In Vitro Study Of Anti-Chronic Myelocytic Leukemia Induced By Dentritic Cell Transfected With Chronic Myelocytic Leukemia Total RNA

Objective:To investigate the specific Cytotoxic T Lymphocyte ( CTL ) immunoreation of choronic myelocytic leukemia-dendritic cell(CML-DC) transfected with CML total RNA in vitro,to provide a theoretic basis of CML vaccine in clinical study.Methods:Bone marrow mononuclear cells (BMMNCs)from 10 cases of chronic myeloid leukemia were used.BMMNCs were incubated with recombined human interleukin-4(rhIL-4),recombined with human granulocyte/ macrophage colony-stimulating-factor(rhGM-CSF) and tumor necrosis factor–a (TNF-a).Using Trizol method to extract CML-BMMNC total RNA,Using CML-BMMNCs to make tumor frozen-thawed antigen.At the five day.Making CML-DCs into four groups:CML-DCs pulsed with total RNA-lipofectamin; CML-DCs pulsed with total RNA; CML-DCs pulsed with tumor frozen-thawed antigen; CML-DCs pulsed with nothing.All of them could induce T cell into specific CTL.Using T cell cultured with IL-2 as normal control group.To test the capacity of CML-DC for stimulating T-lymphocyte proliferation,mixed leukocyte reaction(MLR) was performed by methyl thiazolyl tetrazolium (MTT) assay.The morphological feature of DC was observed by invertmicroscope.The CD1a,CD 83 were analyzed by flow cytometry.Karyotypes of CML-DC were tested by G-banding technique. Bcr-abl of CML-DC was tested by Reversedtransplantase-poly merasechain response(RT-PCR).Results:CML-BMMNC could be induced into CML-DC.CD1a,CD83 on uncultured CML-BMMNCs were all below 5%,however, The expressions of CD1a,CD83 on CML-DC had increased obviously than the uncultured. CD1a expression was (20.15±3.36)%(P<0.01),CD83 expression is ( 25.37±2.72 )%,P<0.01.Bcr-abl and Ph chromosomal all exsisted in cultured CML-DC and uncultured CML-BMMNC.It suggested that CML-DC was from CML.The cytotoxic rates of CML-DCs pulsed with total RNA-lipofectamin; CML-DC pulsed with total RNA; CML-DCs pulsed with tumor frozen-thawed antigen; CML-DCs pulsed with nothing and T cell cultured with IL-2 were 74.67±3.54%,36.45±3.02%,51.07±3.67%,31.52±1.82%,10.62±3.17%。The CTL induced by CML-DCs pulsed with total RNA- lipfectamin was more significantly active than others(P<0.001).The CTL induced by CML-DCs pulsed with nothing was also more effective than T cell co-cultured with IL-2.Conculusions:being incubated recombined with IL-4 ,GM-CSF and TNF-a,CML-BMMNCs could be induced into CML-DC.CD1a,CD83 expressions all increased obviously.the CML-DCs could induce the CTL to get specific anti-tumor activity in vitro.Otherwise,the CTL induced by CML-DC pulsed with total tumor- lipofectamin was most significantly active than others.