DEVELOPMENT OF RECONSTRUCTIVE SURGERY OF SOFT-TISSUES BY INJECTION OF BASEMENT MEMBRANE

皮下注射重组基底膜（Matrigel）结合碱性成纤维细胞生长因子诱导脂肪新生，内源性前体细胞侵入人工形成的基质膜空间，增殖分化形成脂肪组织。由于这种脂肪形成为不移植前脂肪细胞的软组织重建提供了一种新的方法，因此通过光学和电子显微镜检查了早期过程。结缔组织的第一反应是在Matrigel种植体表面形成多层成纤维细胞样细胞。基质植入物近端4 ~ 5层内的细胞细胞质增厚，细胞核增大，与内皮细胞一起侵入基质间隙，形成新生血管。发育良好的溶酶体吞噬合并和消化基质成分似乎是刺激成纤维细胞样细胞成熟的因素，这取决于基质的接近程度。成纤维细胞样细胞经常与毛细血管外表面大面积接触，距离为0.2 ~ 0.5μm，并迅速积聚脂滴。电镜显示发育中的脂肪细胞有组织良好的光滑内质网和线粒体。本研究揭示了脂肪前体细胞的特性，为软组织再生工程提供了可能。作为缺氧调节外周组织胶原代谢的实验模型，我们也在低氧分压下培养了3T3-L1成纤维细胞，发现缺氧使IV型胶原的分泌增加了10倍，加速了细胞的脂肪转化。IV型胶原分泌增强不伴有α1（IV）和α2(IV) mrna的明显升高。脯氨酸4-羟化酶α在缺氧条件下仅增加3倍。

Subcutaneous injection of reconstituted basement membrane (Matrigel) in combination with basic fibroblast growth factor induces de novo adipogenesis in which endogenous precursor cells invade the artificially formed Matrigel space, proliferate and differentiate to form adipose tissue. Since this adipogenesis offers us a novel approach for soft-tissue reconstruction without transplanting preadipocytes, the early process was examined by optical and electron microscopy. Formation of multiple layers of fibroblast-like cells at the surface of Matrigel implant was the first response of connective tissue. The cells within four to five layers proximal to Matrigel implant acquired a thick cytoplasm and an enlarged nucleus, and they invaded Matrigel space together with endothelial cells which caused neovascularization. Phagocytotic incorporation and digestion of Matrigel components by well-developed lysosomes appeared to be a stimulus of fibroblast-like cells to mature depending on proximity to Matrigel. The fibroblast-like cells often contacted to the outer surface of capillary over a large area with a distance of 0.2 to 0.5μm and rapidly accumulated lipid droplets. Electron microscopy of the developing adipocytes showed an well-organized smooth endoplasmic reticulum and mitochondria. This investigation thus revealed the characteristics of adipocyte precursor cells, which can be recruited for regenerative engineering of soft-tissues.As an experimental model for the hypoxic modulation of collagen metabolism in peripheral tissues, we also cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by appreciable increase of α1(IV) and α2(IV) mRNAs. Prolyl 4-hydroxylase α increased only 3-fold under hypoxia.