Effect of conditioned medium from lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells on proliferation and apoptosis of human umbilical vein endothelial cells

Abstract

Objective To investigate the effect of conditioned medium of lipopolysaccharide pretreatment human umbilical cord mesenchymal stem cells (hUCMSC) on proliferation and apoptosis of human umbilical vein endothelial cells (HUVEC). Methods HUVEC was randomly divided into 3 groups: control group, conditioned medium group (CM group) and pretreatment-conditioned medium group (pretreatment-CM group). After 100 ng/mL lipopolysaccharide stimulating HUVEC 12 h, media in control group was replaced for the original medium, and conditioned medium and preconditioning medium were replaced for the CM and lipopolysaccharide pretreatment-CM, respectively. Cell proliferation was detected by MTT. Cell counting were detected by trypan blue exclusion staining. Cell cycle was detected by flow cytometry. Cell apoptosis was assessed by Hoechst staining and flow cytometry. Statistic was analyzed by variance analysis. Results At 24 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.46, 0.52 and 0.56, respectively. At 48 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.35, 0.46 and 0.51, respectively. At 72 h post treatment, the optical density values of control group, CM group and pretreatment-CM group were 0.21, 0.38 and 0.47, respectively. At the same time point post treatment, the proliferation numbers in CM group and pretreatment-CM group were markedly higher than that in control group, as well as that in pretreatment-CM group was higher than that in CM group. The result of cell counting was similar with MTT result. At 24 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (4.554±0.103)×103, (5.251±0.091)×103 and (5.585±0.038)×103, respectively. At 48 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (3.465±0.087)×103, (4.659±0.116)×103 and (5.347±0.099)×103, respectively. At 72 h post treatment, the cell counting of control group, CM group and pretreatment-CM group were (2.079±0.127)×103, (4.063±0.052)×103 and (4.950±0.104)×103, respectively. At the same time point post treatment, the cells numbers in CM group and pretreatment-CM group were markedly higher than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was higher than that in CM group, the difference was statistically significant (P<0.05). Meanwhile, the results of cell cycle were assayed by flow cytometry. At 24 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (17.07±1.15)%, (22.52±1.61)% and (26.19±2.25)%, respectively. At 48 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (13.46±1.74)%, (18.67±2.07)% and (24.58±2.54)%, respectively. At 72 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (8.73±1.61)%, (14.31±1.93)% and (18.43±2.02)%, respectively. At the same time point post treatment, the G2/M+ S ratio in CM group and pretreatment-CM group were markedly higher than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was higher than that in CM group, the difference was statistically significant (P<0.05). Furthermore, the results of Hoechst staining and flow cytometry were showed that cell apoptosis rates of control group, CM group and pretreatment-CM group were (30.78±3.20)%, (23.21±1.72)% and (17.69±1.81)% at 24 h post treatment. At 48 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (26.02±2.06)%, (20.04±1.83)% and (15.03±1.56)%, respectively. At 72 h post treatment, the G2/M+ S ratio of control group, CM group and pretreatment-CM group were (22.41±1.63)%, (15.27±1.14)% and(11.25±1.07)%, respectively. At the same time point post treatment, the apoptosis rates in CM group and pretreatment-CM group were markedly lower than that in control group, the differences were statistically significant (P<0.05), as well as that in pretreatment-CM group was lower than that in CM group, the difference was statistically significant (P<0.05). Conclusion The conditioned medium from 100 ng/mL lipopolysaccharide pretreatment hUCMSC significantly increased HUVEC proliferation activities and deceased cell apoptosis numbers and apoptosis rates, which were induced by lipopolysaccharide. Key words: Lipopolysaccharides; Mesenchymal stem cells; Endothelial cells; Pretreatment; Cell proliferation; Apoptosis