Effects Of Hypoxia On The Expression And Proliferation Of HIF-1α, VEGF, Bcl-2 And Bax Proteins In HepG2 Cells

1 ObjectiveEstablish the hypoxic model of HepG2 cells in vitro with different concentration of cobalt chloride (CoCl2). Observe the effects on HepG2 cells proliferation in varying degrees of hypoxia and different time, and discuss the cause. To survey the expression of HIF-la, VEGF, Bcl-2 and Bax protein in HepG2 cells in varying degrees of hypoxia and discuss the possible mechanism and effect.2 MethodsEstablished different levels of hypoxic model with different concentrations of CoCl2 (0μmol/L,50μmol/L, 100μmol/L,200μmol/L,300μmol/L,500μmol/L), incubated HepG2 cells under hypoxic condition for different time(12h,24h,48h,72h), and used CCK-8 to detect HepG2 cells proliferation.With different concentrations of CoCl2 (0μmol/L,50μmol/L, 100μmol/L,200μmol/L, 300μmol/L,500μmol/L) to simulate the hypoxia condition, incubated HepG2 cells in hypoxia for 48 hours, and use Western Blot to observe the change of the expression of HIF-la,VEGF, Bcl-2, and Bax.3 ResultsWith CCK-8, the results shown that CoCl2 within the certain range (≤100μmol/L) had no obvious impact on HepG2 cells proliferation. HepG2 cells proliferation was inhibited in higher concentration of CoCl2 (≥200μmol/L), and the higher the concentration, the stronger the inhibition degree.We use Western Blot test to investigate the protein expression. The results shown relative grey value of HIF-1α in 50μmol/L, 100μmol/L,200μmol/L,300μmol/L,500μmol/L concentration group was (0.277±0.003), (0.272±0.003), (0.333±0.005), (0.364±0.003), (0.366±0.008), while relative grey value in normoxic group was (0.122±0.002), which had statistical significance (P<0.05); the relative grey value between 50μmol/L and 100μmol/L, 300μmol/L and 500μmol/L concentration was no statistical significance (P>0.05), while the other groups had statistical significance (P< 0.05). Relative grey value of VEGF protein in 50μmol/L, 100μmol/L,200μmol/L,300μmol/L,500μmol/L concentration group was(0.268±0.003), (0.368±0.009), (0.450±0.007), (0.535±0.010), (0.584±0.015), which had statistical significance (P<0.05) copareded with normoxic group; Relative grey value of hypoxia group compared with previous concentration group had statistical significance (P<0.05). The relative order grey value of Bcl-2 protein in 50μmol/L, 100μmol/L,200μmol/L,300μmol/L,500μmol/L concentration group is (0.439±0.003), (0.399±0.005), (0.322±0.004), (0.263±0.001), (0.124±0.002), which had statistical significance (P<0.05) compared with normoxic group (0.445±0.003); relative grey value of hypoxia group compared with previous concentration group had statistical significance (P<0.05). Relative grey value of Bax protein in 50μmol/L, 100μmol/L,200μmol/L,300μmol/L, 500μmol/L concentration group was (0.362±0.005), (0.455±0.007), (0.480±0.009), (0.506±0.006), (0.541±0.014), which had statistical significance (P<0.05) compared with normoxic group (0.243±0.003); Relative grey value of hypoxia group compared with previous concentration group had statistical significance (P<0.05). The expression of HIF-la, VEGF and Bax protein were increased, the levels of which was proportional to the hypoxic levels; But the Bcl-2 protein expression was restrained under hypoxia, which was inversely proportional to the hypoxic levels.4 ConclusionBy different concentration of CoCl2, we successfully established the hypoxic model of HepG2 cells in vitro. CoCl2 within the certain range (≤100μmol/L) had no obvious impact on HepG2 cells proliferation. High concentration of CoCl2 (>200μmol/L) would inhibit the growth of HepG2 cells, and the higher the concentration of CoCl2, the stronger the inhibition degree. Hypoxia can induce the expression of HIF-la protein increased in HepG2 cells, at the same time enhanced the expression of the protein under the HIF-1α in the signal path, such as VEGF and Bax, and Bcl-2 expression is inhibited.