NLS-RARα对白血病细胞NB4增殖和分化的影响及作用机制的研究
发布时间：2019-02-22 20:10
【摘要】：第一部分 NLS-RARα和RARα重组慢病毒载体的构建及其稳定转染细胞株的筛选目的:本实验构建携带目的基因的重组慢病毒,感染白血病细胞NB4并筛选出稳定转染的细胞株。方法:分别以质粒p CMV-HA-NLS-RARα和p CMV-HA-RARα为模板,PCR扩增NLS-RARα和RARα基因,将其分别亚克隆到能表达绿色荧光蛋白的慢病毒载体质粒LV5中,经限制性内切酶酶切和DNA测序鉴定重组载体;用4质粒系统共转染293T细胞;荧光显微镜观察293T细胞绿色荧光蛋白的表达情况,收集病毒上清,浓缩,测定重组慢病毒的滴度。重组慢病毒感染NB4细胞,流式细胞术检测感染效率,用嘌呤霉素筛选出分别稳定转染NLS-RARα和RARα的两组细胞株,Western blot检测稳转细胞株中目的基因的表达。结果:重组慢病毒载体质粒经限制性内切酶酶切和DNA测序分析证实NLS-RARα和RARα分别克隆入穿梭质粒LV5的多克隆位点,荧光显微镜下观察可见293T细胞有大量的绿色荧光,浓缩后重组慢病毒滴度为2×108 Tu/ml。慢病毒对白血病细胞NB4有很高的感染效率,经嘌呤霉素筛选获得了稳定转染目的基因的NB4细胞株。Western blot检测目的基因在稳转细胞株中稳定有效的表达。结论:成功构建LV-NLS-RARα和LV-RARα的重组慢病毒,并且筛选出分别稳定转染NLS-RARα和RARα的NB4细胞株。第二部分 NLS-RARα对白血病细胞NB4增殖和分化的影响及其机制的研究目的:在稳转目的基因的NB4细胞株中,观察NLS-RARα对白血病细胞株增殖和分化的影响及机制。方法:CCK-8法检测NLS-RARα对NB4细胞增殖的影响;用流式细胞术(FCM)检测细胞周期;用维甲酸处理后FCM检测细胞分化相关抗原CD11b;瑞氏染色检测细胞核形态变化;Western blot分别检测NLS-RARα、t-AKT、p-AKT、t-GSK3β、p-GSK3β和c-myc等蛋白的表达情况。结果:CCK-8结果显示NLS-RARα能够明显促进细胞增殖;流式细胞术显示NLS-RARα组细胞S期细胞增多,G2期细胞减少;CD11b表达明显降低,胞核形态变化不明显;Western blot检测NLS-RARα基因在NB4细胞中稳定有效的表达,同时p-AKT、p-GSK3β、c-myc蛋白明显增高,用PI3K抑制剂LY294002处理后p-AKT、p-GSK3β、c-myc明显降低。结论:NLS-RARα可能通过激活PI3K-AKT信号通路,促进NB4细胞的增殖并抑制维甲酸诱导的分化。
[Abstract]:Part one: construction of NLS-RAR 伪 and RAR 伪 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 伪 and RAR 伪 genes were amplified by PCR using plasmids p CMV-HA-NLS-RAR 伪 and p CMV-HA-RAR 伪 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 伪 and RAR 伪 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 伪 and LV-RAR 伪 were successfully constructed, and NB4 cell lines stably transfected with NLS-RAR 伪 and RAR 伪 were screened. The second part: the effect of NLS-RAR 伪 on the proliferation and differentiation of leukemic cell line NB4 and its mechanism. Objective: to observe the effect and mechanism of NLS-RAR 伪 on the proliferation and differentiation of leukemic cell line NB4. Methods: CCK-8 assay was used to detect the effect of NLS-RAR 伪 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 伪, t-AKTnp-AKTnt-GSK3 尾, p-GSK3 尾 and c-myc. Results: CCK-8 results showed that NLS-RAR 伪 could significantly promote cell proliferation, flow cytometry showed that the S phase cells increased and G2 phase cells decreased in NLS-RAR 伪 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 伪 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 伪 may promote the proliferation of NB4 cells and inhibit the differentiation induced by retinoic acid by activating the PI3K-AKT signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7
本文编号：2428576
[Abstract]:Part one: construction of NLS-RAR 伪 and RAR 伪 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 伪 and RAR 伪 genes were amplified by PCR using plasmids p CMV-HA-NLS-RAR 伪 and p CMV-HA-RAR 伪 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 伪 and RAR 伪 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 伪 and LV-RAR 伪 were successfully constructed, and NB4 cell lines stably transfected with NLS-RAR 伪 and RAR 伪 were screened. The second part: the effect of NLS-RAR 伪 on the proliferation and differentiation of leukemic cell line NB4 and its mechanism. Objective: to observe the effect and mechanism of NLS-RAR 伪 on the proliferation and differentiation of leukemic cell line NB4. Methods: CCK-8 assay was used to detect the effect of NLS-RAR 伪 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 伪, t-AKTnp-AKTnt-GSK3 尾, p-GSK3 尾 and c-myc. Results: CCK-8 results showed that NLS-RAR 伪 could significantly promote cell proliferation, flow cytometry showed that the S phase cells increased and G2 phase cells decreased in NLS-RAR 伪 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 伪 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 伪 may promote the proliferation of NB4 cells and inhibit the differentiation induced by retinoic acid by activating the PI3K-AKT signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7
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