摘要
目的观察外源性成纤维细胞生长因子9(fibroblast growth factor 9,FGF9)对体外大鼠肺泡Ⅱ型上皮细胞(ATⅡ细胞)生长的影响。方法体外原代分离、纯化、培养成年大鼠ATⅡ细胞,锥虫(台盼)蓝拒染法测定细胞活力,透射电镜鉴定细胞,碱性磷酸酶(AKP)染色法确定细胞纯度。实验组分别用10,30,50 ng/ml FGF9和200μg/ml肝素处理细胞24 h,对照组为未经药物处理的ATⅡ细胞。选择30 ng/ml FGF9浓度后,再通过噻唑蓝检测法(MTT法)分别检测24,48,72和96 h的细胞存活率。结果原代培养ATⅡ细胞的产量约为2×107个/鼠,细胞活力>90%,电镜下观察到ATⅡ细胞典型细胞结构——板层小体,细胞纯度>90%。采用30和50 ng/ml浓度FGF9刺激细胞24 h,ATⅡ细胞存活率明显增高(P<0.01)。采用30 ng/ml FGF9刺激ATⅡ细胞不同时间(24,48,72和96 h)发现,24,48,72和96 h细胞存活率均明显高于对照组(P均<0.01)。结论利用弹性蛋白酶消化分离和大鼠IgG免疫黏附纯化,可获得高产量、高纯度及高活力的原代ATⅡ细胞。FGF9以浓度和时间依赖的方式促进ATⅡ细胞增殖。
Objective To observe the effect of ectogenic fibroblast growth factor 9（FGF9） on alveolar type Ⅱ epithelial cells（ATⅡ cells） in rats in vitro.Methods ATⅡ cells were isolated,purified and cultured.The cell viability was assessed by trypan blue staining;the cells were identified under a transmitting electron microscopy（TEM） and the purity of the cultured cells was assayed with AKP staining.Different doses of FGF9（10,30 and 50 ng/ml） and heparin of 200 μg/ml treated primary rat ATⅡ cells for 24 h.The control group was left untreated.After proper concentration,the cellular state was analyzed at 24,48,72 and 96 h by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT）assay.Results About 2×107 cells were obtained from one adult rat.The cell viability was over 90%.The lamellar body of ATⅡ cells was observed under TEM and the purity of cultured cells was over 90%.Compared with the control group,the rate of cell survival increased significantly in 30 and 50 ng/ml of FGF9（P〈0.01） and at 24,48,72 and 96 h（P〈0.01） in 30 ng/ml of FGF9.Conclusion Elastase digestion and specific immunosorption to plates coated with IgG are efficient methods of isolation,and purification of ATⅡ cells.FGF9 stimulates ATⅡ cell proliferation in a dose-and time-dependent manner.
出处
《军事医学科学院院刊》
CSCD
北大核心
2010年第6期573-575,共3页
Bulletin of the Academy of Military Medical Sciences
