| 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P450 3A4、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。 |
| 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 |
| DOI：10.16781/j.CN31-2187/R.20230064 |
| 投稿时间：2023-02-20修订日期：2023-04-07 |
| 基金项目:上海市自然科学基金(21ZR1477400),上海市人才发展资金(2021080). |
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| Induction of human gallbladder epithelial cells differentiating into functional hepatocyte-like cells by inhibiting transforming growth factor β and Notch signaling pathways |
| WANG Zi-jun1,DU Han1,XU Shou-jia1,2,LI Jun-lin1,WANG Min-jun1,CHEN Fei1* |
(1. Department of Cell Biology, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Shanghai Baixian Biotechnology Co., Ltd, Shanghai 201318, China
*Corresponding author) |
| Abstract: |
| 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 three-dimensionally 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 factor β (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 markers α1-antitrypsin, cytochrome P450 3A4, 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-β and Notch signaling pathways. |
| Key words: gallbladder epithelial cells transforming growth factor β Notch signaling pathway hepatocytes cell differentiation |
