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Objective To investigate the reversal effect of Topo Ⅱα-shRNA and Topo Ⅱβ-shRNA on Topo Ⅱ gene in K562/AS2 cells. Methods Three pieces of Topo Ⅱα-shRNA and three pieces of Topo Ⅱβ-shRNA were designed,synthesized and transfected into K562/AS2 cells by liposome. Expression level of Topo Ⅱα and Topo Ⅱβ mRNA were determined by real time fluorescence quantitative PCR. The expressions of Topo Ⅱα and Topo Ⅱβ protein were assayed with flow cytometer. Results After treated with Topo Ⅱα-shRNA or Topo Ⅱβ-shRN A for 24 hours,the expression level of Topo Ⅱα mRNA and Topo Ⅱβ mRNA protein in K562/AS2 cells decreased at most (78.22±0.01) %,(31.17±1.27) % (P <0.05),and (57.36±0.01)%,(23.98±1.22) % (P <0.05) respectively. Conclusion The expression of Topo Ⅱ gene can be down-regulated after infected with Topo Ⅱ-shRNA in K562/ AS2 cell line.
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Objective To construct a recombinant adenovirus vector of Hairpin RNA specific for MRP1 gene and study its inhibition of MRP1 gene expression in K562/AS2 cell line resistant to AS_2O_3 (ATO). Methods A MRP1-specific hairpin RNA recombinant adenovirus vector was constructed and used to infected K562/AS2 cells. Expression level of MRP1 mRNA detected by real-time fluorescent quantitative PCR. MRP1 protein detected by flow cytometry. MTT method was used to detected the cytotoxicity of ATO and etoposide. Results MRP1 mRNA and protein expression level in K562/AS2 cells before and after the pAd-MRPl-shRNA adenovirus infection was (34.70±0.28 vs 4.19±0.03, P <0.05) and (26.40±0.16 vs 10.85±0.37, P<0.05), respectively. RR of K562/AS2 to arsenic trioxide and etoposide was (11.4078±0.3183 fold vs 1.6126±0.3015 fold, P<0.05) and (5.9141 ±0.0149 fold vs 1.7664±0.1038 fold, P <0.05), respectively. The reversal fold of ATO and etoposide was (7.2409±1.3668) and (3.3555±0.1886), respectively. Conclusion Successfully constructed pAd-EGFP-U6-shRNA-MRPl adenovirus vector, the vector of infection K562/SA2 cells can inhibit MRP1 gene expression and reverse the resistance of the ATO and etoposide.
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AIM:To explore the changes of the subsets and HLA-DR expression of dendritic cells and their concerning cytokine levels in peripheral blood of patients with breast cancer.METHODS:The subsets of the precussors of dendritic cells(pDC)in the peripheral blood of 57 cases of patients with breast cancer before operation and a week or six months after operation and 20 cases of healthy controls were analyzed by four-color FCM.The levels of IL-12p40,IL-10,IFN-? and IL-4 in the plasmas were tested by ELISA.RESULTS:Among 57 cases of patients with breast cancer,2 cases in Ⅲ phase and 4 cases in Ⅳphase expressed deficiency of pDC,the ratios of pDC1/pDC2 in the other cases inⅠ,Ⅱ,Ⅲ,Ⅳ phase were respectively 1.62?0.59,1.41?0.63,0.91?0.32,0.81?0.29 before operation,which were markedly lower than those in controls(1.94?0.44).The ratios of pDC1/pDC2 in the cases inⅠ,Ⅱ,Ⅲ phase were 1.71?0.47,1.52?0.54,1.04?0.36 a week after operation,which were the same as those in pre-operation,but markedly lower than those in controls.The ratios of pDC1/pDC2 in the cases inⅠ,Ⅱ,Ⅲ phase were 1.92?0.72,1.63?0.65,1.28?0.34 six months after operation,which were markedly higher than those in pre-operation,meanwhile,to compare with controls,those were still lower for patients in Ⅱ,Ⅲ phase except in Ⅰphase.No difference between patients and controls in the expression of HLA-DR of pDCs and the levels of IL-12p40,IL-10,IFN-?,IL-4 in plasmas and the ratios of IL-12p40/IL-10,IFN-?/IL-4 was observed.CONCLUSION:The ratios of pDC1/pDC2 in peripheral blood of patients with breast cancer inⅠ-Ⅳ phase are decreased.Parts of patients in Ⅲ,Ⅳ phase are deficiency of pDCs.HLA-DR expression of DCs and the ability of DCs which secret the concerning cytokines do not change as pDC subsets change.pDC subsets improve markedly inⅡ,Ⅲ phase patients and recover to the normal level inⅠphase patients after operation.
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AIM: To explore the characters of fibroblast-like (F-L) cells cultured from granunocyte clony stimunating factor (G-CSF)-mobilized peripheral blood cell (PBC) harvests. METHODS: The adherent cells in the PBC harvests were cultured for 2 week in the mediums of RPMI-1640/L-DMEM/G-CSF or interleukin-3 (IL-3) plus RPMI-1640, the cultured F-L cells were analyzed by flow cytometry (FC). RESULTS: The adherent non-confluent F-L cells obtained from the four groups were similar in their phenotypes: CD33+, CD11c+, CD64+, CD14+, CD45+, HLA-DR+, CD86+, CD34-, CD38-, CD3-, CD19-, CD56-, CD29-, CD44-, CD105-. The F-L cells are similar to monocytes except CD38-and were distinct from dendritic cells (DC) or mesenchymal stem cells (MSC). CONCLUSION: The cultured F-L cells are macrophages rather than DC or MSC. G-CSF, rhIL-3 enhances their numbers.