胰岛素干预后脂肪来源干细胞分泌功能对HaCaT细胞生物学影响

CYTOBIOLOGICAL EFFECT OF ADIPOSE-DERIVED STEM CELLS TREATED WITH INSULIN ON HaCaT CELLS

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 and culture adipose-derived stem cells （ADSCs）, and to study the effects of the conditioned medium of ADSCs （ADSC-CM） treated with insulin on HaCaT cells. Methods ADSCs were isolated from adi pose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5×10^4cells/mL. The cells were divided into 2 groups： group A in which the cells were incubated in 1×10^-7 mol/L insulin for 3 days, and group B in which the cells were not treated with insulin. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor （HGF）. HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups： group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1×10^-7 mol/L insulin; group D1, 1 mL 2% FBS. Proliferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration ability was measured by in vitro wound-healing assay 0, 12, 24, 36 and 48 hours after culture. Results The level of VEGF in groups A and B was （643.28±63.57） and （286.52± 46.68） pg/mL, respectively, and the level of HGF in groups A and B was （929.95±67.52） and （576.61±84.29） pg/mL, respectively, suggesting differences were significant between two groups （P 〈0.05）. Cell proliferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881±0.039, 0.804±0.041, 0.663±0.027 and 0.652±0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 （P 〈 0.01）, group A1 was significantly higher than group B1 （P 〈 0.05）. The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23%±1.98%, 8.82%±2.59%, 31.70%±8.85% and 29.60%±8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 （P 〈 0.05）, group B1 was significantly higher than group A1 （P 〈 0.05）. The migration distance of HaCaT cells in groups A1, B1, C1 and D1 at 36 hours was （0.1846±0.0192）, （0.1598±0.0294）, （0.0592±0.0176） and （0.0582±0.0123）mm, respectively, whereas at 48 hours, it was （0.2318±0.1740）, （0.2051±0.0121）, （0.0792±0.0081） and （0.0784±0.0117）mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours （P 〈0.01）, group A1 was significantly higher than group B1 （P 〈0.05） at 36 and 48 hours, no significant difference was evident at other time points（P 〉0.05）. Conclusion ADSCs treated with insulin can significantly promote the proliferation and the migration of HaCaT cells and inhibit their apoptosis.