Hedgehog与STAT3信号通路在T细胞淋巴瘤中的作用及其机制研究

发布时间：2018-06-30 04:29

  本文选题：T细胞淋巴瘤 + GL11　； 参考：《山东大学》2016年博士论文

【摘要】：T细胞淋巴瘤是一组具有高度侵袭性及异质性的淋巴系统恶性肿瘤,各亚型在病理特征、临床表现、生物学行为等方面具有明显异质性,可累及淋巴结、肝、脾、骨髓、皮肤等,临床分期多较晚,有时还伴有多种临床综合征(如嗜血综合征),预后差。该病发病率具有明显的地域性和种族特征,在欧美占NHL的10%-15%,在我国比例相对高,约占NHL的25%一35%。相比B细胞淋巴瘤,T细胞淋巴瘤发病率相对较低,缺乏特异性分子遗传学特征病理诊断复杂、缺乏多中心随机临床试验导致相关研究进展比较缓慢,目前已成为淋巴瘤治疗中最具挑战性的研究领域之一。目前尚无有效的标准一线治疗方案,在既往的治疗中CHOP或CHOP样的化疗仍是患者最常用的方案,但相比弥漫大B细胞淋巴瘤(DLBCL),T细胞淋巴瘤缓解率低,缓解持续时间短。虽然增强的联合化疗及干细胞移植在一定程度上可提高疗效,但难治复发仍是突出难题,5年生存率仅30%左右,亟待新的更有效的治疗策略。T细胞淋巴瘤病因和发病机制还不完全明确,一般认为是多种因素相互作用的结果,如感染因素(包括EB病毒、人类T淋巴细胞病毒等)、理化刺激、免疫缺陷、遗传变异等。众所周知,正常的信号传导是人体各项生命活动的基础。许多信号传导通路失调参与细胞恶性转化、肿瘤血管生成、侵袭、迁移等重要过程。国内外研究发现T细胞淋巴瘤中许多信号传导通路(如Notch、Wnt、PI3K/AKT、 P53等)调节异常,并且某些关键靶标或小分子标志物与淋巴瘤的复发、耐药等密切相关。研究信号传导通路在T细胞淋巴瘤中的调控作用,将为探寻新的治疗靶点及改善疾病预后提供依据。Hedgehog(Hh)信号通路是一个高度保守的细胞间信号传导通路,该信号通路的异常活化已见于许多实体肿瘤及血液肿瘤,尤其是该通路上关键转录因子GLI1(glioma-associated oncogene-1)在多种肿瘤中显著上调,表明Hh通路在肿瘤发生中起重要调控作用。STAT3 (Signal transducer and activator of transcription 3)可将上游细胞因子及生长因子受体信号传递到胞内,调节靶基因转录,STAT3持续激活可进而参与肿瘤形成。越来越多的研究表明调节异常的Hedgehog与STAT3信号通路可能通过促进细胞增殖、抑制细胞凋亡、诱发肿瘤血管形成及放化疗抵抗等促进肿瘤形成与生存,目前已成为国内外研究的热点领域。然而上述两大信号传导通路在T细胞淋巴瘤中的作用机制尚不明确亟待研究。本研究探讨了GANT61 (Hh/GLI1抑制剂)及WP1066 (STAT3抑制剂)在T细胞淋巴瘤中的抑制作用及潜在的作用机制,将为该疾病的诊断、预后评估及靶向治疗等提供新的思路与实验依据。第一部分：GANT61在T细胞淋巴瘤细胞中的抗肿瘤作用及其机制研究目的：T细胞淋巴瘤是一类侵袭性高、预后差的淋巴系统恶性肿瘤。由于确切病因不明,目前尚无统一有效的治疗策略。Hh信号通路的异常活化及其与多种癌通路的相互作用已在许多肿瘤中报道。此外研究发现信号传导与转录激活因子3(STAT3)和细胞因子信号转导抑制分子3(SOCS3)表达异常多种实体瘤及血液肿瘤的发病相关,Hh信号通路的促肿瘤作用是否可通过STAT3信号通路介导亦不明确。有研究表明抑制Hh信号通路活性对T细胞淋巴瘤有抑制作用,GLI1抑制剂GANT61是一种很有前景的Hh抑制剂。本研究旨在探讨GANT61对T细胞淋巴瘤细胞的抑制作用及其潜在的作用机制。材料与方法：1.病例标本收集；2.免疫组织化学染色；3.外周血T细胞的分离提取；4.细胞培养；5.慢病毒转染介导的RNAi靶向沉默淋巴瘤细胞株的GLI1基因；6.采用CCK-8法检测药物对细胞增殖的影响；7.采用AnnexinV/7-AAD双染法进行细胞调亡分析；8.细胞蛋白提取、蛋白印迹分析；9.免疫共沉淀；10.统计学分析。结果：1.通过对35例T细胞淋巴瘤患者的石蜡组织切片进行免疫组化染色(以反应性增生的淋巴结组织作为对照)发现：GLI1、p-STAT3、STAT3及SOCS3蛋白在淋巴瘤组织中表达的阳性率分别为28/35(80.0%),26/35(74.2%),32/35(91.4%)；24/35(68.60%)；且p-STAT3及SOCS3的蛋白表达与GLI1呈正相关(P0.05)。2.蛋白质印迹方法分析表明T细胞淋巴瘤细胞株(Jurkat,Karpass299及Myla3676)中GLI1,p-STAT3及SOCS3的蛋白表达显著高于健康志愿者的外周血T细胞,且免疫共沉淀证实上述淋巴瘤细胞内存在Gli1与p-STAT3的物理性结合。3.不同浓度的GANT61分别处理三种T细胞淋巴瘤细胞株48小时后通过CCK8方法检测细胞增殖活性,结果显示该药物对细胞增殖的抑制作用呈现药物浓度依赖性增强,IC50值分别为Jurkat 13.761±0.81μM;Karpass299 6.81±0.91μM; Myla3676 10.23±0.94μM。4.不同浓度的GANT61分别处理三种T细胞淋巴瘤细胞24小时后应用AnexinV-PE/7AAD流式试剂盒检测细胞凋亡率,结果表明三种细胞凋亡率呈药物浓度依赖性增加,同时蛋白印迹分析显示细胞中GLI1、p-STAT3及SOCS3蛋白随药物作用浓度增高而表达下降。5. GLI1-RNAi慢病毒和阴性对照慢病毒分别转染上述三种细胞,与未转染的细胞株相比,转染了GLI1-RNAi慢病毒的细胞GLI11、p-STAT3及SOCS3的蛋白表达明显下调,细胞增殖活性下降,细胞凋亡增加；而转染阴性对照病毒的细胞较未转染组无明显变化。结论：上述研究结果发现T细胞淋巴瘤组织及细胞株中GLI1、p-STAT3、STAT3及SOCS3蛋白表达是显著上调的。应用抑制剂或者RNA干扰方法抑制T淋巴瘤细胞株GLI1表达,可以抑制细胞增殖,诱导细胞凋亡,同时引起p-STAT3和SOCS3蛋白表达的下调。这些结果提示GANT61对T细胞淋巴瘤细胞株的抑制作用可能部分是通过下调p-STAT3和SOCS3的表达介导的。GANT61是很有前景的靶向抗肿瘤药物,Hh与STAT3信号通路的相互作用仍需进一步研究。目的：鼻型NK/T细胞淋巴瘤是一类侵袭性高、预后差的淋巴系统恶性肿瘤,我国发病率远高于西方国家。虽然三分之二病例发病时仅局限于局部病灶,但由于该疾病病因不明、侵袭性高、病程进展快伴放化疗抵抗等原因,目前尚缺乏有效治愈方案,预后差。大量研究表明STAT3信号通路过度激活与许多肿瘤的发生及进展有关。WP1066是一种小分子STAT3抑制剂,可抑制STAT3信号通路的激活,在肾细胞癌、恶性胶质瘤等多种肿瘤细胞的体外实验及动物模型中已经表现出显著的抗肿瘤作用。