| Experimental objectiveIn vitro study of Rho kinase inhibitor fasudil on HL-60 cell proliferation, apoptosis, differentiation and migration elucidate the fasudil inhibition of leukemia transfer mechanism, for clinical admiral fasudil as anti leukemia drugs to provide more theoretical basis. Experimental material and experimental methodsUsage fasudil at different concentrations on HL-60 cells and leukemia cells treated for 96 hours(24, 48, 72 hours, 96 hours) by trypan blue staining method to count the number of living cells, in order to observe the inhibitory effect of fasudil on the proliferation of leukemia cells; HL-60 cells from Tianjin Blood Disease Hospital; usage fasudil in different concentration of HL-60 cells were treated for 48 hours, time interval(6h, 12 h, 24 h, 48h) by flow cytometry instrument to measure the apoptosis rate; usage fasudil in different concentration of HL-60 cells were treated for 72 hours, NBT reduction test is used to calculate percentage of positive cells, using flow cytometry to detect cell surface differentiation antigen CD11 b, CD13; usage fasudil in different concentrations on HL-60 cells to detect the expression of MMP-2,MMP-9m RNA by RT-PCR after 72 hours; the Transwell chamber in vitro tumor cell migration model, usage fasudil in different concentration in Transwell chamber room on HL-60 cells and patient ’s leukemia cells after 12 hours treatment of Transwell cell number counting the lower chamber, to observe the effects of fasudil on leukemia cell migration. The experimental results1. Inhibition method by using trypan blue dye exclusion of the monitoring effect of fasudil on the proliferation of HL-60 cells to different concentrations of fasudil(25, 50, 75umol/l) can inhibit the proliferation of in 24, 48, 72, 96 hours, inhibited the proliferation of strength with time and concentration dependence.2. Inhibition method by using trypan blue dye exclusion of the monitoring effect of fasudil on the proliferation of patient ’s leukemia cells to different concentrations of fasudil(25, 50, 75,100umol/l) can inhibit the proliferation of in 24, 48, 72, 96 hours, inhibited the proliferation of strength with time and concentration dependence.3. Flow cytometry was used to detect the apoptosis rate in different concentrations(50, 100, 150umol/L) on HL-60 cells treated with 6,12,24, 48 h, Annexin V-7AAD double labeled by flow cytometry detection showed effects of fasudil in a certain range of time induced apoptosis of HL-60 cells with the drug concentration and time increased, and showed a time- and dose-dependent manner.At the same time, the apoptosis rate of the experimental group was different, and it was statistically significant(P < 0.05).4. Usage fasudil the 25umol/l, 50umol/l, 75umol/l concentration treatment HL-60 cells for 72 hours, the cells were collected by NBT staining under optical microscope count positive cells percentage. The results show that the with the drug concentration increased the percentage of positive cells increased; usage fasudil the 25umol/l, 50umol/l, 75umol/l concentration treatment HL-60 cells for 72 hours, by flow cytometric detection of HL-60 cell surface differentiation antigen CD11 b, CD13 expression rate, found CD11 b expression rate increases with the increasing of drug concentration, and the expression of CD13 rate decreases, compared control group and the experimental group, with statistical significance(P < 0.05).5.Using RT-PCR detection of MMP-2 and MMP-9m RNA expression, cell usage fasudil in 50, 100, 150umol/l concentration treatment of HL-60 cells with 72 h, results show that compared with the control group, with the increasing of drug concentration, MMP-2, down-regulation of MMP-9m RNA expression.6. Transwell chamber was used for in vitro tumor cell migration model to observe the effect of fasudil on HL-60 cells and leukemia cell transfer of: control group compared with the increase of drug concentration and fasudil on HL-60 cells and leukemia cells migration inhibition increases gradually. There were significant differences between the control group and the experimental group(P < 0.05). Experimental conclusion:Fasudil can inhibit the proliferation of leukemic cells and induce the apoptosis, promote the differentiation of leukemia cells, can inhibit leukemic cells distant metastasis, revealing the role of the Rho kinase inhibitor fasudil in anti leukemia, which lay a theoretical basis for future clinical application of fasudil treatment of leukemia. |
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