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
發(fā)布時(shí)間：2019-04-12 20:21
【摘要】：目的構(gòu)建RNAi-CD59慢病毒載體,研究其對(duì)急性T淋巴細(xì)胞白血病細(xì)胞株Jurkat CD59的沉默效應(yīng),運(yùn)用體內(nèi)和體外實(shí)驗(yàn)探討CD59特異性沉默對(duì)腫瘤細(xì)胞免疫逃逸的作用。方法體外實(shí)驗(yàn):免疫熒光比較正常人T細(xì)胞和Jurkat細(xì)胞上的CD59表達(dá)情況;構(gòu)建 CD59 干擾序列(RNAi-CD59-A、RNAi-CD59-B、RNAi-CD59-C)以及錯(cuò)義序列(RNAi-NC)融合綠色熒光蛋白,以慢病毒為載體轉(zhuǎn)染進(jìn)入人急性T系白血病Jurkat細(xì)胞株,陰性對(duì)照為RNAi-NC,空白對(duì)照組為未經(jīng)任何處理的正常培養(yǎng)的Jurkat細(xì)胞系;運(yùn)用熒光顯微鏡和流式細(xì)胞儀檢測(cè)各組細(xì)胞的轉(zhuǎn)染效率;RT-PCR檢測(cè)各組CD59基因以及凋亡相關(guān)基因Bcl-2/Bax mRNA的表達(dá);Western-blot檢測(cè)各組CD59蛋白以及凋亡相關(guān)蛋白Bcl-2、Bax、Caspase-3、Survivin蛋白表達(dá)水平的變化;激光共聚焦觀察CD59分子的定位;ELISA檢測(cè)各組細(xì)胞培養(yǎng)上清中IL-3、TNF-β的表達(dá);CCK8檢測(cè)各組細(xì)胞增殖效率的改變;流式細(xì)胞儀觀察各組細(xì)胞凋亡水平的變化。體內(nèi)實(shí)驗(yàn):24只BALB/c-nu雌性裸鼠隨機(jī)分為4組(PBS組、Jurkat組、RNAi-NC組、RNAi-CD59-A組),將培養(yǎng)篩選后的細(xì)胞系通過(guò)尾靜脈注射進(jìn)入裸鼠體內(nèi)構(gòu)建裸鼠轉(zhuǎn)移瘤模型,注射前后0周、1周、2周、3周、4周,監(jiān)測(cè)小鼠生長(zhǎng)狀態(tài)、小鼠體質(zhì)量及外周血白細(xì)胞數(shù)量的變化;流式細(xì)胞檢測(cè)技術(shù)觀察小鼠外周血及骨髓中淋巴細(xì)胞的凋亡情況;ELISA檢測(cè)各組小鼠血液上清中IL-3、TNF-β的表達(dá)。結(jié)果體外實(shí)驗(yàn):熒光顯微鏡和FCM觀察轉(zhuǎn)染效率在90.00%以上;RT-PCR結(jié)果顯示沉默組CD59、Bcl-2 mRNA表達(dá)水平降低(P0.05),Bax mRNA表達(dá)水平升高(P0.05);Western-blot結(jié)果顯示沉默組CD59、Bax、Survivin蛋白的表達(dá)量減少,Bcl-2、caspase-3蛋白的表達(dá)量增多(P0.05);激光共聚焦觀察到CD59分子主要定位于細(xì)胞膜;ELISA結(jié)果顯示沉默組IL-3表達(dá)水平降低,TNF-β的表達(dá)水平升高(P0.05);CCK8結(jié)果顯示RNAi-CD59-A組的細(xì)胞增殖效率明顯降低(P0.05);流式細(xì)胞儀觀察到RNAi-CD59-A組的細(xì)胞凋亡率明顯升高(P0.05)。體內(nèi)實(shí)驗(yàn):動(dòng)物模型構(gòu)建成功,與PBS組相比,各模型組裸鼠體質(zhì)量均有一定的下降(P0.05),其中沉默組裸鼠體質(zhì)量下降不如RNAi-NC組和Jurkat細(xì)胞組明顯(P0.05);與PBS組相比,各模型組外周血白細(xì)胞數(shù)均有明顯的升高(P0.05),其中與Jurkat細(xì)胞和RNAi-NC組相比,沉默組外周血白細(xì)胞計(jì)數(shù)減少(P0.05);流式細(xì)胞儀結(jié)果顯示沉默組外周血和骨髓細(xì)胞中的細(xì)胞凋亡率明顯高于未沉默組(P0.05);ELISA結(jié)果顯示小鼠血液上清中沉默組IL-3表達(dá)水平降低,TNF-β的表達(dá)水平升高(P0.05)。結(jié)論RNAi-CD59轉(zhuǎn)染細(xì)胞系及白血病轉(zhuǎn)移瘤模型均構(gòu)建成功,在體內(nèi)外均驗(yàn)證了沉默CD59基因表達(dá)可抑制急性T系白血病的增殖并誘導(dǎo)細(xì)胞凋亡,為臨床急性T系白血病的診斷和治療提供了一個(gè)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(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
本文編號(hào)：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(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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