本研究旨在评估WP1066对鼻型NK/T细胞淋巴瘤细胞的抑制作用,并进一步揭示潜在相关的分子生物学机制。材料与方法：1.病例标本收集；2.免疫组化；3.细胞培养；4.免疫荧光；5.采用CCK-8法检测药物对细胞增殖的影响；6.采用AnnexinV/7-AAD双染法进行细胞调亡分析；7.蛋白提取和western-blot分析；8.RNA提取、逆转录和实时定量PCR；9.统计学分析。结果：1.通过对28例鼻型NK/T细胞淋巴瘤患者的石蜡组织切片进行免疫组化染色分析发现,有21例患者(75.0%)p-STAT3蛋白表达呈阳性;同时通过对免疫组化结果分析发现p-STAT3蛋白表达与Ki-67水平呈正相关(P0.05)。2.CCK-8方法及AnexinV-PE/7AAD流式双染法检测表明STAT3抑制剂WP1066对SNK6细胞具有显著的生长抑制和凋亡促进作用。3.蛋白质印迹及免疫荧光实验均表明STAT3抑制剂WP1066可以降低SNK6细胞中STAT3的磷酸化水平。4.不同浓度的WP1066处理SNK6细胞24小时后,蛋白印迹及RT-PCR分析显示WP1066可抑制SNK6细胞中c-Myc,cyclinD1,及Bcl-2的蛋白及mRNA的表达,且该抑制作用具有药物浓度依赖性。结论：本研究证实STAT3在鼻型NK/T细胞淋巴瘤组织及细胞株中存在组成性活化。STAT3抑制剂WP1066对鼻型NK/T细胞淋巴瘤细胞株SNK6具有抑制增殖、诱导凋亡的作用；其潜在的分子机制可能与WP1066抑制STAT3激活,下调c-Myc, cyclinD1及Bcl-2的mRNA及蛋白表达相关,这将为进一步提高鼻型NK/T细胞淋巴瘤的疗效及改善预后提供新的治疗策略。
[Abstract]:T cell lymphoma is a group of highly invasive and heterogeneous lymphoid malignant tumors. The subtypes have obvious heterogeneity in pathological features, clinical manifestations, biological behavior and so on. They can involve lymph nodes, liver, spleen, bone marrow, skin and so on. The clinical stages are late, and sometimes there are many clinical syndromes (such as bloodthirsty syndrome). The incidence of the disease has obvious regional and racial characteristics. The proportion of NHL 10%-15% in Europe and America is relatively high in our country, the 25% 1 35%. of NHL is compared to B cell lymphoma, the incidence of T cell lymphoma is relatively low. The lack of specific molecular genetic characteristics of the pathological diagnosis is complex, and the lack of multicenter randomized clinical trials leads to related research. Slow progress has become one of the most challenging areas of research in lymphoma treatment. There is no effective standard first-line therapy at present. CHOP or CHOP like chemotherapy is still the most commonly used regimen in previous treatment, but compared to diffuse large B cell lymphoma (DLBCL), T cell lymphoma has a low remission rate and duration of mitigation. Short. Although enhanced combined chemotherapy and stem cell transplantation can improve the curative effect to a certain extent, refractory recurrence is still a prominent problem, the 5 year survival rate is only about 30%. The cause and pathogenesis of.T cell lymphoma need new more effective treatment strategy. Elements (including EB virus, human T lymphocyte virus, etc.), physical and chemical stimulation, immunodeficiency, genetic variation and so on. It is well known that normal signal transduction is the basis of human life activities. Many signal transduction pathways are involved in cell malignant transformation, tumor angiogenesis, invasion, migration and other important processes. Domestic and foreign studies have found T cell lymph nodes Many of the signal transduction pathways (such as Notch, Wnt, PI3K/AKT, P53, etc.) regulate abnormality, and some key targets or small molecular markers are closely related to the recurrence and drug