慢病毒介导WWOX基因过表达对Jurkat和K562白血病细胞生物学特性影响的实验研究

目录中文摘要 (1)英文摘要 (3)前言 (6)第一部分WWOX基因过表达慢病毒载体的构建与鉴定 (8)材料与方法 (8)实验结果 (16)讨论 (21)结论 (22)第二部分慢病毒介导的WWOX过表达对Jurkat及K562白血病细胞生物学特性的影响 (23)材料与方法 (23)实验结果 (29)讨论 (43)结论 (46)第三部分WWOX基因过表达诱导Jurkat及K562细胞凋亡的分子机制研究 (47)材料与方法 (47)实验结果 (53)讨论 (60)结论 (63)参考文献 (64)综述 (69)致谢 (78)慢病毒介导WWOX基因过表达对Jurkat及K562白血病细胞生物学特性影响的实验研究摘要目的：1. 构建WWOX基因过表达慢病毒重组质粒载体并完成相关鉴定；2. 研究上调WWOX基因对人Jurkat及K562白血病细胞生物学特性的影响；3. 初步探讨其生物学特性改变的相关分子机制。

方法：1. 通过PCR扩增获得目的基因WWOX 全长cDNA 片段，纯化后经Age I 酶切消化。

同时用Age I酶对载体质粒进行酶切、回收得到线性化载体片段。

经T4噬菌体DNA连接酶作用将WWOX连接至线性化载体，连接产物经转化、涂板、挑取克隆、扩增培养后，用质粒小量抽提试剂盒提取质粒，酶切后PCR凝胶电泳鉴定连接产物，并行序列测定。

将构建好的融合基因表达载体进行病毒颗粒包装，得到WWOX过表达慢病毒重组质粒载体系统（pGC-FU-WWOX-GFP和pGC-FU-GFP）。

采用qPCR、Western blot等方法对重组载体进行鉴定。

2. 采用CCK-8比色、集落形成实验、DAPI染色、DNA片段电泳、Annexin V PE/7-AAD双荧光标记、Western blot等方法，研究上调WWOX 对Jurkat和K562细胞生物学特性的影响。

3. 间接免疫荧光检测凋亡相关蛋白p53、p73、NF-κBp65及细胞色素C（Cytochrome C，Cyto C）等的表达及定位。

qPCR、Western blot检测Bcl-2、Bax、Cyto C、Caspase-9、Caspase-3、p53、p73及NF-κB p65等凋亡相关蛋白的表达变化。

免疫共沉淀、Western blot检测WWOX 与p73、p53之间的相互作用。

结果：1. 测序鉴定结果与目标序列完全一致，包装后的慢病毒经qPCR测定滴度约为2×108TU/ml。

2. WWOX重组慢病毒质粒感染Jurkat及K562细胞48h 后，观察到GFP稳定表达。

qPCR显示pGC-FU-WWOX组细胞WWOX 在mRNA 水平表达增加。

Western blot 检测到约72kD的融合蛋白（WWOX融合GFP）表达，随时间延长，融合蛋白表达呈增加趋势。

CCK-8比色实验显示，Jurkat和K562细胞中pGC-FU-WWOX组的细胞生长较两对照组明显受抑制（P＜0.05）。

集落形成实验表明，Jurkat和K562细胞中pGC-FU-WWOX组细胞集落形成率较两对照组显著降低（P=0.000）。

DAPI染色显示，Jurkat和K562细胞中pGC-FU-WWOX组细胞核呈现Ⅱb晚期凋亡形态。

DNA片段电泳显示，Jurkat 和K562细胞中pGC-FU-WWOX组出现明显的梯状降解条带，并随时间延长梯状条带生成更加明显。

Annexin V PE/7-AAD双荧光染FCM检测显示，Jurkat和K562 pGC-FU-WWOX组细胞48h、72h、96h的凋亡率明显高于其他两对照组（P ＜0.001），主要为凋亡中、晚期细胞。

3. 间接免疫荧光染色可见，Jurkat和K562细胞中pGC-FU-WWOX组细胞Cyto C在胞质中呈弥散或粗块状分布，而p53、p73及NF-κB p65均定位于胞质，较对照组表达差异不明显。

qPCR及Western blot 显示，Jurkat和K562细胞中pGC-FU-WWOX组较其他两对照组Bcl-2表达呈下降趋势，Cyto C表达增加，同时两细胞均有Caspase-9和Caspase-3的剪接激活，而p53表达变化不明显。

K562 细胞中Bax表达呈上升趋势。

免疫共沉淀后SDS-PAGE电泳染色显示，Jurkat和K562细胞pGC-FU-WWOX组均于约73～74kD处出现浓染的蛋白条带，而其他两对照组蛋白条带染色为阴性。

