Study On The Construction Of Lentiviral Vector Of RNAi And Its Silence Effect On The CD59 Of Acute T Leukemia Cell Lines

Objective To explore the silence effect on the CD59 of the acute T lymphocyte leukemia Jurkat cell lines.,We build RNAi-CD59 lentivirus vector and observe the mechanism of this CD59 on tumor immune escape through vivo and vitro experiments.Methods Experiment in vitro:We applied fluorescence microscope to test the expression of CD59 in normal T cell and T lymphocyte leukemia’s cell line Jurkat.We builded interference sequence about CD59 gene(RNAi-CD59-A RNAi-CD59-B RNAi-CD59-C)and missense sequence(RNAi-NC)which combined with green fluorescent protein,then transfected to acute T lymphocyte Jurkat cell lines by lentivirus and selected stable transfection cell line,we used the RNAi-NC as the negative control group and the Jurkat as the blank control group.We used fluorescence microscope and flow cytometer(FCM)to test the efficiency of transfection.The expression of CD59 Bcl-2/Bax mRNA was measured by the Real-time PCR(RT-PCR);while the Western-Blot were employed to detect the change of expression about CD59,Bcl-2/Bax and Caspase-3/Survivin.we applied confocal to observe the location of CD59 molecular We selected ELISA to test the change of IL-3 and TNF-βin cell culture supernatant.CCK8 was used to analyze the change of cell proliferation;FCM was applied to test the cell apoptosis.Experiment in vivo:24 female BALB/c-nu mice were randomly distributed into four groups(PBS,Jurka,RNAi-NC,RNAi-CD59-A),then the tail vein of nude mice were injected the different cell lines to develop the metastatic tumor model.We weighted the different group of mice and tested the peripheral white blood cells in 0,1,2,3,4 weeks.We used the flow cytometry to check the apoptosis of lymphocytes in peripheral blood and bone marrow in mice.ELISA were applied to observe the expression of IL-3 and TNF-βsupernatant in the blood of mice.Results Experiment in vitro:We got the results that the efficiency of transfection was over 90%.The expression of CD59 and Bcl-2 mRNA was decreased in experimental group while Bax was increased(P<0.05).The experimental group could enhance the expression of Bcl-2,caspase-3(P<0.05),inhibit the protein expression of CD59,Bax and Survivin(P<0.05);confocal observed that CD59 molecules were mainly located in cell membrane,the expression of cell culture supernatant IL-3 was decreased while the TNF-βwas increased when we silence the expression of CD59(P<0.05).The proliferation of cell in the RNAi-CD59-A group was significantly lower than the Jurkat group and the RNAi-NC group(P<0.05);The apoptosis rate in RNAi-CD59-A group was significantly more than the RNAi-NC group and the Jurkat group(P<0.05).Experiment in vivo:The animal model were built successfully.Compared with the PBS group,all model group’s weight was decreased and the count of peripheral blood leukocyte were increased(P<0.05),then the quality of the RNAi-CD59-A group were bigger than the RNAi-NC and the Jurkat group(P<0.05);the count of peripheral blood leukocyte were less than the RNAi-NC and the Jurkatgroup(P<0.05).The apoptosis rate of the RNAi-CD59-A group was significantly more than the RNAi-NC and the Jurkat group(P<0.05).The expression of IL-3 was decreased while the TNF-β was increased(P<0.05).Conclusion The building of RNAi-CD59 stable transfected cell line is successful.We also successfully construct the metastatic tumor model.Silencing the expression of CD59 gene can inhibit the proliferation of acute T lymphocyte Jurkat cell lines and induce the apoptosis in vivo and vitro,it provides a new thought to the clinical therapy.