resistance of lymphoma. The study of the regulatory role of signal transduction pathway in T cell lymphoma will provide a basis for exploring new therapeutic targets and improving the prognosis of the disease (H). (H H) signal pathway is a highly conserved intercellular signal transduction pathway. The abnormal activation of the signal pathway is seen in many solid tumors and blood tumors, especially the key transcriptional factor GLI1 (glioma-associated oncogene-1) in this pathway is significantly up-regulated in a variety of tumors, indicating that the Hh pathway plays an important role in the regulation of.ST in the carcinogenesis. AT3 (Signal transducer and activator of transcription 3) can transmit the upstream cytokines and growth factor receptor signals into the cell, regulate the target gene transcription, and continue to activate STAT3 to participate in the formation of the tumor. More and more studies have shown that the regulation of abnormal Hedgehog and STAT3 signaling pathways may be inhibited by promoting cell proliferation and inhibition. Apoptosis, inducing tumor angiogenesis and radiochemotherapy resistance to promote tumor formation and survival have become a hot field of research at home and abroad. However, the mechanism of the two major signal transduction pathways in T cell lymphoma is still unclear. This study explored the GANT61 (Hh/GLI1 inhibitor) and WP1066 (STAT3 inhibitor). The inhibitory effect and potential mechanism of T cell lymphoma will provide new ideas and experimental basis for the diagnosis, prognosis evaluation and targeting therapy of the disease. Part 1: the anti-tumor effect and mechanism of GANT61 in T cell lymphoma cells: T cell lymphoma is an aggressive, poor prognosis type. There is no unified and effective therapeutic strategy for the abnormal activation of.Hh signaling pathway and its interaction with a variety of cancer pathways in many tumors because of the exact unknown etiology. In addition, the expression of signal transduction and transcription activator 3 (STAT3) and cell factor signal transduction inhibitor 3 (SOCS3) expression are also found. Many kinds of solid tumors are associated with the incidence of hematological tumors, and whether the tumor promoting effect of Hh signaling pathway can be mediated by the STAT3 signaling pathway is also unclear. Studies have shown that inhibition of Hh signal pathway activity has a inhibitory effect on T cell lymphoma. GLI1 inhibitor GANT61 is a promising Hh inhibitor. This study aims to explore GANT61 to T fine. Inhibitory effects and potential mechanisms of cell lymphoma cells. Materials and methods: 1. cases were collected, 2. immunohistochemical staining, 3. peripheral blood T cells isolated and extracted; 4. cell culture; 5. lentivirus transfected RNAi targeted silent lymphoma cell line GLI1 basis; 6. using CCK-8 to detect drug against cells Effect of proliferation; 7. cell