RNAi慢病毒載體的構(gòu)建及對(duì)急性T系白血病細(xì)胞CD59因沉默效應(yīng)的研究
[Abstract]:Aim to construct RNAi-CD59 lentivirus vector and study its silencing effect on acute T lymphocyte leukemia cell line Jurkat CD59. To investigate the effect of CD59 specific silencing on immune escape of tumor cells by in vivo and in vitro experiments. Methods in vitro experiment: immunofluorescence was used to compare the expression of CD59 on normal T cells and Jurkat cells. The fusion green fluorescent protein of CD59 interference sequence (RNAi-CD59-A,RNAi-CD59-B,RNAi-CD59-C) and missense sequence (RNAi-NC) was constructed and transfected into human acute T-line leukemia Jurkat cell line with lentivirus as vector. The negative control group was RNAi-NC, blank control group, and the control group was normal cultured Jurkat cell line without any treatment. Fluorescence microscopy and flow cytometry were used to detect the transfection efficiency, RT-PCR was used to detect the expression of CD59 gene and apoptosis-related gene Bcl-2/Bax mRNA in each group. The expression level of CD59 protein and apoptosis-related protein Bcl-2,Bax,Caspase-3,Survivin protein was detected by Western-blot, the localization of CD59 molecule was observed by laser confocal scanning, the expression of IL-3,TNF- 尾 in cell culture supernatant was detected by ELISA, and the expression of IL-3,TNF- 尾 in cell culture supernatant was detected by ELISA. CCK8 was used to detect the change of cell proliferation efficiency and flow cytometry was used to observe the change of apoptosis level in each group. In vivo experiment: 24 BALB/c-nu female nude mice were randomly divided into 4 groups (PBS group, Jurkat group, RNAi-NC group, RNAi-CD59-A group). 0 weeks, 1 weeks, 2 weeks, 3 weeks and 4 weeks before and after injection, the growth state, body mass and the number of peripheral blood leukocytes were monitored. Flow cytometry was used to observe the apoptosis of lymphocytes in peripheral blood and bone marrow, and ELISA was used to detect the expression of IL-3,TNF- 尾 in the supernatant of mice. Results in vitro, the transfection efficiency was more than 90.00% observed by fluorescence microscope and FCM, and the expression level of CD59,Bcl-2 mRNA in silent group was lower than that in control group (P0.05), Bax mRNA expression level was increased (P0.05). The results of Western-blot showed that the expression of CD59,Bax,Survivin protein decreased and the expression of Bcl-2,caspase-3 protein increased in the silent group (P0.05). The laser confocal observation showed that the CD59 molecule was mainly localized in the cell membrane. The results of ELISA showed that the expression level of IL-3 decreased and the expression of TNF- 尾 increased in silent group (P0.05), and the proliferation efficiency of RNAi-CD59-A group was significantly lower than that of RNAi-CD59-A group (P0.05). The apoptosis rate in RNAi-CD59-A group was significantly increased by flow cytometry (P0.05). In vivo experiment: the animal model was successfully constructed, compared with the PBS group, the body weight of each model group decreased (P0.05), and the body weight of the silent group was lower than that of the RNAi-NC group and Jurkat cell group (P0.05). Compared with PBS group, the number of white blood cells in each model group increased significantly (P0.05), and compared with Jurkat cells and RNAi-NC group, the white blood cell count in silent group decreased (P0.05). The results of flow cytometry showed that the apoptosis rate of peripheral blood and bone marrow cells in the silencing group was significantly higher than that in the non-silent group (P0.05). The results of ELISA showed that the expression of IL-3 decreased and the expression of TNF- 尾 increased in the silent group (P0.05). Conclusion RNAi-CD59 transfected cell lines and leukemia metastatic tumor models were successfully constructed. In vitro and in vivo, the silencing of CD59 gene expression could inhibit the proliferation and induce apoptosis of acute T-lineage leukemia. It provides a new idea for the diagnosis and treatment of acute T-lineage leukemia.
【學(xué)位授予單位】:青島大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2017
【分類號(hào)】:R733.71
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