Nisato Riccardo E, Harrison Jillian A, Buser Raphaele, Orci Lelio, Rinsch Chris, Montesano Roberto, Dupraz Philippe, Pepper Michael S
Department of Morphology, University Medical Center, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.
Am J Pathol. 2004 Jul;165(1):11-24. doi: 10.1016/S0002-9440(10)63271-3.
The study of lymphatic endothelial cells and lymphangiogenesis has, in the past, been hampered by the lack of lymphatic endothelial-specific markers. The recent discovery of several such markers has permitted the isolation of lymphatic endothelial cells (LECs) from human skin. However, cell numbers are limited and purity is variable with the different isolation procedures. To overcome these problems, we have transfected human dermal microvascular endothelial cells (HDMVECs) with a retrovirus containing the coding region of human telomerase reverse transcriptase (hTERT), and have produced a cell line, hTERT-HDLEC, with an extended lifespan. hTERT-HDLEC exhibit a typical cobblestone morphology when grown in culture, are contact-inhibited, and express endothelial cell-specific markers. hTERT-HDLEC also express the recognized lymphatic markers, Prox-1, LYVE-1 and podoplanin, as well as integrin alpha9, but do not express CD34. They also form tube-like structures in three-dimensional collagen gels when stimulated with vascular endothelial growth factors -A and -C. Based on these currently recognized criteria, these cells are LEC. Surprisingly, we also found that the widely studied HMEC-1 cell line expresses recognized lymphatic markers; however, these cells are also CD34-positive. In summary, the ectopic expression of hTERT increases the life span of LECs and does not affect their capacity to form tube-like structures in a collagen matrix. The production and characterization of hTERT-HDLEC will facilitate the study of the properties of lymphatic endothelium in vitro.
过去，由于缺乏淋巴管内皮特异性标志物，淋巴管内皮细胞和淋巴管生成的研究受到了阻碍。最近发现了几种此类标志物，使得从人皮肤中分离淋巴管内皮细胞（LECs）成为可能。然而，细胞数量有限，且不同分离程序所得细胞的纯度存在差异。为克服这些问题，我们用含有人类端粒酶逆转录酶（hTERT）编码区的逆转录病毒转染人真皮微血管内皮细胞（HDMVECs），并建立了一种寿命延长的细胞系hTERT-HDLEC。hTERT-HDLEC在培养时呈现典型的鹅卵石样形态，具有接触抑制特性，并表达内皮细胞特异性标志物。hTERT-HDLEC还表达公认的淋巴管标志物Prox-1、LYVE-1和血小板内皮细胞黏附分子，以及整合素α9，但不表达CD34。当用血管内皮生长因子-A和-C刺激时，它们也能在三维胶原凝胶中形成管状结构。基于这些目前公认的标准，这些细胞属于LEC。令人惊讶的是，我们还发现广泛研究的HMEC-1细胞系也表达公认的淋巴管标志物；然而，这些细胞也是CD34阳性。总之，hTERT的异位表达延长了LEC的寿命，且不影响它们在胶原基质中形成管状结构的能力。hTERT-HDLEC的建立和特性分析将有助于体外研究淋巴管内皮的特性。
