| Acute leukemia is a disease that involves haemopoietic stem cell. One of the feathers of leukemia is that the differentiation of leukemic cells are blocked in immature stage and can't produce functional blood cells,and then lead to anemia,infection,hematosis. As the research on the biological characters of leukemic cells, it is showed that acute leukemic cells can be induced to differentiation under some certain conditions. So inducing differention of leukemic cells is a new way in the treatment of leukemia. Looking for inductive drugs with high-effect and low-toxicity can become a new focus in the field of leukemic treatment.TPA (12-O- tetradecanoylphorbol-13-acetate) is a powerful tumor promoter, at the same time it's one of the most powerful inducers of differentiation. TPA can induce differentiative effects not only on many human and mouse leukemic cell lines, but also on human primary leukemic cells at low concentration(10-6mmol/L~10-8mmol/L) in vitro. TPA can induce them to undergo morphological,functional,some physiological and biochemical changes. But the exact mechanism of TPA induced differentiation is still unkown. To further understand the effect of TPA induced differentiation on human primary leukemic cells and the differentiative potential of leukemic cells,in this paper human primary acute leukemic cells were cultured in vitro with the involvement of TPA,RA,or the combination of TPA and RA. We investigated the feature of TPA induceddifferentiative effect on sorts of human primary acute leukemic cells, and explore the mechanism of induced differentiation of TPA. Therefore, it may provide a theoretical basis for therapy for acute leukemia.Methods and Subjects:1 . Selection of SubjectsWe selected 28 patients with acute leukemia, 18 male and 10 female. They were all diagnosed according to "the hematology diagnosis" witten by Zhang Zhinan and classified by FAB and MIC standard. Among the subjects,there are 14 M2, 4 MS, 4 MI, 5 ALL.And they all were new cases.2. Cell culture and groupingLeukemic cells were obtained from patients 'bone marrow before chemotherapy. We selected single-nuclear cells with lymphocyte-abstracting fluid (leukemic cells>95%), and the cell concentrations were modulated to 5-10 X 105 /ml.Every subject was matched 4 groups: the control group(adding the solvent without TPA or RA), TPA group(adding TPA only), RA group(adding RA only),TPA+RA group(adding both TPA and RA). The final concentration of TPA was 1.6X 10~7mol/L in TPA group, and 1.6 10-8mol/L in TPA+RAgroup. The final concentration of RA was 1 X 10-6mol/L in RA group and TPA+RA group. Then the cells of each group were plated in one 24- well culture plate and cultured in CO2 gas incubator, 4 wells for each group.3. The investigation of primary leukemic cells differention by TPA(1) The growth states of leukemic cells cultured in vitro were observed by the upside-down microscope. After 72 hours' culture,observe the morphological change of cells by Giemsa stain.(2) Cytochemical changesAfter 72 hours' culture, the cells were undergone NAE and NaF restraining tests.(3) Estimating the cell growth activity by MTT method.After 72 hours' culture, each well was added 20 1 MTT and cultured 4 hours in CO2 incubator. Then detected OD values of each well by spectrophotometer.(4) Flow cytometer( FCM ) detected the expression of surface antigen: CD11K CD13,CD33, CD14, CD64.Results:(1)The results of cell growth and morphology showed : After 12-48 hours culture, the cells in TPA group and TPA+ RA group appeared congregation in all the 28 cases in our experiment. All ANLL cases except 2 cases M2 and 2 cases MS of TPA group and except 1 cases M2 of TPA+ RA group adhered to the culture plates The adherent cells took on shapes of shuttle, astroid, racket, and usually had long pseudopodia or filar stick-ups, especially in group.The cells in TPA group and TPA+RA group of 5 cases all appeared congregregation ,but only 2 cases in TPA+RA group became adherent to the culture plates and had filar stick-ups.
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