Silencing HSP70 By The Lentiviral Vector Mediated RNAi Can Sensitize Lymphoma Cells To Apoptosis

Part One:Optimization the GFP Gene Transfection Effeciency of Lymphoma Cells Mediated by the Lentiviral Vectors.Objective:To observe the GFP gene transfection efficiency of lymphoma cells mediated by the lentiviral vectors in different conditions, to optimize the conditions of the lentiviral vector mediated transfection. To provide the basic conditions for further research of lentiviral vector-mediated RNA interference in lymphoma cells.Methods:Firstly, we designed and synthesized the lentiviral vector particles with a green fluorescent protein gene, then we used the lentiviral vector to transfect the lymphoma cells in different groups which were grouped by the cell line(Human Burkitt lymphoma cell line Raji; Human diffuse large B lymphoma cell line Ly8 and Human mantle cell lymphoma cell line Sp53); The multiplicity of infection(1; 10; 30; 50; 60; 100); The transfection reagent(polybrene and enhanced infection solution)and the transfection conditions(centrifugation and not centrifuged).After that we observed the GFP expression of lymphoma cells under the fluorescence microscope at different time, to obtain the transfection efficiency by calculating the ratio of cells expression green fluorescence protein. Human diffuse large B cell lymphoma cell line Ly8 were subcultured after transfection, then we analyzed the ratio of cells with GFP by flow cytometry one month later.Results: 1.The cell line, multiplicity of infection, transfection reagent and the transfection conditions can affect the transfection efficiency in varying degrees. The transfection effect of the Sp53 cell line is better than the Ly8 cell lines, both of them are better than the Raji cell lines; The greater of the MOI, the higher of the transfection efficiency. However, when the MOI reached a certain limit, the transfection efficiency did not increase with the increase of the MOI; The transfection-promoting effect of polybrene is higher than Enhanced Infection Solution; For the suspended cells, the transfection efficiency of centrifugation is greater than not. All the data were analyzed by SPSS16.0 and was considered statistical significant(P<0.05).2.The lentivirus-mediated gene transfer of green fluorescent protein into the lymphoma cells is stable, after 4 weeks, the transfection efficiency could still reach 85.6%.Conclusion:1. The GFP gene transfection efficiency of lymphoma cells mediated by the lentiviral vectors is highest when the cell line is Sp53, the MOI is 60 and using polybrene as the transfection reagent.2.The GFP expression in the transfected cells mediated by the lentiviral vector is stable.Part Two:Silencing HSP70 by the lentiviral vector mediated RNAi can sensitize lymphoma cells to apoptosis.Objective:Observe the biology effect on the mantle cell lymphoma cell lines Sp53 when silencing HSP70 by the lentiviral vector-mediated RNAi. To investigate the role of HSP70 in the apoptosis pathway of mantle cell lymphoma. To provide the new therapy strategy for the mantle cell lymphoma.Methods:Firstly, we designed and synthesized the lentiviral vector particles with a GFP gene and a HSP70 specific siRNA sequence. Secondly, we used the lentiviral particles to transfect the mantle cell lymphoma cell line Sp53 in the optimized conditions that had been tested in Part One. Then we detected the change of HSP70 expression in Sp53 cell line by western blot. Finally, we detected the effect of the silencing HSP70 on the apoptosis of Sp53 cell line by the Annetin-V-PE.Results:1.The expression of HSP70 in the Sp53 cell line.We collected the Sp53 cells 240 hours after the lentiviral vector particles targeting HSP70 transfecting the mantle cell lymphoma cell line Sp53, then detected the expression level of HSP70 by Western blot. Compared with the blank group and the negative control group, the expression of HSP70 was significantly decreased in the Sp53 cells transfected by the lentiviral vector particles targeting HSP70 (P<0.05).It suggests that the lentiviral vector particles targeting HSP70 can inhibit the expression of HSP70 in the post-transcriptional level.2.Targeted silencing HSP70 by RNAi can sensitize lymphoma cells to apoptosis.240 hours after the lentiviral particles transfected the Sp53 cells, the apoptotic cells were combined with the PE labled anti-Annexin-V specifically. Then we observed the ratio of the PE positive cells under the fluorescence microscope. The results shows that targeted silencing HSP70 can sensitize lymphoma cells to apoptosis(P<0.05).Conclusion:1. The lentiviral vector particles targeting HSP70 can inhibit the expression of HSP70 in the post-transcriptional level.2. RNA interference targeting HSP70 can promote the apoptosis of the Sp53 cells.