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类型 基础研究 预答辩日期 2017-12-27
开始(开题)日期 2015-12-28 论文结束日期 2017-11-08
地点 东南大学附属中大医院血液科示教室 论文选题来源 其他项目    论文字数 5.11 (万字)
题目 汉黄芩素对LMP1阳性非霍奇金淋巴瘤生物学行为的影响及其分子机制的研究
主题词 汉黄芩素,淋巴瘤,潜伏膜蛋白1,miR-155,核因子kappaB
摘要 目的 近年来,非霍奇金淋巴瘤(non-Hodgkin`s lymphoma, NHL)因其发病率的逐年上升及发病呈现年轻化趋势,而成为人们关注的焦点。部分因感染EB病毒(Epstein-Barr virus,EBV)而产生的NHL,会表达潜伏膜蛋白1(latent membrance protein 1,LMP1),LMP1是EBV感染后具有致癌性的一个基因,它的致癌作用可能与激活微小RNA-155(microRNA-155,miR-155)的表达相关,但关于二者对NHL预后的影响还未见报道。 汉黄芩素是唇形科植物黄芩的主要活性成分之一,它已在众多血液系统恶性疾病和实体瘤中表现出高效低毒的抗肿瘤特性。然而,目前尚没有关于汉黄芩素用于治疗NHL的研究。 因此,本研究诣在明确LMP1/miR-155在NHL预后中的作用,探索汉黄芩素对LMP1阳性表达NHL的作用机制,尝试为LMP1阳性的NHL的诊治及预后预测找到高效低毒的药物及新的分子标志物。 方法 首先收集2010年5月至2011年12月间于某三甲医院初诊的弥漫大B 细胞淋巴瘤患者的淋巴结石蜡标本,应用microRNA芯片筛查、免疫组化和realtime-qPCR技术检测组织标本中LMP1和miR-155表达情况,收集患者临床资料,使用Log-rank、COX回归模型、Kaplan–Meier曲线等统计学方法对各项指标与患者预后之间的关系进行评估。 其次,通过CCK-8 细胞增殖实验,选取汉黄芩素对Raji细胞无明显细胞毒性的浓度;流式细胞技术(flow cytometry, FCM)分析细胞凋亡及细胞周期;免疫荧光染色从形态学角度观察细胞凋亡情况。分别使用miR-155抑制剂、NF-κB抑制剂和汉黄芩素作用于细胞,观察细胞生物学行为改变和LMP1/ p65/pp65/miR-155/PU.1等信号分子的表达情况,realtime-qPCR检测信号分子mRNA的相对表达量;Western blot检测信号分子的蛋白表达情况;凝胶迁移实验(electrophoretic mobility shift assays, EMSA)及双荧光素酶报告基因检测核蛋白NF-κB的表达。 最后,建立荷瘤裸鼠模型,观察Raji细胞的成瘤性,同时设生理盐水对照组和腹腔给药组(汉黄芩素80mg/kg,隔天一次,连续两周),记录小鼠体重及瘤体生长情况,处死裸鼠,摘取瘤组织,计算瘤体积和重量变化;免疫组织化学染色Ki67了解细胞增殖情况,同时染色p65及PU.1了解信号分子表达情况。 结果 本研究最后入组的患者人数为82例。miR-155芯片分析结果显示,miR-155在不同类型的淋巴瘤中表达均升高,在B细胞淋巴瘤中升高最明显。根据患者是否表达LMP1,我们将患者分为LMP1阳性组(n=41)和LMP1阴性组(n=41);根据患者miR-155表达情况不同,我们将患者分为miR-155高表达组(≤12倍,n=42)和miR-155极高表达组(>12倍,n=40)。研究结果显示,LMP1、miR-155及IPI评分与患者预后相关,LMP1和miR-155是预测患者无进展生存时间(progression-free survival, PFS)的独立因素,LMP1和miR-155高表达提示PFS差;同时,LMP1和IPI评分是预测患者总生存时间(overall survival, OS)的独立因素,LMP1高表达和IPI评分高提示OS差。 体外实验中,使用不同浓度(0-200 μmol/L)汉黄芩素干预Raji细胞24、48和72小时,CCK-8实验结果显示,汉黄芩素可以抑制Raji细胞生长,且这一作用呈时间依赖性和浓度依赖性。选取汉黄芩素毒性较小的浓度处理Raji细胞,FCM分析细胞凋亡发现,对照组(0 μmol/L)与实验组(12.5和50 μmol/L)早期凋亡和晚期凋亡的细胞的总和占所有细胞的比例分别为:(6.44 ± 1.26)%,(8.86 ± 0.74)%和(12.94 ± 2.68)%;FCM分析细胞周期发现,对照组S、G1、G2/M期细胞比例分别为(19.77 ± 1.60)%、(44.30 ± 1.91)%和(32.93 ± 1.78)%,实验组S、G1、G2/M期细胞比例分别为(6.98 ± 1.17)%,(70.04 ± 1.23)%和(22.99 ± 0.65)%,实验组G1期细胞明显增多,S期及G2/M期细胞减少;免疫荧光染色观察到实验组细胞出现细胞核碎裂、固缩的凋亡状态,并且随着汉黄芩素浓度的增加,核碎裂、固缩的细胞核数量增多。 体外实验中还发现,使用miR-155抑制剂作用于Raji细胞后, Raji细胞发生凋亡,对照组与实验组(100 nmol/L)早期凋亡和晚期凋亡的细胞的总和占所有细胞的比例分别为:(8.5 ± 0.74)%和(12.94 ± 4.59)%,同时,realtime-qPCR和Western blot检测到细胞中LMP1表达无明显变化,实验组PU.1的表达较对照组明显上升,具有统计学意义(p <0.05)。使用NF-κB抑制剂PDTC作用于Raji细胞后,Raji细胞发生凋亡,对照组与实验组早期凋亡和晚期凋亡的细胞的总和占所有细胞的比例分别为:(8.5 ± 0.74)%和(11.19 ± 1.71)%,同时,realtime-qPCR和Western blot检测到细胞中LMP1表达无明显变化,实验组miR-155表达较对照组下降、PU.1表达上升,且均具有统计学意义(p <0.05)。另外,与单独用药组(PDTC组、汉黄芩素组)相比,联合用药组(PDTC+汉黄芩素)对miR-155及PU.1表达的影响并没有太明显变化,与单独用药组无统计学差异,提示PDTC与汉黄芩素之间不存在协同作用。 