NLS-RARα對白血病細胞NB4增殖和分化的影響及作用機制的研究
發(fā)布時間：2019-02-22 20:10
【摘要】：第一部分 NLS-RARα和RARα重組慢病毒載體的構(gòu)建及其穩(wěn)定轉(zhuǎn)染細胞株的篩選目的:本實驗構(gòu)建攜帶目的基因的重組慢病毒,感染白血病細胞NB4并篩選出穩(wěn)定轉(zhuǎn)染的細胞株。方法:分別以質(zhì)粒p CMV-HA-NLS-RARα和p CMV-HA-RARα為模板,PCR擴增NLS-RARα和RARα基因,將其分別亞克隆到能表達綠色熒光蛋白的慢病毒載體質(zhì)粒LV5中,經(jīng)限制性內(nèi)切酶酶切和DNA測序鑒定重組載體;用4質(zhì)粒系統(tǒng)共轉(zhuǎn)染293T細胞;熒光顯微鏡觀察293T細胞綠色熒光蛋白的表達情況,收集病毒上清,濃縮,測定重組慢病毒的滴度。重組慢病毒感染NB4細胞,流式細胞術檢測感染效率,用嘌呤霉素篩選出分別穩(wěn)定轉(zhuǎn)染NLS-RARα和RARα的兩組細胞株,Western blot檢測穩(wěn)轉(zhuǎn)細胞株中目的基因的表達。結(jié)果:重組慢病毒載體質(zhì)粒經(jīng)限制性內(nèi)切酶酶切和DNA測序分析證實NLS-RARα和RARα分別克隆入穿梭質(zhì)粒LV5的多克隆位點,熒光顯微鏡下觀察可見293T細胞有大量的綠色熒光,濃縮后重組慢病毒滴度為2×108 Tu/ml。慢病毒對白血病細胞NB4有很高的感染效率,經(jīng)嘌呤霉素篩選獲得了穩(wěn)定轉(zhuǎn)染目的基因的NB4細胞株。Western blot檢測目的基因在穩(wěn)轉(zhuǎn)細胞株中穩(wěn)定有效的表達。結(jié)論:成功構(gòu)建LV-NLS-RARα和LV-RARα的重組慢病毒,并且篩選出分別穩(wěn)定轉(zhuǎn)染NLS-RARα和RARα的NB4細胞株。第二部分 NLS-RARα對白血病細胞NB4增殖和分化的影響及其機制的研究目的:在穩(wěn)轉(zhuǎn)目的基因的NB4細胞株中,觀察NLS-RARα對白血病細胞株增殖和分化的影響及機制。方法:CCK-8法檢測NLS-RARα對NB4細胞增殖的影響;用流式細胞術(FCM)檢測細胞周期;用維甲酸處理后FCM檢測細胞分化相關抗原CD11b;瑞氏染色檢測細胞核形態(tài)變化;Western blot分別檢測NLS-RARα、t-AKT、p-AKT、t-GSK3β、p-GSK3β和c-myc等蛋白的表達情況。結(jié)果:CCK-8結(jié)果顯示NLS-RARα能夠明顯促進細胞增殖;流式細胞術顯示NLS-RARα組細胞S期細胞增多,G2期細胞減少;CD11b表達明顯降低,胞核形態(tài)變化不明顯;Western blot檢測NLS-RARα基因在NB4細胞中穩(wěn)定有效的表達,同時p-AKT、p-GSK3β、c-myc蛋白明顯增高,用PI3K抑制劑LY294002處理后p-AKT、p-GSK3β、c-myc明顯降低。結(jié)論:NLS-RARα可能通過激活PI3K-AKT信號通路,促進NB4細胞的增殖并抑制維甲酸誘導的分化。
[Abstract]:Part one: construction of NLS-RAR 偽 and RAR 偽 recombinant lentivirus vectors and screening of stable transfected cell lines objective: to construct recombinant lentivirus carrying the target gene, infect leukemia cell NB4 and screen stable transfected cell lines. Methods: the NLS-RAR 偽 and RAR 偽 genes were amplified by PCR using plasmids p CMV-HA-NLS-RAR 偽 and p CMV-HA-RAR 偽 as templates, respectively. They were subcloned into the lentivirus vector LV5, which could express green fluorescent protein. The recombinant vector was identified by restriction endonuclease digestion and DNA sequencing. The expression of green fluorescent protein (GFP) in 293T cells was observed by fluorescence microscope, the virus supernatant was collected and concentrated, and the titer of recombinant lentivirus was determined. NB4 cells were infected with recombinant lentivirus, the infection efficiency was detected by flow cytometry, and the expression of target gene was detected by purine mycin screening and stable transfection of NLS-RAR 偽 and RAR 偽 in two groups of cell lines, Western blot, respectively. Results: the recombinant lentivirus vector plasmid was cloned into the shuttle plasmid LV5 by restriction endonuclease digestion and DNA sequencing analysis. A large amount of green fluorescence was observed in 293T cells under fluorescence microscope. The concentration of recombinant lentiviral droplets was 2 脳 10 8 Tu/ml.. Lentivirus has a high infection efficiency on leukemia cell NB4. The stable and effective expression of target gene in stable transformed NB4 cell line. Western blot was obtained by purine mycin screening. Conclusion: the recombinant lentiviruses of LV-NLS-RAR 偽 and LV-RAR 偽 were successfully constructed, and NB4 cell lines stably transfected with NLS-RAR 偽 and RAR 偽 were screened. The second part: the effect of NLS-RAR 偽 on the proliferation and differentiation of leukemic cell line NB4 and its mechanism. Objective: to observe the effect and mechanism of NLS-RAR 偽 on the proliferation and differentiation of leukemic cell line NB4. Methods: CCK-8 assay was used to detect the effect of NLS-RAR 偽 on the proliferation of NB4 cells, flow cytometry (FCM) was used to detect the cell cycle, and FCM was used to detect the cell differentiation related antigen CD11b; stain after retinoic acid treatment. Western blot was used to detect the expression of NLS-RAR 偽, t-AKTnp-AKTnt-GSK3 尾, p-GSK3 尾 and c-myc. Results: CCK-8 results showed that NLS-RAR 偽 could significantly promote cell proliferation, flow cytometry showed that the S phase cells