人羊膜上皮细胞原代培养及肝细胞特异性蛋白的表达

Primary culture and hepatocelluar specific protein expression in human amniotic epithelial cells

目的完善人羊膜上皮细胞(AECs)的原代培养技术,检测肝细胞特异性蛋白在AECs中的表达。方法采用胶原酶-胰蛋白酶联合消化法分离获取AECs并进行原代培养。分别向培养液中添加10、20、40ng/mL表皮生长因子(EGF)和10ng/mL的碱性成纤维细胞生长因子(bFGF),倒置显微镜观察细胞生长和增殖情况,从中选择最适宜AECs原代培养的细胞培养液。以培养液中未添加生长因子的原代培养细胞作为对照。将羊膜组织石蜡切片和AECs细胞爬片进行免疫组化染色,检测4种肝细胞特异性蛋白的表达,分别为白蛋白(Alb)、细胞角蛋白-18(CK-18)、α-1-抗胰蛋白酶(AAT)和α-1-甲胎蛋白(AFP)。结果经胶原酶-胰蛋白酶联合消化法获得大量较纯的AECs,间杂少数间充质细胞。当细胞培养液中添加10ng/mLEGF时,AECs生长和增殖速度显著加快,故最终选择添加10ng/mL EGF配制细胞培养液。免疫化学染色显示,羊膜组织和体外传代培养的AECs中均有Alb、CK-18、AAT和AFP表达。结论胶原酶-胰蛋白酶联合消化及培养液中添加10ng/mL的EGF是AECs较为适宜的原代培养方法。人羊膜组织及体外培养AECs能表达肝细胞特异性蛋白,提示AECs具有肝细胞的部分生物学特性。

Objective To optimize the method of primary culture for human amniotic epithelial cells （AECs） and detect the expression of hepatocelluar specific proteins in AECs. Methods AECs were obtained by collagenase and trypsin digestion method. 10,20,40 ng/mL epidermal growth factor （EGF） and 10 ng/mL basic fibroblast growth factor were added separately to the culture media,inverted microscope was used to observe the cell growth and proliferation,and the optimal condition for primary culture of AECs was obtained. The primary cells cultured without growth factors were served as controls. The expression of hepatocelluar specific proteins such as albumin,cytokeratin-18 （CK-18）,alpha-1 antitrypsin （AAT） and alpha-1 foetoprotein （AFP） in cultured cells and histological sections were detected by immunohistochemical staining. Results The AECs available were relatively pure,with only a few mesenchymal cells. EGF of 10 ng/mL was chosen as an ingredient of culture medium as the growth and proliferation of AECs significantly accelerated with 10 ng/mL EGF. The expression of albumin,CK-18,AAT and AFP was detected in amnion tissues and AECs cultured in vitro. Conclusion Collagenase and trypsin digestion method and culture medium with 10 ng/mL EGF are favourable conditions for primary culture of AECs. Hepatocelluar specific proteins are expressed in human amnion tissues and AECs cultured in vitro,indicating that AECs have some characteristics of hepatocytes.