Biological Characteristics Research Of Side Population Cells In Human NK/T-cell Lymphoma Multi-drug Resistant Cell Line

ObjectiveLymphoma is a group of malignant tumor originating from lymphoid hematopoietic system,which mainly affects lymph nodes or extracorporeal lymphoid organs.According to the differences between the histopathology,genetics,immunological characteristics and clinical characteristics,Lymphoma can be divided into Hodgkins lymphoma(HL)and non-Hodgkins lymphoma(NHL).NK/T cell lymphoma(NKTCL)is about 10% of the NHL in the worldwide and incidence in Asia is obviously higher,especially in China(about 21.38% of all the lymphoma).NKTCL mainly occur in nasal cavity,nose pharynx,sinuses and other relevant organs.Sometimes NKTCL may concomitant with B symptoms,such as weight lose,night sweats,fever and other symptoms.Even though the disease can be effectively relieved through radiotherapy and chemotherapy,the malignancy is high and dangerous,the clinical progress is fast and easy to relapse and drug resistance with poor prognosis.Therefore,during recent years,the research of multidrug resistance become a hot spot within the tumor research.Side population cell(SP cell)is a small amount of cells which is found when using the fluorescent Hoechst-33342 by Goodell on separation of bone marrow stemcells and is occupied by a side of a large amount of cells.SP cells have the ability of multi-directional differentiation,Infinite proliferation and self-renewal and other biological characteristics.SP cells in tumor cells not only have the above biological characteristics,but also have stronger tumorigenic ability and invasive ability than non-SP cells.HKTCL's clinical curative effect is poor and is closely related with cell express multidrug resistance(MDR).These genes lead to the tumor cells are not sensitive to chemotherapy drugs and tumor drug resistance and relapse.Most of the MDR genes are the members of transporter family of ATP-binding cassette.They transport the intracellular substrate into cells by ATP-dependent,active transport,limiting intracellular substrate concentrations,and correlating with multidrug resistance in tumor cells.In that case,this research try to sort NK/T cell lymphoma drug-resistant strains of SP cells,test the biological characteristics and the expression of ABC transporters and explore NKTCL multi-resistant related mechanism.MethodsSNK-6/ADM cell line,resistanting to doxorubicin,was constructed,and was detected multi-drug resistance.SNK-6/ADM-SP was isolated from SNK-6/ADM cell line and the proliferation,cell cycle,cell surface molecular marker of SNK-6/ADM-SP was detected by CCK8 method and flow cytometry And multidrug resistance and other biological characteristics.The expression of ABC-related genes and proteins in SNK-6/ADM-SP cells was determined by Real Time-PCR and Western Blot.Results1.Resistance index(RI)of SNK-6/ADM was 5.158.The inhibition of concentration 50%(IC50)of SNK-6 and SNK-6 / ADM on gemcitabine,adriamycin,cisplatin and peptone enzyme were as follows: 0.076�0.016,0.067�0.00;57.268�0.225,37.49�0.639;5.19�0.541,23.63�1.157;4.569�0.652,5.338�0.403.2.The SNK-6/ADM-SP cell line's IC50 of gemcitabine,adriamycin,cisplatin,and peptone enzyme were 0.089�0.014,63.55�1.847,61.09�1.502,5.534�0.207.Theproliferation and apoptosis rate of SNK-6/ADM-SP is low(p<0.05).The proportion of G0/G1 is(63.3�2.4)%.The proportion of G2/M is(6.377�0.578)%.The proportion of S period is(30.333�1.856)%.The expression of cell colony proporion of CD56+,CD16-,CD34 and CD117-is similar to that of SNK-6 ? SNK-6/ADM.And the multidrug resistance of SNK-6/ADM-SP is higher than that of SNK-6/ADM cell line.3.The expression of ABCG2 mRNA and protein in SNK-6/ADM-SP cells was higher than that in SNK-6 and SNK-6/ADM cells(p <0.05).Conclusion1.The side population cells in human NK/T-cell lymphoma multi-drug resistant cell line are a special class of mature tumor cells,which are more multi-drug resistant.2.The multidrug resistance of NK/T cell lymphoma is associated with high expression of ABCG2 gene and protein.