蛋白印迹在两种细胞的pGC-FU-WWOX组均检测到p73及WWOX的表达，而未检测到p53蛋白的表达。

结论：1. 成功构建WWOX基因慢病毒重组载体。

2. WWOX过表达可明显抑制Jurkat和K562细胞增殖，诱导细胞凋亡。

3. WWOX过表达均可使Jurkat 和K562白血病细胞中Bcl-2表达下调，Cyto C表达增多，同时均可激活Caspase-9和Caspase-3的表达剪接，推测通过激活内源性线粒体途径可能是WWOX抑癌机制之一。

4. Jurkat和K562白血病细胞中，上调的WWOX可直接与p73结合，参与诱导细胞凋亡的过程。

关键词：WWOX基因，慢病毒载体，过表达，白血病，增殖，凋亡，分子机制Effects of lentivirus mediated-WWOX gene overexpression on biological properties of Jurkat and K562 human hematopoieticmalignant cellsAbstractObjective: 1.To construct a lentivirus mediated-WWOX overexpression system and verify its validity and infectious efficiency; 2.To investigate the effects of lentivirus mediated-WWOX gene overexpression on biological properties of Jurkat and K562 leukemia cells; 3.To explore the molecular mechanisms underlying the biological properties changes.Methods: 1.PCR technique was performed to obtain our target gene WWOX clones, the cDNA strands obtained in this process were digested by Age I enzyme. At the same time, pGC-FU vector was treated as in the same manner by Age I enzyme to make it linear enough. The linear WWOX cDNA strands and the linear vectors were bound together with the help of T4DNA ligase and followed by transformation, coated onto agar plate, picked clones and amplification, respectively. The combined plasmids were extracted and sent to sequencing. After sequencing, the lentivirus vectors combined full length WWOX cDNA strands were propagated and harvested after centrifugation, then pGC-FU-WWOX-GFP and pGC-FU-GFP overexpression system was completed. qPCR and Western blot were employed in the following evaluation tests. 2.The effects of WWOX gene overexpression on biological properties of Jurkat and K562 cell lines were estimated by cell counting kit 8(CCK-8), Clone forming assay, DAPI stain, DNA ladder electrophoresis Annexin V PE/7-AAD fluorescence test and Western blot, respectively. 3. Indirect immunofluorescence was employed to observe the expression and location of apoptosis related proteins such as p53, p73, NF-κB p65 and cytochrome C in Jurkat and K562 cells.The expression levels of apoptosis related proteins including Bcl-2, Bax, cytochrome C, Caspase-9, Caspase-3, p53 and p73 were identified by qPCR and Western blot after transfection. Immunoprecipitation and Western blot were used to analyze the mutual effectsbetween WWOX and p73, p53.Results: 1. The sequencing of the reconstruct plasmids were completely correct, and the virus titer measured via qPCR was 2×108TU/ml. 2. Stable green fluorescence was observed after infected for 48 hours in both two kinds of cell lines. The WWOX expression level of the overexpresion groups increased distinctly showed by qPCR and a 72kD fusion protein was also detected by Western blot technique. CCK-8 test showed the cell growth in pGC-FU-WWOX group of both Jurkat and K562 cells were inhibited remarkably than that of two control groups(P＜0.05). Clone forming assay revealed the cloning formation ability of pGC-FU-WWOX group cells of both Jurkat and K562 cells were significantly decreased. There were obvious ultrastructure changes of pGC-FU-WWOX group cells in Jurkat and K562 cells with the morpha of chromatin condensation, shrink in phraseⅡb exhibited by DAPI stain. DNA degradative fragments in pGC-FU-WWOX group of Jurkat and K562 cells were detected by DNA ladder electrophoresis, which also showed apoptosis degree of pGC-FU-WWOX group increased with time prolonged. Annexin V PE/7-AAD fluorescence test via FCM showed cells in pGC-FU-WWOX groups of Jurkat and K562 had a significant high apoptosis rate than that of two control groups (P＜0.001), which most of the cells were in late apoptosis stage. 3. Indirect immunofluorescence unfolded that cytochrome C of pGC-FU-WWOX group located in cytoplasm with a dispersive or block like distribution both in Jurkat and K562 cells, while p73, p53 and NF-κB p65 located in cytoplasm equably with no difference. qPCR and Western blot revealed that compared with the other two control groups the expression of Bcl-2 protein in pGC-FU-WWOX group of Jurkat and K562 cells decreased in a time-dependent manner and cytochrome C increased as well as the activation of Caspase-9and Caspase-3 compared with two control groups, while p53 changed unconspicuously. Bax protein of pGC-FU-WWOX group ascended only observed in K562 cells. SDS-PAGE of pGC-FU-WWOX groups in Jurkat and K562 cells exhibits a high concentration protein mixture on 73～74kD of the marker after immunoprecipitation, while the control groups were negative.At the same time, WWOX and p73 proteins were positive in the immunoprecipitation mixture of thetarget gene groups with the help of Western blot, while p53 protein was undetected.Conclusions: 1. Lentivirus mediated-WWOX overexpression system was constructed successfully. 2. Overexpression of WWOX gene exibits an antitumor effect by inhibiting the proliferation of Jurkat and 562 cells and induc ing their apoptosis. 3. Overexpression of WWOX gene in Jurkat and 562 cells could down-regulate the expression of Bcl-2 and increase cytochrome C as well as activate both Caspase-9 and Caspase-3. By activating the endogenous mitochondria pathways is one of the possible mechanisms in its tumor suppression activities of WWOX in Jurkat and K562 cells. 4. WWOX could combine with p73 directly in its apoptosis inducing activities in Jurkat and K562 leukemic cells.Keywords: WWOX gene; Lentivirus vector; Overexpression; Leukemia; Proliferation; Apoptosis; Molecular mechanism前言白血病是一类异质性的造血干细胞恶性克隆性疾病，通常会出现染色体或基因异常。