Effect of conditioned medium from TNF-α pretreatment hUCMSC on proliferation and apoptosis of HUVEC

Abstract

Objective To investigate effect of conditioned medium from 100 ng/mL TNF-α pretreatment human umbilical cord mesenchymal stem cell (hUCMSC) on human umbilical vein endothelial cell (HUVEC) injury and apoptosis induced by TNF-α. Methods HUVEC were randomly divided into 3 groups: control group, conditioned medium group and pretreatment-conditioned medium group. After 100 ng/mL TNF-α stimulating HUVEC 12 h, media in control group was replaced for the original medium, and conditioned medium and preconditioning medium were replaced for the conditioned medium and TNF-α pretreatment-conditioned medium, respectively. Cell proliferation was detected by MTT. Cell cycle was detected by flow cytometry. Cell apoptosis was assessed by AO-EB staining and flow cytometry. Data was analyzed using repetitive measure analysis of variance method. Results At 24, 48 and 72 h post treatment, the optical density values of control group were 0.51, 0.43 and 0.32, and those in conditioned medium and pretreatment-conditioned medium groups were 0.56, 0.51, 0.45 and 0.59, 0.56, 0.52, respectively. Meanwhile, the result of cell cycle, which was assayed by flow cytometry, showed similar tendency with MTT result. At 24 h, 48 h and 72 h post treatment, the G2/M+ S ratio of control group were (22.34±1.03)%, (10.66±1.07)% and (9.58±0.82)%, and those in conditioned medium and TNF-α pretreatment-conditioned medium groups were (27.95±1.85)%, (18.59±1.48)%, (13.02±1.91)% and (31.17±1.29)%, (22.44±2.28)%, (16.28±1.11)%, respectively. Three groups of comparative difference was statistically significant (F=58.793, P<58.793). Furthermore, the results of flow cytometry were showed that cell apoptosis rates of control group were (18.95±1.01)%, (16.66±1.51)% and (11.37±1.01)%, and those in conditioned medium and TNF-α pretreatment- conditioned medium groups were (12.04±1.13)%, (10.88±1.39)%, (6.88±1.63)% and (8.16±1.44)%, (6.97±1.34)%, (2.97±1.44)% at 24, 48 and 72 h post treatment. Three groups of comparative difference was statistically significant(F=119.372, P<0.05). The results of flow cytometry were similar with those of AO-EB. Conclution The conditioned medium from 100 ng/mL TNF-α pretreatment hUCMSC significantly increased HUVEC proliferation activities and deceased cell apoptosis numbers and apoptosis rates, which were induced by TNF-α. Key words: Tumor necrosis factor-alpha; Mesenchymal stem cells; Umbilical veins; Endothelial cells; Cell proliferation; Apoptosis