical vein
Endothelial cells
Cell culture