为探究汉黄芩素对NF-κB分子通路中的影响,我们使用EMSA直接检测核蛋白NF-κB的表达情况。结果显示,汉黄芩素具有与PDTC相同的效应,能够抑制NF-κB的表达,双荧光素酶报告基因检测经不同浓度汉黄芩素处理后的细胞核蛋白NF-κB的表达情况,提示汉黄芩素是直接作用于NF-κB,从而引起细胞凋亡等一系列作用。 裸鼠体内实验,对照组瘤体积增长速度显著快于用药组(p <0.05),而裸鼠治疗前后体重并无明显变化(p >0.05)。免疫组织化学染色后可见汉黄芩素可以下调Ki67、p65的表达,上调PU.1的表达,与对照组相比,差别具有统计学意义(p <0.05)。 结论 LMP1和miR-155可作为预测淋巴瘤患者可靠的预后新指标,而且预测结果较为可靠,应在淋巴瘤初诊时将这两个生物学指标加入监测范围以评估患者病情。 汉黄芩素可以通过下调NF-κB的表达来抑制肿瘤生长,通过LMP1/NF-κB/miR-155/PU.1途径诱导细胞凋亡。由此可见,汉黄芩素可成为治疗LMP1阳性淋巴瘤的药物。
英文题目 The effect of Wogonin on LMP1 (+) non-Hodgkin`s lymphoma and its molecular mechanism
英文主题词 Wogonin,Lymphoma,LMP1,miR-155,NF-κB
英文摘要 BACKGROUND The morbidity of non-Hodgkin`s lymphoma (NHL) increased consistently, especially among young adults, which has always been in the spotlight during the recent years. Parts of the NHLs have the expression of latent membrance protein 1 (LMP1) due to the infection of Epstein-Barr virus (EBV). LMP1 is an oncogene that can promote the activation of microRNA-155 (miR-155). However, the effect of LMP1 and miR-155 on the prognosis of NHL still remains controversial. Wogonin (5, 7-dihydroxy-8-methoxyflavone) is extracted from Scutellaria baicalensis Georgi (Huangqin), a perennial labiatae. During the past two decades, wogonin has been identified as a potent apoptositic inducer for cancer cells with minor side effects. But there is no research about the treatment of wogonin on NHL up to now. In this study, we focused on the prognostic role of bio-markers LMP1 and miR-155 in NHL and the anticancer effect of wogonin alongside with the underlying mechanisms for targeted therapy in patients with LMP1 (+) NHL. This will facilitate the introduction of the two bio-markers LMP1 and miR-155 and one novel drug, wogonin, to clinical use, which is planned in the near future. METHODS First, formalin fixed paraffin embedded samples and clinic information of newly diagnosed diffuse large B cell lymohoma (DLBCL) from May 2010 to December 2011 were collected from our center. Microarray analysis, immunohistochemistry staining (IHC) and realtime-qPCR were used to evaluate the expression of LMP1 and miR-155. The association between bio-markers or clinic features with patients` outcomes was assessed by Log-rank statistical test, Cox proportional hazard model, and Kaplan–Meier method. Then, cell proliferation was studied by CCK8 assay and the most reasonable concentration was chosen for the further researches. Flow cytometry was used to analyze the apoptosis and the cycle arrest of Raji cells. Further, we also used immunofluorescent staining to detect the morphologic changes of the apoptotic cells. Moreover, we treated the Raji cells with miR-155 inhibitor, PDTC and wogonin