【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(xì)胞株的篩選目的:本實(shí)驗(yàn)構(gòu)建攜帶目的基因的重組慢病毒,感染白血病細(xì)胞NB4并篩選出穩(wěn)定轉(zhuǎn)染的細(xì)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(kuò)增NLS-RARα和RARα基因,將其分別亞克隆到能表達(dá)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(cè)序鑒定重組載體;用4質(zhì)粒系統(tǒng)共轉(zhuǎn)染293T細(xì)胞;熒光顯微鏡觀察293T細(xì)胞綠色熒光蛋白的表達(dá)情況,收集病毒上清,濃縮,測(cè)定重組慢病毒的滴度。重組慢病毒感染NB4細(xì)胞,流式細(xì)胞術(shù)檢測(cè)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(xì)胞株,Western blot檢測(cè)穩(wěn)轉(zhuǎn)細(xì)胞株中目的基因的表達(dá)。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(cè)序分析證實(shí)NLS-RARα和RARα分別克隆入穿梭質(zhì)粒LV5的多克隆位點(diǎn),熒光顯微鏡下觀察可見293T細(xì)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(duì)白血病細(xì)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(xì)胞株。Western blot檢測(cè)目的基因在穩(wěn)轉(zhuǎn)細(xì)胞株中穩(wěn)定有效的表達(dá)。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(xì)胞株。第二部分 NLS-RARα對(duì)白血病細(xì)胞NB4增殖和分化的影響及其機(jī)制的研究目的:在穩(wěn)轉(zhuǎn)目的基因的NB4細(xì)胞株中,觀察NLS-RARα對(duì)白血病細(xì)胞株增殖和分化的影響及機(jī)制。方法:CCK-8法檢測(cè)NLS-RARα對(duì)NB4細(xì)胞增殖的影響;用流式細(xì)胞術(shù)(FCM)檢測(cè)細(xì)胞周期;用維甲酸處理后FCM檢測(cè)細(xì)胞分化相關(guān)抗原CD11b;瑞氏染色檢測(cè)細(xì)胞核形態(tài)變化;Western blot分別檢測(cè)NLS-RARα、t-AKT、p-AKT、t-GSK3β、p-GSK3β和c-myc等蛋白的表達(dá)情況。結(jié)果:CCK-8結(jié)果顯示NLS-RARα能夠明顯促進(jìn)細(xì)胞增殖;流式細(xì)胞術(shù)顯示NLS-RARα組細(xì)胞S期細(xì)胞增多,G2期細(xì)胞減少;CD11b表達(dá)明顯降低,胞核形態(tài)變化不明顯;Western blot檢測(cè)NLS-RARα基因在NB4細(xì)胞中穩(wěn)定有效的表達(dá),同時(shí)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(hào)通路,促進(jìn)NB4細(xì)胞的增殖并抑制維甲酸誘導(dǎo)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.7

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