人腹膜间皮细胞的体外培养及Ⅰ型胶原、TGF-β1表达的检测  

Culture of human peritoneal mesothelial cells in vitro and detection of typeⅠ collagen and TGF-β1 expressions

作　　者：朱桂松[1] 何劲松[2] 

机构地区：[1]南京市中医院ICU,江苏省210001 [2]南京大学医学院附属鼓楼医院肾脏病科

出　　处：《江苏医药》2013年第18期2132-2135,共4页Jiangsu Medical Journal

摘　　要：目的建立人腹膜间皮细胞(hPMCs)体外培养模型,并检测hPMCs中Ⅰ型胶原和转化生长因子β1(TGF-β1)的表达。方法用0.2%胰蛋白酶-0.02%乙二胺四乙酸二钠消化人大网膜组织,进行hPMCs原代培养并传代。采用形态学及免疫组化方法对培养细胞进行鉴定,RT-PCR检测hPMCs中Ⅰ型胶原和TGF-β1 mRNA表达。结果原代hPMCs在光镜下呈成串葡萄样,融合后形成铺路鹅卵石样外观;电镜下可见细胞表面存在大量微绒毛,细胞浆内有丰富的内质网和线粒体。免疫组化鉴定细胞抗角蛋白抗原、抗波形蛋白抗原阳性,抗白细胞CD45抗原和抗第Ⅷ因子相关抗原阴性。hPMCs亦表达Ⅰ型胶原和TGF-β1 mRNA。结论胰蛋白酶消化法是一种简单实用分离hPMCs的方法,可为进一步研究腹膜纤维化的防治及Ⅰ型胶原、TGF-β1在腹膜透析中的作用提供理论依据。Objective To establish a model for the culture of human peritoneal mesothelial cells （hPMCs） in vitro and detect the expressions of type I collagen and TGF-β1 in hPMCs. Methods Primary hPMCs were isolated from human omentum tissues by 0. 2% trypsin-0. 02% EDTANa2 digestion method and then subcultured, hPMCs were identified by morphology and immunohistochemistry. The mRNA expressions of type Ⅰ collagen and TGF-β1 in hPMCs were detected by RT-PCIL Results Primary hPMCs showed cluster of grape-like appearance, and confluent hPMCs presented cobblestone shape under light microscope. Numerous microvilli on cell surface and abundant endoplasmic reticulum and mitochondria in cytoplasm were observed under electron microscope. Cytokeratin and vimentin antigens were positive, while leukocyte CD45 and factor Ⅷ-related antigens were negative by immunohistochemistry identification. Moreover, the mRNA expressions of type Ⅰ collagen and TGF-β1 were expressed in hPMCs. Conclusion Trypsinization is a simple and pragmatic method to isolate and culture hPMCs, which will provide the basis for investigating the prevention of peritoneal fibrosis and role of type Ⅰ collagen and TGF-β1 in peritoneal dialysis.

关 键 词：腹膜间皮细胞 Ⅰ型胶原 转化生长因子Β1 

分 类 号：R331[医药卫生—人体生理学]