apoptosis was analyzed by AnnexinV/7-AAD double staining, 8. cell protein extraction, Western blot analysis, 9. immunoprecipitation, 10. statistical analysis. Results: 1. by immunohistochemical staining of paraffin section of 35 cases of T cell lymphoma (reactive proliferative lymph node tissue as control) It was found that the positive rates of GLI1, p-STAT3, STAT3 and SOCS3 protein expression in lymphoma tissues were 28/35 (80%), 26/35 (74.2%), 32/35 (91.4%) and 24/35 (68.60%), and the protein expression of p-STAT3 and SOCS3 was positively correlated with GLI1 (P0.05) The protein expression of GLI1, p-STAT3 and SOCS3 was significantly higher than that of T cells in the peripheral blood of the healthy volunteers, and the immunoprecipitation proved that there was a physical binding of Gli1 and p-STAT3 in the lymphoma cells in the above-mentioned lymphoma cells with the GANT61 of different concentrations of.3., respectively, to treat three kinds of T cell lymphoma cell lines after 48 hours to detect cell proliferation through CCK8 method. The results showed that the cell proliferation activity was detected by CCK8 method. The inhibitory effect of the drug on cell proliferation was enhanced by drug concentration, IC50 value was Jurkat 13.761 + 0.81 M, Karpass299 6.81 + 0.91 mu M, Myla3676 10.23 + 0.94 M.4. GANT61 treated three T cell lymphoma cells respectively for 24 hours, and the apoptosis rate was detected by AnexinV-PE/7AAD flow kit. The results showed that the three kinds of apoptosis rates were increased in drug concentration, and Western blot analysis showed that the expression of GLI1, p-STAT3 and SOCS3 protein in the cells decreased with the increase of drug action concentration, the expression of.5. GLI1-RNAi lentivirus and negative control lentivirus were transfected to the above three cells respectively. Compared with the untransfected cell lines, the transfection of GLI1-RNAi was slow. The protein expression of GLI11, p-STAT3 and SOCS3 decreased obviously, the cell proliferation activity decreased and the cell apoptosis increased, but the cells transfected with negative control virus had no obvious changes in the untransfected group. Conclusion: the results showed that the expression of GLI1, p-STAT3, STAT3 and SOCS3 protein in T cell lymphoma tissues and cell lines was significantly higher. Inhibition of T lymphoma cell line GLI1 expression by inhibitor or RNA interference method can inhibit cell proliferation, induce apoptosis, and induce down regulation of p-STAT3 and SOCS3 protein expression. These results suggest that the inhibitory effect of GANT61 on T cell lymphoma cell lines may be mediated by down regulation of p-STAT3 and SOCS3 expression. .GANT61 is a promising anticancer drug. The interaction of Hh and STAT3 signaling pathways still needs further study. Objective: nasal type NK/T cell lymphoma is a kind of aggressive and poor prognosis lymphatic