| Objective :To culture human MHCC97H hepatocellular carcinoma cells by using serum-free suspension culture method and make them into the ball and to show the characteristics of stem cells,so as to provide a way for MHCC97H hepatocellular stem cell sorting.And to explore the effect of arsenic trioxide on MHCC97H hepatocellular carcinoma stem cells and its intrinsic mechanism.Method:By using the vitro clonal formation experiment,the expressi on of CD133 and CD90CD45 on the surface of hepatocellular carcinoma stem cells were detected by immun of luores cence staining and WB respectively.The stem cells of MHCC97H cells were identified by stem cell identification.The CCC8 method was used to detect the OD of MHCC97H hepatocarcinoma stem cells.The MHCC97H hepato carcinoma stem cells were inoculated into 96 plates.After treatment at different concentrations(0μM,2μM,4μM,6μM,8μM)of arsenic trioxide for 12,24 and 48 hours,The cell morphology was observed by an inverte d microscope and 10 μl of CCK8 solution was added to each well.Then incubated the cells for 12 hours in an incubator and the absorbance at 450 nm was measured by a microplate reader.Repeat each time three times to calculate the average.The cells were collected with the best concentra tion of arsenic trioxide for 12,24 and 48 hours,and the apoptosis of MH CC97H hepatocellular carcinoma cells was detected by flow cytometry.The expression of Bcl-2 protein was detected by Western Blot,and the expression of anti-apoptotic gene Bcl-2 was detected by Real-time PCR.Results:1.MHCC97H hepatocellular carcinoma cells can be cultured in to stem cells in serum-free suspension medium.The cells grew into balls and the spheroids grew rapidly in the serum-free medium.2.Immunofluorescence results showed that most of the MHCC97H hepatocarcinoma cells showed red fluorescence,that is,CD133 positive expression,while the adherent cell expression was negative.WB results showed that the relative expression of CD90CD45 protein in MHCC97H hepatocellular carcinoma stem cells was higher than that in adherent cells.The number of clonal proliferation in vitro was significantly higher than that in adherent cells.3.Under the microscope,the morphology and number of MHCC97H he patocellular stem cells decreased gradually with the increase of arsenic trioxide concentration.After 24 hours of drug administration,CCK8 results showed that the OD value was 0.846 ± 0.007 and 0.723 ± 0.076 at the concentration of 2-4 uM,and the OD value of control group(0 uM)was 0.831± 0.050.Compared the results with these two groups,the difference indicated a slight decrease in the mean P> 0.05.The OD value was 0.633 ± 0.021 and 0.570 ± 0.074 at the concentration of 6-8uM,and the results had statistical meaning(p<0.05)compared with the control group(0 uM).There was no significant difference between 6uM and 8uM.The results showed that arsenic trioxide could inhibit the proliferation of MHCC97H cells in vitro,and it was proved that the concentration of of 6-8uM could be the best concentration group of arsenic trioxide.4.The results of flow cytometry showed that the apoptotic rates were 3.063 ± 0.073,3.537 ± 0.042,4.110 ± 0.159 at 12 hours,24 hours and 48 hours.Compared with the apoptotic rate at 0 hour(0.097 ± 0.032),the difference had statistical meaning(P<0.05).The results of flow cytometry showed that the apoptosis rate of hepatocellular carcinoma stem cells was time-dependent.5.The results of Western Blot showed that the relative expression of CD90 protein was 0.160 ± 0.010,0.130 ± 0.010 and 0.100 ± 0.010 at 12 hours,24 hours and 48 hours.Compared with the control group(0.167 ± 0.032),the expression of CD90 was slightly decreased after 12 hous(P<0.05).While the expression of CD90 was significantly decreased after 24 hours and 48 hours(P<0.05).The expression of CD45 was negative.Indicating that arsenic trioxide can inhibit the expression of surface markers of liver cancer stem cells.6.The results of Western Blot and Real-time PCR showed that the relative expression of Bcl2 protein was 0.27 ± 0.021 and 0.77 ± 0.025 and 0.27 ± 0.035 respectively at 12,24 and 48 hours respect ively.The relative expression levels of RNA were 2.740 ± 0.257,3.060 ± 0.308,3.70 ± 0.235 at 12,24 and 48 hours.Compared with the control group(0 hours),the protein and RNA expression of Bcl-2 decreased slightly after 12 hours,the difference was not statisti cally significant(p> 0.05).While after 24 hours and 48 hours,the difference was statistically significant(P<0.05).Indicating that arsenic trioxide can promote the apoptosis of hepatocellular stem cells by down-regulating the expression of Bcl-2.Conclusion: 1.MHCC97H hepatocarcinoma cells can cultured hepatocellular carci noma stem cells in serum-free suspension culture medium.2.Arsenic trioxide can inhibit the proliferation and promote apoptosis of hepatocarcinoma stem cells in vitro.3.Arsenic trioxide promotes the apoptosis of hepatocarcinoma st em cells by down-regulating the expression of Bcl-2. |
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