ABSTRACT
Objective To explore the effect of ginsenoside Rg1 on leukemia stem cells through comparing the biological senescence characteristics of HSCs in the patients with leukemia and healthy people,and provide new ideas and methods for leukemia prevention and treatment.Methods Fifteen cases of normal bone marrow in normal group and sixteen cases of chronic myeloid leukemia in leukemia group were divided into control group and Rg1 group,respectively.The control group used the conventional culture.The Rg1 group used the culture system with 10 μg/mL ginsenoside Rg1,other conditions were the same as control group.The bone marrow mononuclear cell of all groups were extracted after 2 days,and the CD34 +/CD38-cells population was isolated and purified by immunomagnetic adsorption cell sorting(MACS).The purity of the cells and cell cycles phase were detected by flow cytometry.Cell viability was detected by trypan blue staining.The percentage of positive cells was detected by SA-β-gal staining.CCK-8 detected the CD34 +/CD38-proliferation ability of each group.Results The ratio of CD34 +/CD38-cell population was (1.76 ± 0.34) ％ in every 1 × 106 BMNCs before sorting;the proportion of CD34 +/CD38-cell population per 1 × 106 cells after immunomagnetic sorting was (91.15 ± 2.41)％.The positive rate of SA-β-gal staining in human bone marrow CD34 +/CD38-cells of leukemia Rg1 group was significantly higher than that in leukemia control group,the difference was not significant (P ＞ 0.05);meanwhile there was no significant difference between normal control group and normal Rg1 group,but higher than that in leukemia control group,the difference was significant(P ＜ 0.05).CCK-8 results showed that the proliferation of CD34 +/CD38-cells was significantly increased in leukemia control group than those in the other groups.The survival rate of CD34 +/CD38-cells in human bone marrow was 99.1％ in all groups.Cell cycle phase results showed that the G1 arrest of CD34 +/CD38-cells in leukemia control group was significantly lower than those in the other three groups.Conclusion CD34 +/CD38-cells in chronic myeloid leukemia patients may be caused by some chronic myeloid leukemia.Ginsenoside Rg1 can effectively delay the process of aging.
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Objective To investigate the inhibitory effects of docosahexaenoic acid (DHA)and 5-fluorouracil (5-FU)in combination on human gastric cancer cell line AGS in vitro .Methods Human gastric cancer line AGS was treated with different concentrations of DHA and 5-FU alone or in combination.The inhibition of cell proliferation was evaluated by MTT assay.Dose of median (IC50 )of drugs (alone or in combination)and the combination index (CI)were calculated using the median-effect equation and the combination index equation of Chou-Talalay.Flow cytometry was used to detect the cell cycle distribution.The expression of mitochondrial respiratory membrane protein complex in AGS cells was analyzed with Western blot.Results DHA and 5-FU alone or in combination could markedly suppress the proliferation of AGS in significantly time-dependent and dose-dependent manners (P <0.05).IC50 values with DHA or 5-FU administered for 24 h and 48 h were 5 1.60 μg/mL (DHA:24 h),34.82 μg/mL (DHA:48 h),45.90 μg/mL (5-FU:24 h),and 1 6.86 μg/mL (5-FU:48 h), respectively.DHA remarkably strengthened the inhibitory effect of 5-FU and decreased IC50 of 5-FU by 3.56 -2.1 5 folds.The combination of DHA and 5-FU showed synergism.Flow cytometry showed that AGS cells treated with DHA and 5-FU were arrested in G0/G1 phase and the proportion of AGS cells in G0/G1 phase increased compared with that in the control group,DHA group and 5-FU group,while the proportion of the cells in S phase decreased significantly (P < 0.05 ).Western blot showed after treatment with DHA and 5-FU for 48 h,the expression of mitochondrial respiratory membrane protein complex was significantly decreased compared with control group,DHA group and 5-FU group (P <0.05).Conclusion DHA could act synergistically with 5-FU in inhibiting the growth of gastric carcinoma cells,and meanwhile decrease the dose of 5-FU.The mechanism may be associated with cell cycle arrest in G0/G1 phase and interference in the energy metabolism of AGS cells due to inhibition of the expression of mitochondrial oxidative respiratory chain complexes by the two compounds.
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To observe the effects of basic fibroblast growth factor (bFGF) on human adenoid cystic carcinoma ACC-2 cell line proliferation and ERK, cyclin D1/p21waf/ciplsignaling pathways, human adenoid cystic carcinoma cells (ACC-2) were cultured and the influence of bFGF of different concentrations on cell proliferation was determined by MTT. Protein was detected by im muno-precipitation and ERK activity by using ERK agent kit. P-ERK1/2 and down-stream cyclin D1, p21waf/ciplexpression were detected by Western blotting and the interfering role of mitogen pro- tein-activated kinase (MEK) suppressor U0126 in the afore-mentioned indicators was examined. MTr demonstrated ACC-2 cell proliferation was substantially enhanced by bFGF, immuo-precipitation displayed ERK activity was up-regulated by bFGF, and immuno-imprinting also showed p-ERK1/2, cyclin D1 expression was greatly enhanced and p21waf/ciplexpression was inhibited by bFGE U0126 suppressed the effect of bFGE It is concluded that bFGF can promote the proliferation of human adenoid cystic carcinoma ACC-2 cells, and its pathways are associated with the up-regulated activity and expression of p-ERK1/2, inhibited p21waf/cipl expression and enhanced cyclin DI expression.
ABSTRACT
Flowcytometry is a very important technique for the analysis of cell properties, with the advantages of simultaneous multiparameter analysis of large cell population in a short time. Recent advances in computer science and techniques in cell preparation and staining make it more valuable for the study of cell biology and its clinical application. This study was performed to establish the techniques of flowcytometry analysis of osteosarcoma cells, to evaluate the results of the characteristics of the DNA and specific cell cycle phase of osteosarcoma cells obtained by preparation of paraffin-embedded tissue blocks, and to analyze any possible difference between cell populations lacated apart from each other in the tumor mass for making a base for further clinical application. Paraffin-embedded tissue blocks were obtained from 10 cases of primary osteosarcoma, which had undergone amputation without chemotherapy or radiotherapy. Tissue blocks obtained from the most superficial and the deepest portions of the tumor mass from the skin surface were selected respectively in each cases. To evaluate the technique and results obtained, analysis of the whole sample were performed twice in a separate setting. Satisfactory DNA histogram was obtained from 14 of 20 tissue blocks, with the values of distribution in the specific cell cycle phases. DNA aneuploidy was found in 2 cases with a DNA index of 1.6 and 1.3, and no difference in DNA ploidy by the location in tumor mass. The S-phase and G2+M phase fraction were 13.2±8.5 and 6.2±3.1 respectively, reflecting the increased cell proliferation compared with normal cell population. There was no statistically significant difference of these values between superficial and deep portions, but the difference was 9.0±9.7 with a maximum of 26.6, much greater than the difference 3.3±3.6, between the first and second set of analysis. Flowcytometry is a very useful technique in the analysis of the DNA and cell cycle phase properties, and the characteristics of DNA and cell proliferation status of osteosarcoma cells were successfully evaluated by this technique. Unsatisfactory DNA histograms were thought to be the result of inappropriate samples. To adequately evaluate the changes in the tumor mass, standardization in obtaining tumor tissue about the location in the tumor mass is suggested for future studies with flowcytometry about the properties of tumor cells.