Purification and Functional Analysis of Adfesion Related Molecules Between Hematopoietic Stem Cells and Marrow Stromal Cells.

为了研究基质造血中的新型粘附分子，我们制备了针对人骨髓基质细胞株KM-101、KM-102和骨髓造血细胞株（CMK）的单克隆抗体（MAb）。将抗体直接加入含有KM细胞系和K562的共培养板中筛选。其中一种抗体增强了K562与基质细胞系之间的附着。该抗体识别135千道尔顿膜分子，其Fab片段明显抑制基质细胞与K562细胞之间的粘附。免疫组化染色显示，该单抗在多种组织间质细胞和血管内皮、平滑肌细胞以及包括肾集合小管、结肠和胰管在内的部分上皮细胞中均有阳性反应。在神经细胞和骨骼肌中没有发现阳性反应。分子的分布与CD44抗原相似，只有少数例外，但分子大小有很大不同。^<125>I在各种血细胞表面膜上的直接结合表明，外周血白细胞具有较低的放射性标记，而相对不成熟的造血细胞系则含有两次或更多的放射性抗体。用10ng/ml TPA处理造血细胞系3天后，可调节直接结合量。这些结果表明，一些外源刺激调节了135k道尔顿膜分子的表达。结合分子在各种间质和薄壁组织中的分布，Mab C-F-9识别的分子可能是实质和间质信号转导之间的信号分子之一，也是一种粘附相关蛋白。

In order to investigate novel adhesion molecule (s) in stromal hematopoiesis, we raised monoclonal antibodies (MAb) against human marrow stromal cell lines KM-101 and KM-102 and myeloid hematopoietic cell line (CMK). The antibodies were screened by the direct addition into coculture plates containing KM cell line and K562. One of the antibodies enhanced the attachment between K562 and stromal cell lines. This antibody recognized the 135-Kilodalton membrane molecules and its Fab fragment markedly inhibited the adhesion between stromal cells and K562 cells. Immunohistochemical staining of this Mab revealed positive reaction in stromal cells of wide variety of tissues and vascular endothelia, smooth muscle cells, and some of epithelial cells including renal collecting tubules, colon and pancreatic duct. No positive reaction was found in neural cells or skeletal muscles. The distribution of the molecules are similar to CD44 antigen with a few exception, but the size of the molecules are much different. Direct binding of ^<125>I on the surface membrane of various kind of blood cells showed that peripheral white blood cells had low radiolabelling while relatively immature hematopoieticcell lines incorporated twice or more radioactive antibodies. The amount of the direct binding was modulated when the hematopoietic cell lines were treated with 10ng/ml TPA 3 days in advance. These results indicates that some sosts of exogenous stimuli modulate the expression of 135 K dalton membrane molecule. Taking together with the distribution of the molecules among a wide varieties of stroma and parenchyma, the molecules recognized by Mab C-F-9 may be one of signaling molecules between parenchymal and stromal signal transduction, as well as an adhesion related protein.