| Objective: To establish a ADM-resistant Kasumi-1 cell line(Kasumi-1/ADM), and investigate the effect of CD40 L gene overexpression on the proliferation, apoptosis and drug resistance of Kasumi-1/ADM cells.Methods:⑴The Kasumi-1/ADM cell line was established by a drug concentraction gradient method. ⑵The proliferative inhibitory rates of Kasumi-1 and Kasumi-1/ADM cells were detected by CCK-8 assay, both IC50 of two type cells and resistance index were calculated based on the inhibitory rates. ⑶The overexpression CD40L-lentivirus vector was used to transfect Kasumi-1/ADM cells, the transfection efficiency was tested by flow cytometry, and the overexpression of CD40 L gene was verified by q RT-PCR. ⑷The transfected cells were divided into three groups:normal Kasumi-1/ADM, blank vector transfected Kasumi-1/ADM, CD40 L overexpression transfected Kasumi-1/ADM. The effects of CD40 L gene overexpression on proliferation and toxicity in Kasumi-1/ADM cells were detected by CCK-8 assay. Cell apoptosis was analyzed by Hoechst33342/PI double staining method. The m RNA expressions of drug resistance and proliferation related genes including MDR1, MRP and PCNA were detected by q RT-PCR.Results:⑴The human acute myeloid leukemia Kasumi-1/ADM cell line were successfully induced against ADM. The IC50 values of both Kasumi-1 and Kasumi-1/ADM cells were 0.88±0.04μg/m L, 6.00±0.17μg/m L after 24 h treatment of ADM, respectively,and the RI was 6.85±0.26(P<0.05). ⑵The overexpression lentivirus vector of CD40 L gene was successfully transfected Kasumi-1/ADM cells, the transfective efficiencies of blank vector transfected cells and CD40 L overexpression transfected cells were 95.6% and96.9% respectively. The gene expression of CD40 L in non-transfected normal group, blank vector transfected group and CD40 L transfected group were 1.00±0.07, 1.03±0.19 and215.09±28.83, respectively. The gene expression of CD40 L in Kasumi-1/ADM cells was increased significantly after transfection in comparison to both normal and blank vector transfected groups(P<0.05). ⑶After transfection, the proliferatibecapacity of Kasumi-1/ADM cells was remarkedly inhibited compared with other both groups(P<0.05).⑷The 24h-IC50 value against DNR in non-transfected normal group, blank vector transfected group and CD40 L transfected group were 5.819±0.101, 5.915± 0.102 and1.502 ± 0.050, respectively. The 24h-IC50 value against DNR in CD40 L overexpression transfected cells was significant lower than that of other both groups(P<0.05). ⑸The apoptosis rates without DNR treatment for non-transfected normal group, blank vector transfected group and CD40 L transfected group were 1.72±0.11%, 1.60±0.09% and7.33±0.45%, respectively(P<0.05). After DNR treatment for 24 h, their apoptosis rates were9.17±0.93%, 9.10±0.26% and 35.87±1.70%, respectively(P<0.05). ⑹The gene expression of MDR1 without DNR treatment for non-transfected normal group, blank vector transfected group and CD40 L transfected group were 1.02±0.23, 1.00±0.35 and 0.04±0.01,respectively. Their MRP gene expressions were 1.04±0.31, 1.00±0.20 and 0.13±0.01,respectively. Their PCNA gene expressions were 1.04±0.35,1.03±0.45 and 0.49±0.18,respectively. After DNR treatment for 24 h, their MDR1 gene expressions were 1.13±0.28,1.08±0.28 and 0.03±0.01, respectively. Their MRP gene expressions were 1.21±0.52,1.11±0.32 and 0.07±0.01, respectively. Their PCNA gene expressions were1.00±0.45,1.08±0.06 and 0.42±0.15, respectively. After transfection, the relative expression levels of MDR1, MRP and PCNA genes were significantly decreased in the CD40 L overexpression Kasumi-1/ADM cells(P<0.05).Conclusion:⑴The Kasumi-1/ADM cell line against ADM was successfully established.⑵The overexpression of CD40 L gene can inhibit the proliferation of Kasumi-1/ADM cells,increase the sensibility of Kasumi-1/ADM cells to DNR, andr result in the downregulation expression of MDR1, MRP and PCNA genes. |
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