Human lipogenesis with basic fibroblast growth factor gene transfected preadipocytes for breast reconstruction.

1)从乳腺癌手术中获得的脂肪组织中分离出包括前脂肪细胞在内的人基质细胞，体外培养传代。经第10代传代后，基质细胞的增殖能力得以保留。碱性成纤维细胞生长因子(BFGF)促进基质细胞增殖。细胞产量超过培养开始时细胞数量的1000倍。培养的基质细胞经过3代传代后，对脂肪细胞的成熟活性维持在初始水平，之后逐渐下降。这一结果提示，骨髓基质细胞的最佳成脂细胞来源是传代培养的基质细胞。2)与胎牛血清(FCS)相比，自体血清培养能促进基质细胞的增殖。淋巴清扫后腋腔积液对体外培养的基质细胞增殖的影响与FCS相似。这一结果表明，培养的基质细胞可以在体内增殖。3)S…传代将更多的含碱性成纤维细胞生长因子培养的基质细胞植入含有碱性成纤维细胞生长因子微球的胶原盘支架裸鼠背部皮下。组织学观察支架植入部位的成脂情况。2×10~(-6)培养基质细胞移植组血管生成面积最大，但表达人波形蛋白的成脂面积不到成脂面积的1%。在8×10~(-6)培养的基质细胞移植组中，人波形蛋白阳性脂肪组织的面积占整个脂肪生成的15%，而培养的基质细胞移植组的脂肪生成面积小于2×10~(-6)。4)首先，用腺病毒载体转染人b-FGF基因，结果表明，在没有外源性b-FGF的情况下，转b-FGF基因的基质细胞在体外增殖并分化为成熟的脂肪细胞。为了将来这种成脂方法的临床应用，我们正在研究一种安全有效的非病毒基因递送系统。较少

1) Human stromal cells including preadipocytes which were separated from fatty tissue obtained at breast cancer operation were incubated and passed in vitro. Proliferation of stromal cells was preserved through 10^<th> passage. Basic fibroblast growth factor (bFGF) enhanced the proliferation of stromal cells. Cell yield exceeded 1000 times of the cell number at the beginning of culture. Cultured stromal cells maintained maturation activity to adipocyte at the initial level through 3^<rd> passage and declined thereafter. This result suggested that the optimal cell source for lipogenesis was 3^<rd> passage of cultured stromal cells.2) Proliferation of stromal cells was enhanced when they were cutltured with autorogous serum compared with fetal calf serum (FCS). Effusion of axillary cavity after dissection of lymph nodes showed similar effect on the proliferation of stromal cells in vitro as FCS. This result suggested cultured stromal cells would proliferate in vivo.3) 3^<rd> passage of s … More tromal cells cultured with bFGF was implanted with collagen disk scaffold incorporated by the microspheres containing bFGF into subcutaneous tissue of the back of nude mice. Adipogenesis at the implanted site of scaffold was evaluated histologically. Area of angiogenesis in 2 x 10^6 cultured stromal cell implant group was largest among groups implanted different number of cultured stromal cells, but area of adipogenesis expressing human vimentin was less than 1% of whole adipogenesis. In 8 x 10^6 cultured stromal cell implant group, area of human vimentin positive adipose tissue was 15% of whole adiopogenesis, although the area of whole adipogenesis was less than 2 x 10^6 cultured stromal cell implant group.4) Initialially, b-FGF gene transfected using adenovirus vector, and obtained the result that stromal cells transfected b-FGF gene proliferated in vitro without exogenous b-FGF and differentiated into mature adipocytes. For future clinical application of this adipogenesis method, we are investigating a safe and efficient non-viral gene delivery system. Less