malignant tumor. The incidence rate of our country is much higher than that in western countries. Although 2/3 cases are limited to local lesions, but they are only localized. Due to the unknown cause of the disease, high invasiveness, rapid progression of the disease and chemotherapy resistance, there is still a lack of effective cure and poor prognosis. A large number of studies have shown that the excessive activation of STAT3 signaling pathway is associated with the occurrence and progress of many tumors and.WP1066 is a small molecule STAT3 inhibitor, which can inhibit the activation of STAT3 signaling pathway and is fine in kidney. The aim of this study is to assess the inhibitory effect of WP1066 on nasal NK/T cell lymphoma cells and to further reveal the potential related molecular biological mechanisms. Materials and methods: 1. cases collection; 2. immunization. Histochemistry; 3. cell culture; 4. immunofluorescence; 5. CCK-8 method was used to detect the effect of drug on cell proliferation; 6. using AnnexinV/7-AAD double staining method for cell apoptosis analysis; 7. protein extraction and Western-blot analysis; 8.RNA extraction, reverse transcription and real-time quantitative PCR; 9. statistical analysis. Results 1. through 28 cases of nasal type NK/T cells drenched cells The immunohistochemical staining of paraffin tissue sections of the patients with boma found that the expression of p-STAT3 protein in 21 patients (75%) was positive, and the expression of p-STAT3 protein was positively correlated with the level of Ki-67 (P0.05).2.CCK-8 method and AnexinV-PE/7AAD flow double staining method to show that the STAT3 inhibitor WP1066 pair was found. SNK6 cells have significant growth inhibition and apoptosis promoting effect of.3. Western blot and immunofluorescence experiments all showed that STAT3 inhibitor WP1066 could reduce the phosphorylation level of STAT3 in SNK6 cells and.4. with different concentrations of WP1066 treated SNK6 cells for 24 hours. Western blot and RT-PCR analysis showed that WP1066 could inhibit the SNK6 cells. D1, and the expression of Bcl-2 protein and mRNA, and the inhibitory effect has a drug concentration dependence. Conclusion: This study confirmed the existence of STAT3 in nasal NK/T cell lymphoma tissues and cell lines, the existence of a constituent activated.STAT3 inhibitor WP1066, which inhibits proliferation and induces apoptosis in nasal NK/T cell lymphoma cell strain, and its potential to induce apoptosis. The molecular mechanism may be associated with WP1066 inhibition of STAT3 activation and down regulation of mRNA and protein expression of c-Myc, cyclinD1 and Bcl-2, which will provide a new therapeutic strategy for further improvement of the efficacy of nasal NK/T cell lymphoma and the improvement of prognosis.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.1
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