The Impact Of Rh-LIF And Daunorubicin On DAMI Cell Line

Leukaemia is a malignant disease in human hematopoietic tissue and is also a disease of malignant tumor that rate of the incidence is the highest in childhood. It is one of the main causes of the death under the five yeas old. The growth and development of the leaukemia with abnormal cell apoptosis have close relation ship .Apoptosis is a process of the initiative dissolution by self under the gene guidance,and it is a normal cell clearnning pathyway in the incidence progress of the normal embryogey and the growth of tissue and organ in adult. When the pathway of apoptosis was suprresed or blocked, cell immortalization would happen and result in canceration. Apoptosis involves in a series of gene regulation. The genes inducing apoptosis includes families of the Bax,p53 and so on. The apoptosis theory not only opens up new field for cause and pathogenesy of leukemia but also provides new thinking for the treatment of leukemia.Now we know many traditional chemotherapy to treat leukemia as daunorubicin ,methotrexate and so on. They can induce apoptosis of leukemia cell and cure leukemia. Chemotherapy is almost only one choice to cure leukemia, For example of acute lymphoblastic leukemia in the children, valid single chemical druges were found in 1940s, which rate of relief and survival were very low. Combination chemotherapy and maintenance therapy were used to cure acute leukemia and in the 1950s and at the early 1960s,which the rate of disease free survival in five years had arrived to 10%～20%.Because of the occurrence of new medicine to cure leukemia the rate of disease free survival in five years had reached to 50%～80%. The occurrence of some cytokines provid new strategy for leukemia therapy in recent years.In order to explore impact of the cytokines and chemical drugs on the leukemia cell,human acute myeloid leukemia DAMI cell was used as the target cell. Cell proliferation,index of the early apoptosis,cell cycle and expression of p73 protein were detected by cck-8,flow cytometer and immunochistochemistry assay. To investigate the effect of single rh-LIF or DNR or two drugs together were used in DAMI cell line. This studying provides the base of theory and experiment for clinical therapy of leukemia.Methods:DAMI cells were incubated at 37℃in RPMI 1640 containing 10% fetal bovine serum in a 5%CO₂ incubator,reproduce one time every two to three days. We used exponentially growing DAMI cells in experiment. By means of cell proliferation assay to observe variation of proliferation and viability in different dose rh-LIF or DNR or two drugs together for 6h,12h,24h,48h on DAMI cell line,Flow cytometer were perfomed to defect index of early apoptosis - Annexin V.We detected the changes of p73 protein expression before and after drugs ,cell cycle analysised apoptosis cycle. All the data were caculated by SPSS satistical software 13.0 ,all the data were expressed as mean±SD.t test was used for comparing variancebetween two groups,otherwise rank-sum test. Size of testα=0.05.Results: 1. Variation of cell line proliferation were observed in different concentration rh-LIF(1, 5, 10, 20, 40ng/ml) for 6h,12h,24h,48h.Low concentration rh-LIF(1ng/ml) inhibit proliferation quiety(p>0.05), while high concertration (≥5ng/ml) can inhibit cell inhibit cell proliferation (p<0.05).Flow cytometer detect index of early apoptosis ,when concentration is 1ng/ml ,no difference obviously(p> 0.05),>5ng/ml (p<0.05).With rh-LIF concentration increase gradually,the rate of p73 protein positive expression increased obviously (p < 0.05).with drugs concentration and action time have correlation.cell cycle analysis showes the most cell were blocked in the phase of G₀/G₁ .2 Variation of cell line proliferation were observed in different concentration DNR(0.5, 1, 2, 4, 8μmol/1) for 6h,12h,24h,48h.when DNR (0.5μmol/l) cellproliferation inhibition is quietly (p>0.05), when concentration is( 1, 2, 4, 8μmol/l) that cell proliferation inhibition with concentration increase gradually. And for inhibition of cell proliferation is obviously (p<0.05).0.5μmol/1 cell start apoptosis and with chang of time the rate of apoptosis increase obviously in 0.5, 1, 2μmol/l (p<0.05), the rate of apoptosis is descend in 4, 8μmol/l,immunochistochemistry assayshows that the rate of positive expression of p73 protein increasly have correlation obviously with time. The rate of positive expression of p73 protein have degressiontrend in 4, 8μmol/l. cell cycle showes DAMI cell were stoped in the phase ofG₀/G₁.3 rh-LIF combined DNR inhibit DAMI cell line proliferation and the rate of cell apoptosis to show that it compare with single rh-LIF was used in cell line,Effection of rh-LIF combined DNR is powerful (p<0.05),and with compared with single DNR was used in cell line no significant difference.cell cycle shows DAMI cell was stopped in the phase of G₀/G₁.Conclutions: 1 rh-LIF and DNR may inhibit the DAMI cell proliferation and viability and have dose and time dependent relation; over-expression of P73 may be one of mechanisms influencing apoptosis of DAMI cells.2 The cytokines- rh-LIF with chemical drugs no manifest reciprocal enhance apoptosis in DAMI cells.