Barreca A, De Luca M, Del Monte P, Bondanza S, Damonte G, Cariola G, Di Marco E, Giordano G, Cancedda R, Minuto F
Cattedre di Endocrinologia e di Fisiopatologia Endocrina-DiSEM, Università di Genova, Italy.
J Cell Physiol. 1992 May;151(2):262-8. doi: 10.1002/jcp.1041510207.
Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.
从皮肤活检中分离出的人角质形成细胞，在经辐照的成纤维细胞饲养层上进行体外培养，可重建适合移植到大面积烧伤患者身上的复层鳞状上皮。由于来自3T3-J2细胞的条件培养基可部分替代完整的饲养层，我们研究了以旁分泌方式起作用的胰岛素样生长因子可能的参与情况。在角质形成细胞生长的经致死性辐照的3T3-J2成纤维细胞饲养层条件培养基中（在酸性条件下经Sephadex G-50凝胶色谱法后）测量了胰岛素样生长因子。饲养层条件培养基中存在免疫反应性（IR）胰岛素样生长因子-I、胰岛素样生长因子-II和胰岛素样生长因子结合活性。角质形成细胞条件培养基显示几乎检测不到IR胰岛素样生长因子-I和胰岛素样生长因子-II，这表明角质形成细胞无法合成胰岛素样生长因子肽。重组胰岛素样生长因子-I和胰岛素样生长因子-II以及来自3T3-J2细胞的条件培养基，导致培养的角质形成细胞中3H-胸腺嘧啶掺入量呈剂量依赖性增加。胰岛素样生长因子和3T3-J2条件培养基的刺激作用被识别胰岛素样生长因子-I和胰岛素样生长因子-II但不识别胰岛素的单克隆抗体Sm 1.2以及作为I型胰岛素样生长因子受体特异性拮抗剂的单克隆抗体α IR-3所抑制。源自胎鼠的3T3-J2细胞和成人皮肤成纤维细胞同样能够维持角质形成细胞生长，并且在这两种情况下添加Sm 1.2单克隆抗体都会导致角质形成细胞数量减少50%。当使用不产生胰岛素样生长因子的BALB/c 3T3细胞作为饲养层时，角质形成细胞数量与用3T3-J2细胞以及人成纤维细胞加Sm 1.2单克隆抗体时观察到的数量相似。胰岛素样生长因子-I和胰岛素样生长因子-II将BALB/c 3T3细胞的生长促进活性恢复到3T3-J2细胞和正常人成纤维细胞的水平。我们的结果表明，胎鼠3T3-J2细胞和人成纤维细胞合成胰岛素样生长因子肽，而角质形成细胞则不合成。成纤维细胞衍生的胰岛素样生长因子以旁分泌方式刺激角质形成细胞生长，表明它们在皮肤生长和伤口愈合过程中对角质形成细胞增殖的调节作用。
