INVITRO PARACRINE REGULATION OF HUMAN KERATINOCYTE GROWTH BY FIBROBLAST-DERIVED INSULIN-LIKE GROWTH-FACTORS
INVITRO PARACRINE REGULATION OF HUMAN KERATINOCYTE GROWTH BY FIBROBLAST-DERIVED INSULIN-LIKE GROWTH-FACTORS
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DOI：
10.1002/jcp.1041510207
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发表时间：
1992-05-01
影响因子：
5.6
通讯作者：
MINUTO, F
中科院分区：
文献类型：
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作者：
BARRECA, A;DELUCA, M;MINUTO, F
从皮肤活检中分离出的人角质形成细胞，在体外培养于经辐射的成纤维细胞饲养层上，可重建适合移植到患有大面积烧伤创面的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以部分替代完整的饲养层，因此我们研究了以旁分泌方式起作用的胰岛素样生长因子的可能参与。 在用经致死照射的3T3-J2成纤维细胞饲养层调节的培养基中（在酸性条件下进行Sephadex G-50凝胶色谱法后）测量IGF，角质形成细胞在其上生长。 饲养层调节的培养基中存在免疫反应性 (IR) IGF-I、IGF-II 和 IGF 结合活性。 角质形成细胞条件化的培养基显示出几乎不可检测量的IR IGF-I和IGF-II，表明角质形成细胞无法合成IGF肽。 重组 IGF-I 和 IGF-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 H-3-胸苷掺入量呈剂量依赖性增加。 IGF 和 3T3-J2 条件培养基的刺激作用受到 MoAb Sm 1.2 和 MoAb α-3 的抑制，MoAb Sm 1.2 识别 IGF-I 和 IG-II，但不识别胰岛素，MoAb α-3 是 I 型 IGF 受体的特异性拮抗剂。 胎鼠来源的 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 MoAb 都会导致角质形成细胞数量减少 50%。 当使用不产生IGF的BALB/c 3T3细胞作为饲养层时，角质形成细胞数量与用3T3-J2和人成纤维细胞加Sm 1.2 MoAb观察到的相似。 IGF-I和IGF-II将BALB/c 3T3生长促进活性恢复至3T3-J2和正常人成纤维细胞的水平。 我们的结果表明，胎鼠 3T3-J2 和人成纤维细胞合成 IGF 肽，而角质形成细胞则不合成。 成纤维细胞衍生的 IGF 以旁分泌方式刺激角质形成细胞生长，表明它们在调节皮肤生长的角质形成细胞增殖和伤口愈合中发挥作用。
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 H-3-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 IG-II but not insulin, and by the MoAb alpha-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 of skin growth and in wound healing.