increased and G2 phase cells decreased in NLS-RAR 偽 group, CD11b expression decreased significantly, and nuclear morphologic changes were not obvious. Western blot was used to detect the stable and effective expression of NLS-RAR 偽 gene in NB4 cells. At the same time, p-AKTna-p-GSK3 尾 and c-myc protein were significantly increased. After treated with PI3K inhibitor LY294002, p-AKTna-p-GSK3 尾 and c-myc were significantly decreased. Conclusion: NLS-RAR 偽 may promote the proliferation of NB4 cells and inhibit the differentiation induced by retinoic acid by activating the PI3K-AKT signaling pathway.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.7
本文編號：2428576
[Abstract]:Part one: construction of NLS-RAR 偽 and RAR 偽 recombinant lentivirus vectors and screening of stable transfected cell lines objective: to construct recombinant lentivirus carrying the target gene, infect leukemia cell NB4 and screen stable transfected cell lines. Methods: the NLS-RAR 偽 and RAR 偽 genes were amplified by PCR using plasmids p CMV-HA-NLS-RAR 偽 and p CMV-HA-RAR 偽 as templates, respectively. They were subcloned into the lentivirus vector LV5, which could express green fluorescent protein. The recombinant vector was identified by restriction endonuclease digestion and DNA sequencing. The expression of green fluorescent protein (GFP) in 293T cells was observed by fluorescence microscope, the virus supernatant was collected and concentrated, and the titer of recombinant lentivirus was determined. NB4 cells were infected with recombinant lentivirus, the infection efficiency was detected by flow cytometry, and the expression of target gene was detected by purine mycin screening and stable transfection of NLS-RAR 偽 and RAR 偽 in two groups of cell lines, Western blot, respectively. Results: the recombinant lentivirus vector plasmid was cloned into the shuttle plasmid LV5 by restriction endonuclease digestion and DNA sequencing analysis. A large amount of green fluorescence was observed in 293T cells under fluorescence microscope. The concentration of recombinant lentiviral droplets was 2 脳 10 8 Tu/ml.. Lentivirus has a high infection efficiency on leukemia cell NB4. The stable and effective expression of target gene in stable transformed NB4 cell line. Western blot was obtained by purine mycin screening. Conclusion: the recombinant lentiviruses of LV-NLS-RAR 偽 and LV-RAR 偽 were successfully constructed, and NB4 cell lines stably transfected with NLS-RAR 偽 and RAR 偽 were screened. The second part: the effect of NLS-RAR 偽 on the proliferation and differentiation of leukemic cell line NB4 and its mechanism. Objective: to observe the effect and mechanism of NLS-RAR 偽 on the proliferation and differentiation of leukemic cell line NB4. Methods: CCK-8 assay was used to detect the effect of NLS-RAR 偽 on the proliferation of NB4 cells, flow cytometry (FCM) was used to detect the cell cycle, and FCM was used to detect the cell differentiation related antigen CD11b; stain after retinoic acid treatment. Western blot was used to detect the expression of NLS-RAR 偽, t-AKTnp-AKTnt-GSK3 尾, p-GSK3 尾 and c-myc. Results: CCK-8 results showed that NLS-RAR 偽 could significantly promote cell proliferation, flow cytometry showed that the S phase cells increased and G2 phase cells decreased in NLS-RAR 偽 group, CD11b expression decreased significantly, and nuclear morphologic changes were not obvious. Western blot was used to detect the stable and effective expression of NLS-RAR 偽 gene in NB4 cells. At the same time, p-AKTna-p-GSK3 尾 and c-myc protein were significantly increased. After treated with PI3K inhibitor LY294002, p-AKTna-p-GSK3 尾 and c-myc were significantly decreased. Conclusion: NLS-RAR 偽 may promote the proliferation of NB4 cells and inhibit the differentiation induced by retinoic acid by activating the PI3K-AKT signaling pathway.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.7
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