Ou L D, Zhang A J, Li A, Tao S J, Xu M M, Li Q, Jin P S
Graduate School, Xuzhou Medical University, Xuzhou 221002, China.
Department of Plastic Surgery, the Affiliated Hospital of Xuzhou Medical University, Xuzhou 221000, China.
Zhonghua Shao Shang Za Zhi. 2019 Dec 20;35(12):859-865. doi: 10.3760/cma.j.issn.1009-2587.2019.12.006.
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. (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 test, and Wilcoxon rank sum test. (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 (=48.777, 92.485, <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 ((HSF)=-20.304, -43.516, (HaCaT)=-15.060, -8.684, <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 ((HSF)=-3.374, -6.809, -18.036, (HaCaT)=-4.793, -6.028, -8.141, <0.05 or <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 (=-2.06, -2.07, -2.07, =-15.811, <0.05 or <0.01). One patient was satisfied with the clinical outcome, and the rest 14 patients were completely satisfied with the clinical outcomes. 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.
观察人基质血管成分凝胶(SVF - GEL)中细胞因子的含量,以及SVF - GEL对体外表皮细胞和真皮细胞生物学行为的影响及临床疗效。(1)采用作者所在单位接受腹部抽脂术的女性抽脂吸出物制备SVF - GEL。将SVF - GEL(1 mL)和高糖杜氏改良 Eagle 培养基(DMEM,1 mL)分别与含10%胎牛血清、10 g/L青霉素和10 g/L链霉素的高糖DMEM培养24 h,分别记为SVF - GEL组和阴性对照组,每组6个样本。采用酶联免疫吸附测定法测定上清液中表皮生长因子(EGF)和血管内皮生长因子(VEGF)的含量。(2)将5×10⁵处于对数生长期的人皮肤成纤维细胞(HSF)和HaCaT细胞接种于Transwell小室中培养12 h。将所有含细胞的Transwell小室分为SVF - GEL组(加入0.5 mL SVF - GEL共培养)和对照组(加入0.5 mL高糖DMEM共培养),每组HSF和HaCaT细胞各9个样本。培养24 h后进行划痕试验,分别在划痕后0 h(即刻)、24 h和48 h观察残余划痕宽度。在划痕后24 h和48 h测量细胞迁移距离。培养24 h、48 h和72 h后,使用细胞计数仪计数活细胞数量。(3)2018年6月至2019年6月,对15例面部凹陷性瘢痕患者进行临床应用SVF - GEL治疗,其中男性2例,女性13例,年龄19~42岁。术后6个月观察SVF - GEL的存活情况及有无并发症。比较手术前和术后6个月瘢痕的凹陷程度、颜色和柔韧性。采用温哥华瘢痕量表(VSS)评估手术前和术后6个月瘢痕的颜色、血管分布和柔韧性,并计算总分。术后6个月统计完全满意或满意的患者人数。数据采用析因设计方差分析、配对样本t检验和Wilcoxon秩和检验进行处理。(1)SVF - GEL组和阴性对照组中EGF的含量分别为(316.6±12.8)和(3.4±0.6)pg/mL,VEGF的含量分别为(568.67±12.19)和(4.93±0.16)pg/mL,两组间差异有统计学意义(F = 48.777,92.485,P < 0.01)。(2)SVF - GEL组和对照组中HSF和HaCaT细胞的残余划痕宽度均随时间逐渐减小,其中SVF - GEL组在划痕后24 h和48 h的残余划痕宽度小于对照组。在划痕后24 h和48 h,SVF - GEL组中HSF和HaCaT细胞的迁移距离大于对照组((HSF)t = -20.304,-43.516,(HaCaT)t = -15.060,-8.684,P < 0.01)。培养24 h、48 h和72 h后,SVF - GEL组中HSF和HaCaT细胞的活细胞数量明显多于对照组((HSF)t = -3.374,-6.809,-18.036,(HaCaT)t = -4.793,-6.028,-8.141,P < 0.05或P < 0.01)。(3)术后6个月,移植到患者体内的SVF - GEL存活良好,无并发症发生,瘢痕凹陷程度明显改善,色素沉着减轻,质地变软。与手术前相比,15例面部凹陷性瘢痕患者术后6个月瘢痕颜色、血管分布和柔韧性的VSS评分及总分明显降低(t = -2.06,-2.07,-2.07,F = -15.811,P < 0.05或P < 0.01)。1例患者对临床疗效满意,其余14例患者对临床疗效完全满意。SVF - GEL含有细胞因子EGF和VEGF,可增强HSF和HaCaT细胞的迁移能力和增殖能力,对凹陷性瘢痕临床应用有明显效果。
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