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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 human in vitro culture model of human peritoneal mesothelial cells (hPMCs) and to detect the expression of type Ⅰ collagen and transforming growth factor β1 (TGF-β1) in hPMCs. Methods Human omental tissue was digested with 0.2% trypsin-0.02% disodium EDTA and primary cultured hPMCs were passaged. The cultured cells were identified by morphological and immunohistochemical methods. The expression of type I collagen and TGF-β1 mRNA in hPMCs was detected by RT-PCR. Results Primary hPMCs were clustered into grape clusters under light microscope. The appearance of paving cobblestone after fusion was observed. Under electron microscope, a large number of microvilli were found on the surface of cells, and the endoplasmic reticulum and mitochondria were abundant in cytoplasm. Immunohistochemistry identified cellular anti-keratin antigens, anti-vimentin antigens positive, anti-leucocyte CD45 antigens and anti-factor Ⅷ related antigens negative. hPMCs also express type I collagen and TGF-β1 mRNA. Conclusion Trypsin digestion method is a simple and practical method for the isolation of hPMCs, which can provide a theoretical basis for the further study on prevention and treatment of peritoneal fibrosis and the role of TGF-β1 in peritoneal dialysis.