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

皮下注射重组基底膜（Matrigel）与碱性成纤维细胞生长因子组合诱导从头脂肪形成，其中内源性前体细胞侵入人工形成的Matrigel空间，增殖并分化形成脂肪组织。由于这种脂肪形成为我们提供了一种新的方法，不移植前脂肪细胞的软组织重建，早期过程进行了检查，通过光学和电子显微镜。在Matrigel植入物表面形成多层成纤维细胞样细胞是结缔组织的第一反应。Matrigel植入物近端4 ~ 5层细胞胞质增厚，核增大，与内皮细胞一起侵入Matrigel间隙，形成新生血管。通过发育良好的溶酶体吞噬掺入和消化基质胶组分似乎是成纤维细胞样细胞成熟的刺激，这取决于与基质胶的接近程度。成纤维细胞样细胞常与毛细血管外表面大面积接触，距离0.2 ~ 0.5μm，并迅速聚集脂滴。发育中的脂肪细胞的电子显微镜显示组织良好的滑面内质网和线粒体。作为低氧调节外周组织胶原代谢的实验模型，我们还在低氧分压下培养了3 T3-L1成纤维细胞，发现低氧使IV型胶原分泌增加10倍，并加速了细胞的脂肪转化。IV型胶原分泌的增加并不伴随着α1（IV）和α2（IV）mRNA的明显增加。脯氨酰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.