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目的分离培养脂肪来源干细胞(adipose-derived stem cells,ADSCs),探讨胰岛素刺激前后其分泌因子的变化及对人角质形成细胞株HaCaT细胞生物学影响。方法从腹部外科手术患者自愿捐献皮下脂肪组织中分离、培养ADSCs,取第3代ADSCs调整密度为5×104个/mL,采用1×10-7mol/L胰岛素刺激作为A组,以未加胰岛素刺激作为B组,培养3d后收集两组ADSCs培养上清液(conditioned medium of ADSCs,ADSC-CM),ELISA法测定其VEGF、肝细胞生长因子(hepatocyte growth factor,HGF)含量。取人角质形成细胞株HaCaT细胞培养,将第4代细胞根据培养液不同分为4组。A1组:含A组ADSC-CM和2FBS各0.5mL;B1组:含B组ADSC-CM和2FBS各0.5mL;C1组:1mL含终浓度为1×10-7mol/L胰岛素的2FBS;D1组:仅含2FBS1mL。培养3d采用MTT法测定HaCaT细胞增殖情况;培养12hAnnexinV-FITC双染测定细胞凋亡情况;培养0、12、24、36、48h采用体外细胞划痕法测定其迁移能力。结果A组VEGF、HGF含量分别为(643.28±63.57)、(929.95±67.52)pg/mL,B组分别为(286.52±46.68)、(576.61±84.29)pg/mL,组间比较差异有统计学意义(P<0.05)。细胞增殖测定A1、B1组吸光度值分别为0.881±0.039、0.804±0.041,与C1组(0.663±0.027)及D1组(0.652±0.042)比较,差异有统计学意义(P<0.01);A1组高于B1组(P<0.05)。A1、B1组凋亡率分别为5.23±1.98、8.82±2.59,均低于C1组(31.70±8.85)和D1组(29.60±8.41),差异有统计学意义(P<0.05),但B1组凋亡率高于A1组(P<0.05)。A1、B1、C1和D1组36h迁移距离分别为(0.1846±0.0192)、(0.1598±0.0294)、(0.0592±0.0176)及(0.0582±0.0123)mm,48h分别为(0.2318±0.1740)、(0.2051±0.0121)、(0.0792±0.0081)及(0.0784±0.0117)mm,两时间点A1组和B1组距离明显大于C1组和D1组(P<0.01),A1组大于B1组(P<0.05);其他时间点迁移距离差异无统计学意义(P>0.05)。结论胰岛素干预后ADSCs能更有效促进HaCaT细胞增殖、迁移和抑制凋亡。
Objective To isolate adipose-derived stem cells (ADSCs) and investigate the changes of their secretory factors before and after insulin stimulation and the biological effects on human keratinocyte line HaCaT cells. Methods ADSCs were isolated and cultured from subcutaneous adipose tissue of abdominal surgery patients. ADSCs of the third generation were adjusted to a density of 5 × 10 4 cells / mL and stimulated with 1 × 10 -7 mol / L of insulin as group A, After being cultured for 3 days, two groups of ADSCs conditioned medium (ADSC-CM) were collected and the VEGF and the content of hepatocyte growth factor (HGF) were measured by ELISA. The human keratinocyte cell line HaCaT cells were cultured, and the fourth generation cells were divided into 4 groups according to the culture medium. Group A1: 0.5 mL each with group A ADSC-CM and 2 FBS; Group B1: 0.5 mL each with group B ADSC-CM and 2 FBS; Group C1: 1 mL 2FBS containing final concentration of 1 x 10-7 mol / L insulin; Group: contains only 2FBS1mL. The proliferation of HaCaT cells was detected by MTT assay after 3 days of culture. The apoptosis of HaCaT cells was detected by double staining with Annexin V-FITC for 12 hours. The migration ability of HaCaT cells was evaluated by cell scratch assay at 0, 12, 24, 36 and 48 hours. Results The levels of VEGF and HGF in group A were (643.28 ± 63.57) and (929.95 ± 67.52) pg / mL, respectively, and those in group B were (286.52 ± 46.68) and (576.61 ± 84.29) pg / mL, Significance (P <0.05). The cell proliferation assay A1, B1 absorbance values were 0.881 ± 0.039,0.804 ± 0.041, C1 group (0.663 ± 0.027) and D1 group (0.652 ± 0.042), the difference was statistically significant (P <0.01); A1 group Higher than B1 group (P <0.05). The apoptosis rates in A1 and B1 groups were 5.23 ± 1.98 and 8.82 ± 2.59, respectively, which were significantly lower than those in C1 group (31.70 ± 8.85) and D1 group (29.60 ± 8.41) (P <0.05) The apoptosis rate was higher than A1 group (P <0.05). The migration distances of group A1, group B1, group C1 and group D1 were (0.1846 ± 0.0192), (0.1598 ± 0.0294), (0.0592 ± 0.0176) and (0.0582 ± 0.0123) mm, respectively 0.0121), (0.0792 ± 0.0081) and (0.0784 ± 0.0117) mm respectively. The distance between A1 group and B1 group was significantly greater than that of C1 group and D1 group (P <0.01) There was no significant difference in migration distance at time point (P> 0.05). Conclusion ADSCs can effectively promote the proliferation, migration and apoptosis of HaCaT cells after insulin intervention.