胚胎干细胞向胆管上皮细胞定向分化的体外实验

背景：在胚胎干细胞向肝细胞诱导分化的培养条件基础上增加向胆管上皮细胞分化的培养条件，使其向胆管上皮细胞方向分化在理论上是可行的。 目的：验证在细胞生长因子胚胎干细胞体外定向分化为胆管上皮细胞的可行性。设计：单一样本观察。 单位：中山大学附属第一医院。 材料：选用BALB／c系小鼠胚胎干细胞（BALB／c—ES）由中山大学实验动物中心建系和保存。分化生长因子、酸性成纤维细胞生长因子、肝细胞生长因子、上皮生长因子、角化细胞生长因子均为美国Sigma公司产品，实验用抗小鼠细胞角蛋白7（CK7）和细胞角蛋白19（CK19）购自丹麦DAKO公司，间接免疫荧光试剂盒为美国Biodesign产品，IX70-S8F2倒置相差荧光显微镜为日本OLYMPUS产品。 方法：实验于2004—10／2005-06在中山大学附属第一医院中心实验室完成。①将小鼠胚胎干细胞进行拟胚体分化，在培养系统中按不同时间段分别添加分化生长因子、酸性成纤维细胞生长因子、肝细胞生长因子、上皮生长因子、角化细胞生长因子，使胚胎干细胞向胆管上皮细胞方向分化。以培养液中不添加上述细胞生长因子为对照组，使胚胎干细胞自然分化。②采用倒置相差荧光显微镜动态观察细胞生长及三维的环状结构形成情况。③于细胞分化第10天开始采用免疫细胞化学方法检测胆管上皮细胞标记物CK7、CK19，γ-谷胺酰转肽酶（GGT）表达变化，同时观察γ-谷胺酰转肽酶阳性细胞的形态学特点。 主要观察指标：①细胞生长及三维环状结构形成情况。②胆管上皮细胞标记蛋白CK7、CK19的表达。③胆管上皮细胞标记酶GGT表达及其阳性细胞形态学情况。结果：①细胞生长及三维的环状结构形成情况：胚胎干细胞悬浮培养10h后即可见小的拟胚体形成并悬浮在培养液中。分化第10天在拟胚体细胞分化群落中出现～种三维的环状结构，细胞呈同心圆层状排列，内层细胞排列紧密，继续培养，内层细胞逐渐排列疏松，分化20d左右时细胞活力达到最好，其后活力开始下降，分化36d时三维结构裂解并呈条索状悬浮于培养液中。对照组环状结构于第13天出现，细胞结构于27d裂解。②CK7，CK19表达：胆管上皮细胞环状结构形成当日即有CK7表达，分化第13天时CK19表达，其后两者同时表达于三维结构细胞中并随培养时间增加而表达逐渐增强。对照组细胞在分化第13，15天开始出现CK7，CK19表达。③胆管上皮细胞标记酶GGT表达及其阳性细胞形态学变化：加入生长因子后，细胞环状结构形成初始即表达GGT，说明此结构中含有胆管上皮细胞。在环状结构形成初始，细胞间连接紧密，不能分清单个细胞结构，随着培养时间增长，单细胞结构逐渐清晰。GGT阳性细胞呈多角型或方形排列，核位于中央，细胞器较少，胞质染色较浅，符合立方上皮细胞特点，体现出胆管上皮细胞的形态学特点。 结论：胚胎干细胞在细胞生长因子的作用下可定向分化为胆管上皮细胞并可以形成类胆管样结构。

Biliary epithelial cell differentiation from embryonic stem cells in vitro

BACKGROUND: Previous research demonstrated that embryonic stem (ES) cells can be induced to differentiate into bepatocytes. It is feasible to induce ES cells to differentiate into biliaty epithelial (BE) cells by specific induction conditions based on the previous research.OBJECTIVE: To validate the feasibility to induce ES cells to differentiate into BE cells in the presence of cell growth factors.DESIGN: Single sample observation.SETTING: First Hospital of Sun Yat-sen University.MATERIALS: Undifferentiated BALB/C-ES cell line was provided by the Experimental Animal Center of Sun Yat-sen University; Transforming growth factor, acidic flbroblast growth factor, hepatocyte growth factor, epidermal growth factor, and keratinocyte growth factor by Sigma, USA. First antibody: anti-mouse cytokeratin 7 (CK7) and CKI9 by DAKO, Denmark; indirect immunofluorescence from Biodesign, USA; and inverted contrast fluorescence microscope by OLYMPUS IX70-SSF2, Japan.METHODS: The experiment was performed at Central Laboratory of the First Affiliated Hospital of Sun Yat-sen University from October 2004 to June 2005. During the culture of embryonic bodies (EBs) derived from ES cells, some growth factors such as Transforming growth factor, acidic fibroblast growth factor, hepatocyte growth factor and epidermal growth factor were respectively added into medium to induce BE cells differentiation. ES cells in culture condition with no added growth factors served as control. The differentiation status and three-dimensional duct-like structure formation of ES cells was observed dynamically by inverted microscope. The markers of BE cells such as CK7, CK19 and gamma-glutamyltransferase (GGT) were detected by immunocytochemistry and GGT-positive cells were observed.MAIN OUTCOME MEASURES: ①Cell growth and three-dimensional duct-like structure formation; ②expressions of the marker proteins of BE cells such as CK7 and CK19; ③expressioa of GGT, the marker enzyme of BE cells, and GGT-positive cell morphology.RESULTS: Small EBs formed and floated in culture suspension after 10 hours of ES cell culture. Many three-dimensional duct-like structures appeared in EBs culture system within 10 differentiation days. Cells were arranged in cocentric circle and lamellar shape; the inner layer cells aligned tightly but became sparse gradually. The cells had best viability on day 20, and disintegrated in strands in culture solution on day 36. Duct-like structure was found on day 13 in control group and disintegrated on day 27. CK7 was expressed on the day of duct-like structure formation, and CK19 expression was found on day 13. Both expressions were elevated with culture time thereafter. CK7 and CK19 were expressed in control group on days 13 and 15, respectively. GGT expressed in duct-like structures after growth factors were added, indicating there was BE cells in these structures. At early stage of duct-like structure formation, cells connected too closely to identify single cell structure, but cell structure became clear in further culture. GGT-positive cells had morphological characters consistent with mouse normal BE cells such as: the cells were polygonal or quadrate shapes; the nucleus was big and round and situated at the center of cells; the organdies were lacking in cytoplasm.CONCLUSION: ES cells can differentiate into BE cells in presence of certain growth factors and form bile duct-like structures.