抑制转化生长因子β和Notch信号通路诱导人胆囊上皮细胞分化为功能性肝细胞样细胞  

Induction of human gallbladder epithelial cells differentiating into functional hepatocyte-like cells by inhibiting transforming growth factorβand Notch signaling pathways

作　　者：王紫君 杜涵 徐守佳 李俊霖 王敏君 陈费 WANG Zi-jun;DU Han;XU Shou-jia;LI Jun-lin;WANG Min-jun;CHEN Fei(Department of Cell Biology,College of Basic Medical Sciences,Naval Medical University(Second Military Medical University),Shanghai 200433,China;Shanghai Baixian Biotechnology Co.,Ltd,Shanghai 201318,China)

机构地区：[1]海军军医大学(第二军医大学)基础医学院细胞生物学教研室,上海200433 [2]上海拜羡生物科技有限公司,上海201318

出　　处：《海军军医大学学报》2023年第5期542-550,共9页Academic Journal of Naval Medical University

基　　金：上海市自然科学基金(21ZR1477400);上海市人才发展资金(2021080)。

摘　　要：目的通过小分子化合物改变细胞命运,诱导人胆囊上皮细胞(hGBEC)分化为具有功能的肝细胞样细胞。方法在基质胶中对原代hGBEC进行三维培养,生长培养基中添加B27添加剂、N2添加剂、N-乙酰半胱氨酸、表皮生长因子、肝细胞生长因子等。添加小分子化合物/蛋白因子对细胞进行诱导分化,筛选出关键作用因子。采用PCR、qPCR和免疫荧光染色检测干细胞标志物及肝细胞相关标志物的表达情况,通过脂肪BODIPY-493染色、糖原过碘酸希夫染色和白蛋白ELISA检测评估细胞的肝样功能。结果三维培养的hGBEC表达造血干细胞抗原CD133、上皮细胞黏附分子、肝细胞核因子4α等肝脏干细胞和肝前体细胞标志物。TGF-β信号通路抑制剂和Notch信号通路抑制剂是诱导hGBEC分化的关键作用因子。分化条件下所得细胞表达肝细胞功能标志物α1-抗胰蛋白酶、细胞色素P4503A4、白蛋白和延胡索酰乙酰乙酸水解酶,可以贮存糖原,具有合成脂肪的能力,能够分泌白蛋白。结论hGBEC可在体外长期培养,通过抑制TGF-β和Notch信号通路可初步诱导其分化为具有部分肝功能的肝细胞样细胞。Objective To induce human gallbladder epithelial cells(hGBECs)differentiating into hepatocyte-like cells through adding small molecule compounds which could change cell fate.Methods The primary hGBECs were threedimensionally cultured in Matrigel by adding B27 supplement,N2 supplement,N-acetyl-L-cysteine,epidermal growth factor and hepatocyte growth factor in the medium.Small molecule compounds/proteins were added to induce cell differentiation,and the key molecules were screened out.Polymerase chain reaction(PCR),quantitative PCR,and immunofluorescence staining were used to detect the expression of stem cell markers and liver cell related markers.The liver function was evaluated by lipid BODIPY-493 staining,glycogen periodic acid-Schiff staining,and albumin enzyme-linked immunosorbent assay.Results Three-dimensionally cultured hGBECs expressed hepatic stem/progenitor cells-related markers,such as hematopoietic stem cell antigen CD133,epithelial cell adhesion molecule,and hepatocyte nuclear factor 4α.Transforming growth factorβ(TGF-β)and Notch signaling pathway inhibitors were the key molecules for inducing hGBEC differentiation.The cells obtained under differentiation conditions expressed liver cell functional markersα1-antitrypsin,cytochrome P4503A4,albumin and fumarate acetate hydrolase.The cells could store glycogen,synthesize fat and secrete albumin.Conclusion hGBECs can be cultured in vitro for a long time,and hGBECs can be induced to differentiate into hepatocyte-like cells with partial liver function by inhibiting TGF-βand Notch signaling pathways.

关 键 词：胆囊上皮细胞 转化生长因子Β NOTCH信号通路 肝细胞 细胞分化 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学] R575.3[医药卫生—基础医学]