摘要
目的获得活力稳定、高度纯化的体外培养大鼠血管平滑肌细胞,为肿瘤研究提供实验材料;并探讨低氧对抗肿瘤药物研究新靶点:缺氧诱导因子-1α(HIF-1α)的影响。方法无菌下取SD大鼠心肺组织,然后分离出肺动脉段,分离肺动脉中膜层,用组织块贴壁法培养,倒置相差显微镜观察细胞形态、普通免疫组织化学法(SP法)进行细胞鉴定。随后,将体外培养的大鼠PASMCs,设计常氧组、低氧组H2,H6,H12,H24,用Western blotting法检测HIF-1α的蛋白表达。结果用组织贴块法成功培养大鼠PASMCs,得到生长稳定、纯度高的PASMCs,且培养与纯化可以同时进行,可获得稳定传代的细胞;HIF-1α蛋白在常氧组有少量表达,在低氧条件下,其表达明显增加。结论成功建立体外大鼠肺动脉平滑肌细胞原代培养模型;低氧条件下促进其中HIF-1α的表达;这提示HIF-1α蛋白的表达可能是肿瘤细胞适应缺氧环境的重要原因。
Objective To establish a simple and effective primary culture method of mouse vascular smooth muscle cell. Obtaining a lasting and purified cell line of vaseular smooth musele cells（VSMCs） by primary cullture,and to provide these test material srelated researeh. synchronously, observe the expression of Hypoxia Inducible Factor-1α（HIF-1α）： A New Anticancer Drug Target under hypoxia. Method The lung of SD rats was gotten from chest under aseptic condition. Pulmonary artery was isolated and pulmonary artery tissue was planted with the adherent method of tissue explants. The cellular morphology and their typical growth condition were observed under inverted phase contrast microscope. Morphology of the isolated cells were observed by phase-contrast microscopy and identified by immunocytochemistry and immunofluorescence assay using smooth muscle-α-actin antibody. Cultured rat PASMC were divided into normoxic group; H2,H6,H12 and H24 hypoxic group. The protein expression of HIF-1α was measured by Westen blotting. Results： The primary culture PASMCs with the adherent method of tissue explants can stably grow. Culture and purification can perform in the same time; HIF-1α expression was poorly positive in normoxic group and HIF-1α expression was obviously positive after exposing under hypoxia. Conclusion One primary culture method in vitro cell culture mode of aorta and pulmonary artery vaseular smooth musele cells（VSMCs） was builded; hypoxia promoted the expression of HIF-1α; the expression of HIF-1α may be a cause of tumor cell adapted to hypoxia; hypoxia may plays a key role in in the development of human tumour.
出处
《肿瘤药学》
CAS
2011年第2期114-117,共4页
Anti-Tumor Pharmacy
基金
国家自然科学基金(编号:30570815
30270581)
湖南省自然科学基金(编号:05jj30073)
