| Objective: Acute promyelocytic leukemia(APL) is one subtype of acute myeloid leukemia(AML). Acute promyelocytic leukemia(APL)is a specific type of acute myeloid leukemia characterized by the t(15;17) translocation that fuses the PML gene on chromosome 15 to the retinoic acid receptor a(RARα) gene on chromosome 17 to form the fusion gene and leukemogenic protein PML/RARα. This is the molecular basis of APL leukemogenesis and effective treatment of all-trans retinoic acid(ATRA) and arsenic trioxide (As2O3) on APL. The treatment of all-trans retinoic acid(ATRA) and arsenic trioxide (As2O3) on the acute promyelocytic leukemia(APL) had been widely accepted by the world.CD44 adhesion molecule is a cell surface transmembrane glycoprotein. It displays many variant isoforms generated by alternative splicing of exons, including CD44s and CD44v. It has reported that CD44 cell surface adhesion molecule is strongly expressed on leukemic blasts in all acute myeloid leukemia subtypes. It has been found through in vitro study that CD44 monoclonal antibodies can inhibit proliferation of all acute myeloid leukemia cell lines and induce terminal differentiate or apoptosis of some cell lines to some extent. It has been reported that high expression of CD44v3, CD44v6 and CD44v10 was detected in bone marrow and peripheral blood of patients with AML. CD11b is a member of the Adhesion molecule integrin family, which was the mark of APL cells-differentiation. CD11b is expressed on mature monocytes, NK cells, granulocyte. PML/RARαfusion gene is the specific genetic abnormalities, with acute promyelocytic cells into mature myeloid differentiation or apoptosis, its expression gradually weakened. So PML/ RARαfusion gene can be used as the judging criteria of prognosis and treatment efficacy on APL.In this study, ATRA and As2O3 was acted on human acute promyelocytic leukemia(APL) NB4 cell lines. We observed the changes in cell morphology with Optical microscope, cell inhibitory growth rate investigated by MTT assay, differentiation and apoptosis rate by Flow cytometry(FCM). Moreover expression of PML/RARα, CD44v3, CD44v6 and CD44v10 mRNA on NB4 cells was detected by RQ-PCR. We study the expression trends of different CD44v molecular and PML/RARαon NB4 cells under differentiation and apoptosis,and the relationship between them. Thus, theoretic evidence of CD44v as a leukemia diagnosis, prognostic, and effective indicators in clinical application will be provided.Materials and method: Human Acute promyelocytic leukemia cell line NB4 cells were cultivated in vitro and then treated with ATRA, As2O3, and ATRA+As2O3. Changes in cell morphology was observed by Optical microscope. Cell inhibitory growth rate was investigated by MTT assay. Differentiation and apoptosis rate were detected by Flow cytometry(FCM). Expression of PML/RARα, CD44v3, CD44v6 and CD44v10 mRNA in NB4 cells was detected by RQ-PCR. The results were analyzed by SPSS13.0, and we established the standard of statistic significance asα=0.05.Results: 1 Morphological changes on NB4 cells treated with ATRA, As2O3: NB4 cells was induced to differentiate with ATRA and undergo apoptosis with As2O3. Apoptosis is characterized by nuclear condensation, cell shrinkage, membrane blebbing, nuclear fragmentation and apoptotic body.2 ATRA, As2O3, ATRA+As2O3 on NB4 cell growth inhibition: Cell inhibitory growth rate of NB4 cells after treated with ATRA for 24h, 48h, 72h, 96h were(1.75±1.47)%, (2.91±1.94)%, (7.80±0.56)%, (14.5±4.84)% respec -tively. Cell inhibitory growth rate of NB4 cells after treated with As2O3 for 24h, 48h, 72h, 96h were (3.31±1.02)%, (6.41±0.74)%, (9.43±1.92)%, (20.59±3.88)% respectively. Cell inhibitory growth rate of NB4 cells after treated with ATRA+ As2O3 for 24h, 48h, 72h, 96h were (2.56±1.43)%, (16.45±2.51)%, (20.5±2.56)%, (32.45±2.07)% respectively. Statistical analysis showed that Cell inhibitory growth rate in experiment groups after treated NB4 cells for 24h, 48h, 72h, 96h were significantly increased(P<0.05), at the same time, there was a significant difference among intervention groups (P<0.05). Cell inhibitory growth rate of ATRA+As2O3 group was higher than other groups. But there showed no significant difference between ATRA group and As2O3 group(P>0.05).3 ATRA, As2O3, ATRA+As2O3 on NB4 cells