Objectives To investigate the effects of Tongxinluo and Homocysteine (Hcy) on proliferation of human umbilical vein endothelial cells (Huvecs) in vitro.
Methods Isolation,culture and identification of Huvecs:With the informed consents of puerperants, the normal fetal umbilical cords were obtained through uterine-incision delivery in Third Affiliated Hospital, Sun Yat-sun University. Huvecs were isolated by Percoll density gradient centrifugation from fetal umbilical cords with digestion of collagenase type perfusion, and then suspended in Medium 199 supplemented with 20% FBS, 2 mM L-glutamine, 50 μg/ml endothelial cell growth factor, 100 μg/ml heparin and 100μg/ml streptomycin cultured in 0.05 volume fraction of CO2 incubator at 37°. The culture solution was changed after 24 hours, Huvecs cultured to 80% confluence, the passage was developed with 0.25% trypsinase and 0.02% EDTA. After the Huvecs were identified by flow cytometry with the cell marker CD34, The second or third passage was used for study. MTT assay was used to detect cell viability: Huvecs during the logarithmic phase were inoculated in 96-well plates with Medium 199 in 0.05 volume fraction of CO2 incubator at 37° for 48h, at 80% confluence, incubated in serum-free medium,and divided into two groups, eachgroup including eight parallel wells, blank control group was untreated, he latter cultured and added with Hcy of the concentrations of 0.1, 0.25, 0.5, and 1.0 mmol/L respectively,or Hcy 0.5 mmol/L+Tongxinluo of the concentrations of 0.1, 0.5, 1.0, and 2.0mg/ml. After the cells were cultured for 24 hr, 5 g/L MTT solution (20μL) was added, then dimethyl sulfoxide was used for vibration and dissolvement. Optical density of each hole was detected by using enzyme-linked immunosorbent assay to analyse different concentrations of Hcy and Tongxinluo on the proliferation of human mammary stemscells.
Results Hcy of different doses inhibited the viability of Huvecs dose-dependently, the Optical density were 0.526 ± 0.017, 0.439 ± 0.010, 0.258 ± 0.008, and 0.209 ± 0.004 respectively, lower than that (0.578 ± 0.023) of blank control group (all P<0.01), accordingly,the inhibition rates of Hcy were 0.099 ± 0.079, 0.239 ± 0.046, 0.637 ± 0.007, and 0.553 ± 0.029 respectively (P<0.05, P<0.01); the growth rates of Hcy-induced Huvecs treatment by Tongxinluo of the concentrations of 0.1, 0.5, 1.0, and 2.0mg/ml were 0.682 ± 0.312,0.710 ± 0.040, 0.855 ± 0.130, 0.784 ± 0.031.
Conclusions Hcy can inhibited the viability of Huvecs dose-dependently, and this effect is most prominent at the concentrations of 0.5 mmol/L for 24 hours; Tongxinluo can enhance the proliferation activity of Huvecs, and this effect is most prominent at 1.0mg/ml for 24 hours.