Kadison A S, Maldonado T S, Crisera C A, Longaker M T, Gittes G K
New York University Medical Center, New York, New York, USA.
J Surg Res. 2000 May 15;90(2):126-30. doi: 10.1006/jsre.2000.5876.
Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membrane-rich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro.
We microdissected embryonic pancreases at Embryonic Days (E)9.5-11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10. 5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel.
Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel.
These data validate our in vitro system as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis.
早期胚胎胰腺上皮细胞具有向内分泌或外分泌谱系分化的能力。最近的研究表明这些未分化细胞具有多能性。在肾被膜下培养的分离胰腺上皮细胞主要形成胰岛。然而，当这些相同的上皮细胞在富含基底膜的凝胶（基质胶）中培养时，它们大多形成导管。目前，尚无体外胰腺导管形成的模型，因此，导管形态发生的机制从未被描述过。本研究的目的是通过表征在体外培养的分离未分化胰腺上皮细胞中两种导管标志物碳酸酐酶II（CAII）和囊性纤维化跨膜传导调节因子（CFTR）的表达来提供这样一个模型。
我们在胚胎第（E）9.5 - 11.5天显微解剖胚胎胰腺，并对E9.5全胰腺、E10.5和E11.5上皮细胞以及在基质胶中培养7天的E11.5上皮细胞进行CAII和CFTR的逆转录聚合酶链反应（RT-PCR）。接下来，我们对在基质胶中培养7天的E11.5上皮细胞进行CAII和CFTR的原位杂交以及CAII的免疫组织化学检测。
通过RT-PCR检测，早期未分化的胚胎胰腺上皮细胞不表达CAII和CFTR。然而，当E11.5上皮细胞在基质胶中培养7天时，随着导管形成，两种标志物的基因表达上调。此外，免疫组织化学检测在培养7天后的导管中可见CAII，原位杂交检测在导管中可见CAII和CFTR。
这些数据验证了我们的体外系统可作为研究正常胰腺导管分化机制的模型，并可能有助于我们理解胰腺导管癌发生过程中的异常机制。