separately. The expression of LMP1/miR-155/p65/pp65/PU.1 was evaluated by realtime-qPCR and Western blot, while that of NF-κB was analyzed by electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assay. At last, in vivo, the effect of wogonin on growth of tumor in Raji xenografts and mice body weight was observed between the groups of control and wogonin-treated (80mg/kg, abdominal abministration every other day for two weeks). IHC was performed to detect the expression of Ki67, p65 and PU.1. RESULTS Pathological tissues from 82 patients were available for this study. The results from microarray said that lymphomas had a high expression of miR-155, especially B cell lymphoma. Patients were separated into two groups named LMP1-positive group (n=41) and LMP1-negative group (n=41) according to the expression of LMP1; Meanwhile, patients were separated into two groups named miR-155 very high expression group (>12 folds, n=40) and miR-155 high expression group (≤12 folds,n=42) according to the expression of miR-155. The results showed that LMP1, miR-155 and IPI were related to the prognosis of lymphoma patients. LMP1 and miR-155 were the independent factors for PFS, while IPI and LMP1 for OS. The expression of LMP1 and miR-155 and high IPI score indicated a poor outcome for patients. In vitro, we treated Raji cells with different concentration of wogonin (0-200 μmol/L) for 24, 48 and 72 hours, separately. The results from CCK-8 assay showed that wogonin could inhibit the growth of Raji cells in a time and dose-depended manner. The rate of cell apoptosis was (6.44 ± 1.26)%, (8.86 ± 0.74)% and (12.94 ± 2.68)% for 0 , 12.5 and 50 μmol/L wogonin. In addition, the results from FCM demonstrated that the cells numbers of S phase, G1 phase and G2/M phase for the group of control and wogonin-treated were (19.77 ± 1.60)%、(44.30 ± 1.91)%、 (32.93 ± 1.78)% and (6.98 ± 1.17)%,(70.04 ± 1.23)%, (22.99 ± 0.65)% separately, so the cell cycle was arrested at G1 phase. The morphological changes of the nucleolus in apoptostic cells were observed under a florescence microscope. Raji cells without treatment of wogonin exhibited a pale blue florescence, whereas those treated with wogonin exhibited a nuclear fragmentation in a dose-depended manner. In vitro, we also observed that the apoptostic rate of Raji cells was (8.5 ± 0.74)% and (12.94 ± 4.59)% after treatment without or with miR-155 inhibitor. Meanwhile, the expression of LMP1 remained similar between the two groups, while that of PU.1 was statistically higher (p <0.05) in the wogonin-treated group than that in the control group according to the results from realtime-qPCR and Western