Objective: To observe content of cytokine in human stromal vascular fraction gel (SVF-GEL) and effect of SVF-GEL on biological behaviors of epidermal and dermal cells in vitro and clinical efficacy of SVF-GEL. Methods: (1) SVF-GEL was prepared using liposuction aspirates harvested from females who received abdomen liposuction in author's unit. SVF-GEL (1 mL) and high-glucose Dulbecco's modified eagle medium (DMEM, 1 mL) were respectively cultured for 24 h with high-glucose DMEM containing 10% fetal calf serum, 10 g/L penicillin, and 10 g/L streptomycin, denoted as SVF-GEL group and negative control group, with 6 samples in each group. Content of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) in the supernatant was determined by enzyme-linked immunosorbent assay. (2) A number of 5×10(5) human skin fibroblasts (HSF) and HaCaT cells in logarithmic phase were inoculated and cultured in Transwell chambers for 12 h. All Transwell chambers containing cells were divided into SVF-GEL group (0.5 mL SVF-GEL was added for co-culture) and control group (0.5 mL high-glucose DMEM was added for co-culture), with 9 samples in each group for HSF and HaCaT cells. Scratch assay was performed after culture for 24 h, and residual scratch width was observed at post scratch hour (PSH) 0 (immediately), 24, and 48. Cell migration distance was measured at PSH 24 and 48. After culture for 24, 48, and 72 h, the number of living cell was counted using cell counter. (3) From June 2018 to June 2019, SVF-GEL was applied clinically to treat 15 patients with depressed scars on face, including 2 males and 13 females, aged 19 to 42 years. Survival condition of SVF-GEL and whether complications or not were observed 6 months after surgery. Before surgery and 6 months after surgery, depressed degree, color, and pliability of scar were compared. Vancouver Scar Scale (VSS) was employed to access color, vascularity, and pliability before surgery and 6 months after surgery, and total score was calculated. The number of patients with complete satisfaction or satisfaction was counted six months after surgery. Data were processed with analysis of variance of factorial design, paired samples t test, and Wilcoxon rank sum test. Results: (1) The content of EGF in SVF-GEL group and negative control group was (316.6±12.8) and (3.4±0.6) pg/mL, and the content of VEGF in SVF-GEL group and negative control group was (568.67±12.19) and (4.93±0.16) pg/mL, with statistically significant differences between the two groups (t=48.777, 92.485, P<0.01). (2) Residual scratch widths of HSF and HaCaT in SVF-GEL group and control group were decreased gradually along with time elapse, in which those in SVF-GEL group at PSH 24 and 48 were less than those in control group. At PSH 24 and 48, cell migration distances of HSF and HaCaT in SVF-GEL group were more than those in control group (t(HSF)=-20.304, -43.516, t(HaCaT)=-15.060, -8.684, P<0.01). After culture for 24, 48, and 72 h, the number of living cell of HSF and HaCaT in SVF-GEL group was significantly more than that in control group (t(HSF)=-3.374, -6.809, -18.036, t(HaCaT)=-4.793, -6.028, -8.141, P<0.05 or P<0.01). (3) Six months after surgery, SVF-GEL grafted into patients survived well without complications, and depressed degree of scar ameliorated obviously with lightened pigmentation and softer texture as compared with before surgery. Compared with those before surgery, VSS scores of color, vascularity, and pliability, and total score of 15 patients with depressed scars on face were obviously decreased 6 months after surgery (Z=-2.06, -2.07, -2.07, t=-15.811, P<0.05 or P<0.01). One patient was satisfied with the clinical outcome, and the rest 14 patients were completely satisfied with the clinical outcomes. Conclusions: SVF-GEL contains cytokines EGF and VEGF, which can enhance cell migration ability and proliferation ability of HSF and HaCaT cells and have obvious effects on depressed scars for clinical application.
目的： 观察人脂肪来源干细胞胶(SVF－GEL)中细胞因子含量及其体外对表皮、真皮细胞生物学行为的影响和临床疗效。 方法： (1)收集笔者单位行脂肪抽吸术获取的女性腹部脂肪抽吸液制备SVF－GEL。取1 mL SVF－GEL和1 mL DMEM高糖培养基，均用含体积分数10%胎牛血清、10 g/L青霉素、10 g/L链霉素的DMEM高糖培养基培养24 h，分别设为SVF－GEL组和阴性对照组，样本数均为6，采用酶联免疫吸附测定法检测上清液中表皮生长因子(EGF)、血管内皮生长因子(VEGF)含量。(2)选取处于对数生长期的5×10(5)个人皮肤成纤维细胞(HSF)、HaCaT细胞分别接种于Transwell小室常规培养12 h，将所有培养细胞的Transwell小室分为2组，SVF－GEL处理组在Transwell小室中加入0.5 mL SVF－GEL与细胞共培养，对照组在Transwell小室中加入0.5 mL DMEM高糖培养基与细胞共培养，2种细胞各组样本数均为9。培养24 h后行划痕试验，观察划痕后0(即刻)、24、48 h划痕剩余宽度变化，测量划痕后24、48 h的细胞迁移距离；培养24、48、72 h采用细胞计数仪计数活细胞。(3)2018年6月—2019年6月，笔者单位应用SVF－GEL填充15例患者面部凹陷性瘢痕，其中男2例、女13例，年龄19～42岁。术后6个月，记录SVF－GEL存活情况、有无并发症。术前及术后6个月，对比患者瘢痕凹陷程度、色泽、柔软性，采用温哥华瘢痕量表进行色泽、血管、柔软性评分并计算总分。术后6个月记录十分满意、满意的患者数量。对数据行析因设计方差分析、配对样本t检验及Wilcoxon秩和检验。 结果： (1)SVF－GEL组、阴性对照组EGF含量分别为(316.6±12.8)、(3.4±0.6)pg/mL，VEGF含量分别为(568.67±12.19)、(4.93±0.16)pg/mL，组间比较，差异均有统计学意义(t＝48.777、92.485，P<0.01)。(2)HSF、HaCaT各组细胞划痕剩余宽度随时间延长逐渐缩小，其中SVF－GEL处理组划痕后24、48 h的划痕剩余宽度均小于相应对照组。SVF－GEL处理组划痕后24、48 h HSF、HaCaT细胞迁移距离明显多于相应对照组(t(HSF)＝－20.304、－43.516，t(HaCaT)＝－15.060、－8.684，P<0.01)；培养24、48、72 h，SVF－GEL处理组HSF、HaCaT活细胞数量明显多于相应对照组(t(HSF)＝－3.374、－6.809、－18.036，t(HaCaT)＝－4.793、－6.028、－8.141，P<0.05或P<0.01)。(3)术后6个月，患者移植的SVF－GEL均存活良好，无并发症，瘢痕凹陷程度较术前显著改善，色素沉着较术前显著减轻，瘢痕质地较术前柔软。术后6个月，15例患者面部凹陷性瘢痕的色泽、血管、柔软性评分及总分均明显低于术前(Z＝－2.06，－2.07，－2.07，t＝－15.811，P<0.05或P<0.01)。14例患者对疗效感到十分满意，1例患者对疗效感到满意。 结论： SVF－GEL含有细胞因子EGF、VEGF，可增强HSF、HaCaT细胞迁移和增殖能力，临床治疗凹陷性瘢痕的效果明显。.
Keywords: Cell migration assays; Cell proliferation; Cicatrix; Epidermal growth factor; Stromal vascular fraction gel; Vascular endothelial growth factors.