RNAi慢病毒載體的構建及對急性T系白血病細胞CD59因沉默效應的研究
發(fā)布時間：2019-04-12 20:21
【摘要】：目的構建RNAi-CD59慢病毒載體,研究其對急性T淋巴細胞白血病細胞株Jurkat CD59的沉默效應,運用體內和體外實驗探討CD59特異性沉默對腫瘤細胞免疫逃逸的作用。方法體外實驗:免疫熒光比較正常人T細胞和Jurkat細胞上的CD59表達情況;構建 CD59 干擾序列(RNAi-CD59-A、RNAi-CD59-B、RNAi-CD59-C)以及錯義序列(RNAi-NC)融合綠色熒光蛋白,以慢病毒為載體轉染進入人急性T系白血病Jurkat細胞株,陰性對照為RNAi-NC,空白對照組為未經任何處理的正常培養(yǎng)的Jurkat細胞系;運用熒光顯微鏡和流式細胞儀檢測各組細胞的轉染效率;RT-PCR檢測各組CD59基因以及凋亡相關基因Bcl-2/Bax mRNA的表達;Western-blot檢測各組CD59蛋白以及凋亡相關蛋白Bcl-2、Bax、Caspase-3、Survivin蛋白表達水平的變化;激光共聚焦觀察CD59分子的定位;ELISA檢測各組細胞培養(yǎng)上清中IL-3、TNF-β的表達;CCK8檢測各組細胞增殖效率的改變;流式細胞儀觀察各組細胞凋亡水平的變化。體內實驗:24只BALB/c-nu雌性裸鼠隨機分為4組(PBS組、Jurkat組、RNAi-NC組、RNAi-CD59-A組),將培養(yǎng)篩選后的細胞系通過尾靜脈注射進入裸鼠體內構建裸鼠轉移瘤模型,注射前后0周、1周、2周、3周、4周,監(jiān)測小鼠生長狀態(tài)、小鼠體質量及外周血白細胞數量的變化;流式細胞檢測技術觀察小鼠外周血及骨髓中淋巴細胞的凋亡情況;ELISA檢測各組小鼠血液上清中IL-3、TNF-β的表達。結果體外實驗:熒光顯微鏡和FCM觀察轉染效率在90.00%以上;RT-PCR結果顯示沉默組CD59、Bcl-2 mRNA表達水平降低(P0.05),Bax mRNA表達水平升高(P0.05);Western-blot結果顯示沉默組CD59、Bax、Survivin蛋白的表達量減少,Bcl-2、caspase-3蛋白的表達量增多(P0.05);激光共聚焦觀察到CD59分子主要定位于細胞膜;ELISA結果顯示沉默組IL-3表達水平降低,TNF-β的表達水平升高(P0.05);CCK8結果顯示RNAi-CD59-A組的細胞增殖效率明顯降低(P0.05);流式細胞儀觀察到RNAi-CD59-A組的細胞凋亡率明顯升高(P0.05)。體內實驗:動物模型構建成功,與PBS組相比,各模型組裸鼠體質量均有一定的下降(P0.05),其中沉默組裸鼠體質量下降不如RNAi-NC組和Jurkat細胞組明顯(P0.05);與PBS組相比,各模型組外周血白細胞數均有明顯的升高(P0.05),其中與Jurkat細胞和RNAi-NC組相比,沉默組外周血白細胞計數減少(P0.05);流式細胞儀結果顯示沉默組外周血和骨髓細胞中的細胞凋亡率明顯高于未沉默組(P0.05);ELISA結果顯示小鼠血液上清中沉默組IL-3表達水平降低,TNF-β的表達水平升高(P0.05)。結論RNAi-CD59轉染細胞系及白血病轉移瘤模型均構建成功,在體內外均驗證了沉默CD59基因表達可抑制急性T系白血病的增殖并誘導細胞凋亡,為臨床急性T系白血病的診斷和治療提供了一個全新的思路。
[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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.71
本文編號：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.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.71
【參考文獻】
相關期刊論文 前10條
1 叢蓓蓓;高美華;王冰;王麗娜;邵志偉;;脂筏微域內CD59趨引CBP蛋白介導T細胞下游信號的轉導研究[J];免疫學雜志;2015年01期
2 王麗娜;高美華;王冰;叢蓓蓓;;LAT介導GPI錨固蛋白CD59對Jurkat細胞的生物學效應研究[J];免疫學雜志;2014年12期
3 李園園;高美華;叢蓓蓓;王麗娜;王冰;張蓓;李營;梁潔;;活化/抑制CD59對T淋巴細胞增殖的影響[J];現(xiàn)代生物醫(yī)學進展;2014年19期
4 高美華;鐘丹丹;張蓓;;CD59-CD2對T細胞信號轉導的作用[J];免疫學雜志;2011年09期
5 高美華;梁巖;;RNA干擾沉默Spl基因抑制前列腺癌細胞中CD59的表達[J];免疫學雜志;2010年12期
6 韓國雄;李文倩;馮建明;;白血病常用化療藥物毒副反應分析[J];現(xiàn)代預防醫(yī)學;2010年22期
7 ;CD59 Silencing via Retrovirus-Mediated RNA Interference Enhanced Complement-Mediated Cell Damage in Ovary Cancer[J];Cellular & Molecular Immunology;2009年01期
8 林東軍;范蕊芳;劉相富;;runx3啟動子區(qū)域甲基化在急性白血病中意義的初步研究[J];中國實驗血液學雜志;2008年02期
9 唐旭東;唐由君;;急性白血病患者預后相關的臨床病理學因素[J];腫瘤防治雜志;2005年21期
10 祝毓琳,張英,朱平,楊英,杜金偉,劉靜;常見白血病融合基因篩查在白血病診斷與分型中的意義[J];北京大學學報(醫(yī)學版);2005年03期
，本文編號：2457362
本文鏈接：http://www.sikaile.net/yixuelunwen/zlx/2457362.html
最近更新
教材專著
