RNAi慢病毒载体的构建及对急性T系白血病细胞CD59因沉默效应的研究
发布时间：2019-04-12 20:21
【摘要】：目的构建RNAi-CD59慢病毒载体,研究其对急性T淋巴细胞白血病细胞株Jurkat CD59的沉默效应,运用体内和体外实验探讨CD59特异性沉默对肿瘤细胞免疫逃逸的作用。方法体外实验:免疫荧光比较正常人T细胞和Jurkat细胞上的CD59表达情况;构建 CD59 干扰序列(RNAi-CD59-A、RNAi-CD59-B、RNAi-CD59-C)以及错义序列(RNAi-NC)融合绿色荧光蛋白,以慢病毒为载体转染进入人急性T系白血病Jurkat细胞株,阴性对照为RNAi-NC,空白对照组为未经任何处理的正常培养的Jurkat细胞系;运用荧光显微镜和流式细胞仪检测各组细胞的转染效率;RT-PCR检测各组CD59基因以及凋亡相关基因Bcl-2/Bax mRNA的表达;Western-blot检测各组CD59蛋白以及凋亡相关蛋白Bcl-2、Bax、Caspase-3、Survivin蛋白表达水平的变化;激光共聚焦观察CD59分子的定位;ELISA检测各组细胞培养上清中IL-3、TNF-β的表达;CCK8检测各组细胞增殖效率的改变;流式细胞仪观察各组细胞凋亡水平的变化。体内实验:24只BALB/c-nu雌性裸鼠随机分为4组(PBS组、Jurkat组、RNAi-NC组、RNAi-CD59-A组),将培养筛选后的细胞系通过尾静脉注射进入裸鼠体内构建裸鼠转移瘤模型,注射前后0周、1周、2周、3周、4周,监测小鼠生长状态、小鼠体质量及外周血白细胞数量的变化;流式细胞检测技术观察小鼠外周血及骨髓中淋巴细胞的凋亡情况;ELISA检测各组小鼠血液上清中IL-3、TNF-β的表达。结果体外实验:荧光显微镜和FCM观察转染效率在90.00%以上;RT-PCR结果显示沉默组CD59、Bcl-2 mRNA表达水平降低(P0.05),Bax mRNA表达水平升高(P0.05);Western-blot结果显示沉默组CD59、Bax、Survivin蛋白的表达量减少,Bcl-2、caspase-3蛋白的表达量增多(P0.05);激光共聚焦观察到CD59分子主要定位于细胞膜;ELISA结果显示沉默组IL-3表达水平降低,TNF-β的表达水平升高(P0.05);CCK8结果显示RNAi-CD59-A组的细胞增殖效率明显降低(P0.05);流式细胞仪观察到RNAi-CD59-A组的细胞凋亡率明显升高(P0.05)。体内实验:动物模型构建成功,与PBS组相比,各模型组裸鼠体质量均有一定的下降(P0.05),其中沉默组裸鼠体质量下降不如RNAi-NC组和Jurkat细胞组明显(P0.05);与PBS组相比,各模型组外周血白细胞数均有明显的升高(P0.05),其中与Jurkat细胞和RNAi-NC组相比,沉默组外周血白细胞计数减少(P0.05);流式细胞仪结果显示沉默组外周血和骨髓细胞中的细胞凋亡率明显高于未沉默组(P0.05);ELISA结果显示小鼠血液上清中沉默组IL-3表达水平降低,TNF-β的表达水平升高(P0.05)。结论RNAi-CD59转染细胞系及白血病转移瘤模型均构建成功,在体内外均验证了沉默CD59基因表达可抑制急性T系白血病的增殖并诱导细胞凋亡,为临床急性T系白血病的诊断和治疗提供了一个全新的思路。
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
本文编号：2457362
[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.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
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