人脂肪来源间充质干细胞条件培养基对病理性瘢痕成纤维细胞生物学行为的影响

目的探讨人脂肪间充质干细胞条件培养基(human adipose-derived mesenchymal stem cells conditioned media,hADSCs-CM)对病理性瘢痕成纤维细胞增殖、迁移能力的影响。方法分离培养hADSCs和病理性瘢痕成纤维细胞,取第3代细胞用于后续实验。于培养12、24、48 h收集hADSCs条件培养基作为实验组条件培养基(12 h-CM、24 h-CM、48 h-CM),无hADSCs的空白无血清低糖DMEM培养基置于培养箱内12、24、48 h作为对照组条件培养基(12 h-CC、24 h-CC、48 h-CC)。选取增生性瘢痕成纤维细胞(HSFs)、瘢痕疙瘩成纤维细胞(KFs)以及正常皮肤成纤维细胞(NFs),分别以实验制备的培养基进行处理,每组培养基3个复孔,以CCK-8实验检测HSFs及KFs的增殖能力,以细胞划痕实验检测条件培养基处理后细胞迁移能力的变化,流式细胞仪分析细胞的周期分布、凋亡及乳酸脱氢酶(LDH)水平变化情况。实验数据以Graphpad 7.0软件进行分析,多组样本均数比较应用One-Way ANOVA,P<0.05为差异有统计学意义。结果CCK-8结果显示,24 h-CM、48 h-CM处理后24 h KFs、HSFs的增殖速度较对照组开始出现明显减慢,HSFs增殖趋势也出现了类似的变化趋势,NFs无明显的增殖趋势变化;实验组条件培养基对HSFs增殖的抑制作用在48 h达到峰值:24 h-CM KFs增殖活性为1.81±0.10,24 h-CC KFs为2.36±0.05(t=4.24,P<0.001);24 h-CM HSFs增殖活性为1.52±0.10,24 h-CC HSFs为1.96±0.15(t=8.98,P=0.001);48 h-CM KFs增殖活性为1.65±0.10,48 h-CC KFs为2.57±0.10(t=26.64,P<0.001);48 h-CM HSFs增殖活性为1.29±0.20,48 h-CC HSFs为1.94±0.10(t=11.14,P<0.001);之后抑制作用渐弱。细胞划痕实验结果表明,与对照组相比,24、48 h收集的hADSCs-CM处理后HSFs和KFs的迁移能力下降。细胞周期检测结果显示,在KFs中,12 h-CM、24 h-CM、48 h-CM组细胞处于G0/G1期的细胞比例较对照组升高,而处于S期的细胞比例则较对照组下降,在HSFs中也观察到了类似的现象。细胞凋亡检测结果显示各组NFs、KFs、HSFs未见明显凋亡。LDH漏出检测结果显示各组间无明显差异。结论hADSCs条件培养基可能通过其中含有的分泌因子抑制病理性瘢痕成纤维细胞的增殖、迁移,但不诱导其凋亡。

Study on the effects of conditioned medium of adipose-derived mesenchymal stem cell on biological phenotype of hypertrophic scar fibroblasts and keloid fibroblasts

Objective To investigate the effects of human adipose-derived mesenchymal stem cells conditioned media(hADSCs-CM)on the proliferation and migration of pathological scar fibroblasts.Methods Human adipose-derived mesenchymal stem cells(hADSCs)and pathological scar fibroblasts were isolated and cultured.The hADSCs were cultured in DMEM for 12,24,and 48 h to collect the experimental group conditioned medium.The DMEM medium without hADSCs was placed in the incubator for 12,24,and 48 h to collect control group medium.Three kinds of fibroblasts including hypertrophic scar(HSFs),keloid(KFs)and normal skin were selected and treated with the prepared culture system.Each group of culture medium was in triptriplicate.The proliferation ability was measured by CCK-8 assay,and wound healing test to detect the cell migration ability after conditioned medium treatment,and the cell cycle distribution and apoptosis were detected by flow cytometry.The experimental data were analyzed by Graphpad 7.0 software.One-Way ANOVA was used for comparison among multiple groups of samples.P<0.05 was considered statistically significant.Results The result of CCK-8 showed that the cell viability of KFs after treatment for 24 h,the cell viability of 24 h-CM and 48 h-CM groups begun to be inhibited compared with 24 h-CC and 48 h-CC.There was a similar trend in the proliferation of HSFs;and the inhibition reached a peak at 48 h:cell proliferation rate of 24 h-CM-KFs(1.81±0.10)was significantly inhibited compared with 24 h-CC-KFs(2.36±0.05)(t=4.24,P<0.001);for HSFs,24 h-CM groups’proliferation rate(1.52±0.10)have significant difference when compared to 24 h-CC-HSFs(1.96±0.15)(t=8.98,P=0.001);treated with 48 h-CM,the proliferation rate of HSFs and KFs were significantly lower than that of 48 h-CC groups respectively(t=11.14,P<0.001;t=26.64,P<0.001).The wound healing test showed that the migration ability of hypertrophic scar and keloid fibroblasts decreased after treatment with hADSCs conditioned medium at 24 and 48 h compared with the control group.There was no significant difference between the groups in the LDH leak detection.The result of cell cycle tests showed that KFs after treatment with CM groups,the proportion of cells in the G0/G1 phase was higher than that in the control groups,while the proportion of cells in the S phase was lower than that in the control groups.And a similar phenomenon was observed in HSFs.Conclusions The conditioned medium of hADSCs may inhibit the proliferation and migration of pathological scar fibroblasts through secretory factors,without inducing apoptosis of pathological scar fibroblasts.