expression of CD11b: The CD11b expression of NB4 cells exposed to ATRA for 48h, 72h, 96h was (32.47±2.95)%, (84.93±1.45)%, (90.9±0.35)%, respectively; The CD11b expression of NB4 cells exposed to As2O3 for 48h, 72h, 96h was (10.97±1.33)%, (15.7±0.87)%, (18.9±0.30)%, respectively; The CD11b expression of NB4 cells exposed to ATRA+As2O3 for 48h, 72h, 96h was (18.57±0.65)%, (21.17±0.65)%, (26.53±0.83)%, respectively. Statistical analysis showed that with the length of the medicine exposure, the CD11b expression was gradually increased. During NB4 cells differentiation, the CD11b expression of NB4 cells was time-dependently increased(P<0.05). Among control group, As2O3 and ATRA+As2O3 group, the CD11b expression on NB4 cells treated by ATRA was the highest and the difference was statistically significant(P<0.05).4 As2O3, ATRA+As2O3 on apoptosis of NB4: The early apoptosis percentage(AP) of NB4 cells in ATRA group for 24h, 48h, 72h, 96h were(0.57±0.06)%, (0.9±0.20)%, (1.07±0.35)%, (1.2±0.3)%, respectively; And AP in As2O3 group for 24h, 48h, 72h, 96h were(9.53±0.59)%, (13.77±0.70)%, (18.27±0.35)%, (16.23±1.15)%, respectively; AP in ATRA+As2O3 group for 24h, 48h, 72h, 96h were(2.27±0.32)%, (3.93±0.25)%, (7.83±0.40)%, (9.00±0.75)%, respectively; By one-way ANOVA, with the increasing length of the As2O3 exposure, AP was gradually increased, and the difference was statistically significant (P<0.05) except 72h and 96h. And at the same time, there was a significant difference among intervention groups, AP in As2O3 group is higher than other groups(P<0.05). 5 The expression of PML/RARα, CD44v3, CD44v6, CD44v10 mRNA on NB4 cells treated with ATRA, As2O3, ATRA+As2O3: RQ-PCR showed: The RQ value of PML/RARαon NB4 cells after treated with ATRA+As2O3 for 48h, 72h, 96h were 0.6983±0.0025, 0.2717±0.0035, 0.1910±0.0036 respectively; The RQ value of CD44v3 on NB4 cells after treated with ATRA+As2O3 for 48h, 72h, 96h were 0.4543±0.0047, 0.4127±0.0040, 0.1980±0.0070 respectively; The RQ value of CD44v6 on NB4 cells after treated with ATRA+As2O3 for 48h, 72h, 96h were 0.5027±0.0040, 0.3230±0.0020, 0.2787±0.0020 respectively; The RQ value of CD44v10 on NB4 cells after treated with ATRA+As2O3 for 48h, 72h, 96h were 0.4657±0.0035, 0.4260±0.0598, 0.1487±0.0035 respectively; With the increasing length of the drug exposure, The expression of PML/RARα, CD44v3, CD44v6, CD44v10 mRNA were significantly decreased (P<0.05). At the same time, there was a significant difference among intervention groups (P<0.05). The expression of PML/RARα, CD44v3, CD44v6, CD44v10 mRNA of ATRA+As2O3 group was lower than other groups (P<0.05). It can be obtained by correlation analysis following Scatter diagram:The trend of PML/RARαand CD44v6 on NB4 cells after treated with ATRA, As2O3, ATRA+As2O3 is closely positive correlated. Correlation coefficient were 0.98(P<0.01), 0.951 (P<0.01), 0.999 (P<0.01) respectively.Conclusions: 1 ATRA and As2O3 may inhibit the proliferation of NB4 cells with a time dependent manners. At the same time, there was a significant difference among intervention groups Cell inhibitory growth rate of ATRA+As2O3 group was higher than other groups (P<0.05).2 The expression of CD11b on NB4 cells was time-dependently increased after treated with ATRA, As2O3 and ATRA+As2O3 (P<0.05). At the same time the CD11b expression on NB4 cells treated with ATRA was higher than other groups and the difference was statistically significant(P<0.05).3 NB4 cells can be induced to undergo apoptosis by As2O3 and ATRA+As2O3 with a time dependent manner. And at the same time, there was a significant difference among intervention groups, AP in As2O3 group is higher than other groups(P<0.05). 4 The expression of PML/RARα, CD44v3, CD44v6 and CD44v10 mRNA was decreased with a time dependent manners on NB4 cells treated with ATRA, As2O3, ATRA+As2O3. At the same time, there was a significant difference among intervention groups (P<0.05). The expression of PML/RARα, CD44v3, CD44v6, CD44v10 mRNA after treated with ATRA+As2O3 was lower than other groups (P>0.05). It can be obtained by correlation analysis following Scatter diagram: The trend of PML/RARαand CD44v6 on NB4 cells after treated with ATRA, As2O3, ATRA+As2O3 is closely positive correlated. |
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