blot. Futhermore, the apoptostic rate of Raji cells was (8.5 ± 0.74)% and (10.19 ± 1.71)% after treated without or with PDTC, and the expression of LMP1 remained similar between the two groups, while that of miR-155 was lower and PU.1 was higher in the wogonin-treated group than that in the control group, and there were both statistically significant (p <0.05). In addition, there was no difference between the combination of PDTC with wogonin-treated group and PDTC-treated group or wogonin-treated group about the expression of miR-155 and PU.1, indicating that there was no synergistic reaction between PDTC and wogonin. To investigate the effect of wogonin on NF-κB, we measured the nucleoprotein by EMSA and the results demonstrated that wogonin could inhibit the expression of NF-κB as well as PDTC. The data from dual luciferase reporter assay confirmed the role of wogonin as a NF-κB inhibitor. In vivo study revealed that wogonin could suppress tumor growth (p <0.05), but did not affect the body weight of mice (p >0.05). Moreover, the results of IHC showed significant lower levels of Ki67 and p65 and higher levels of PU.1 in wogonin-treated tumor tissue than that of control tumor tissue (p <0.05). CONCLUSION Both LMP1 and miR-155 are new and reliable bio-markers for prognostic prediction of NHL, which shall be analyzed to evaluate the prognosis of patients with LMP1 (+) lymphoma as integral bio-markers. Wogonin could suppress tumor growth and induce cell apoptosis by inhibiting the expression of NF-κB through LMP1/NF-κB/miR-155/PU.1 pathway. Wogonin could be a potential targeted therapeutic agent for LMP1(+) NHL.
学术讨论
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斯坦福大学影像科Rao实验室 2017.01.13 美国加利福利亚州帕洛阿尔托 吴雪 Analysis of prognostic risk factors in patients with diffuse large B-cell lymphoma in China
斯坦福大学影像科Rao实验室 2017.03.25 美国加利福利亚州帕洛阿尔托 吴雪 How I treat CLL up front
东南大学血液科实验组 2017.10.11 江苏南京 吴雪 C-SNAT4探针在PET/CT中的应用
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学术会议
会议名称时间地点本人报告本人报告题目
第二届中德血液病论坛:血液系统恶性肿瘤的造血干细胞移植及细胞治疗 2015.05.04-06 德国海德堡 Effect of Splenectomy on Pancytopenia after Hematopoietic Stem Cell Transplantation in three CML patients----Experience from Our Center
国际白血病会议 2016.08.14 江苏南京 Wogonin as a targeted therapeutic agent for lymphoma involved in LMP1/NF-κB/miR-155/PU.1 pathway
     
代表作
论文名称
Wogonin as a targeted therapeutic agent for lymphoma involved in LMP1/NF-κB/miR-155/PU.1 pathway
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
陈苏宁 正高 教授,主任医师 博导 苏州大学第一附属医院
沈群 正高 教授,主任医师 博导 江苏省中医院
黄培林 正高 教授 博导 东南大学
葛峥 正高 教授,主任医师 博导 东南大学附属中大医院
王雪梅 正高 教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
张孝平 其他 讲师 东南大学附属中大医院