Modulation of vocal fold scar fibroblast by adipose derived stem cell

瘢痕成纤维细胞（SFs）从2周前电灼的雪貂声带中分离出来（N=2）。从雪貂吸脂腹腔下脂肪中分离脂肪源性干细胞（ASCs） （N=2）。在共培养实验中，将两种细胞组合在Transwell板上6天，去除上腔后进行1或3天的单培养。然后对底室的细胞和培养基进行测定。我们通过酶联免疫吸附法（ELISA）检测了(1)透明质酸（HA）、胶原蛋白和肝细胞生长因子（HGF）的产生；(2)α-平滑肌肌动蛋白（α-SMA）的表达；(3)细胞增殖和(4)细胞凋亡（流式细胞术检测2、3和4）。其他实验考察了HGF对SFs的影响以及在共培养系统中HGF中和的影响。我们发现，共培养导致SF胶原蛋白的产生（p< 0.05）、增殖（p< 0.05）和α-SMA的表达（p< 0.05）显著降低，而HA的产生增加（p< 0.05）。共培养也增加了ASCs的HGF分泌（p< 0.05）。中和HGF可消除ASCs对SF胶原合成的抑制作用（p< 0.05）。我们得出结论，ASCs影响SFs采用较少纤维化的特征。看来HGF至少是造成这种效果的可溶性因子之一。植入式ASCs作为HGF的长期、内在来源，有可能改善声带瘢痕

Scar fibroblasts (SFs) were isolated from ferret vocal folds electrocauterized two weeks previously (N=2). Adipose-derived stem cells (ASCs) were isolated from ferret lipoaspirated subcutaneous abdominal fat (N=2). For co-culture experiments, the two cell types were combined in Transwell plates for 6 days, followed by 1 or 3 days of monoculture after removing the upper chamber. Assays were then performed on cells and media from the bottom chamber. We measured (1) the production of hyaluronic acid (HA), collagen and hepatocite growth factor (HGF) via enzyme-linked immunosorbent assays (ELISA) (2) the expression of α-smooth muscle actin (α-SMA), (3) cell proliferation and (4) apoptosis of SFs (2, 3 & 4 via flow cytometry). Other experiments examined the effects of HGF on SFs and the effects of HGF neutralization in the co-culture system. We demonstrated that co-culture led to significant decreases in SF collagen production (p<.05), proliferation (p<.05), and α-SMA expression (p<.05), while HA production increased (p<.05). Co-culture also increased HGF secretion from ASCs (p<.05). Neutralization of HGF abolished the inhibitory effects of ASCs on SF collagen synthesis (p<.05). We concluded that ASCs influence SFs to adopt a less fibrotic profile. It appears that HGF is at least one of the soluble factors responsible for this effect. Implanted ASCs could potentially ameliorate vocal fold scar by acting as a long-term, intrinsic source of HGF