發(fā)布時(shí)間：2018-06-30 04:29

  本文選題：T細(xì)胞淋巴瘤 + GL11��； 參考：《山東大學(xué)》2016年博士論文

【摘要】：T細(xì)胞淋巴瘤是一組具有高度侵襲性及異質(zhì)性的淋巴系統(tǒng)惡性腫瘤,各亞型在病理特征、臨床表現(xiàn)、生物學(xué)行為等方面具有明顯異質(zhì)性,可累及淋巴結(jié)、肝、脾、骨髓、皮膚等,臨床分期多較晚,有時(shí)還伴有多種臨床綜合征(如嗜血綜合征),預(yù)后差。該病發(fā)病率具有明顯的地域性和種族特征,在歐美占NHL的10%-15%,在我國比例相對(duì)高,約占NHL的25%一35%。相比B細(xì)胞淋巴瘤,T細(xì)胞淋巴瘤發(fā)病率相對(duì)較低,缺乏特異性分子遺傳學(xué)特征病理診斷復(fù)雜、缺乏多中心隨機(jī)臨床試驗(yàn)導(dǎo)致相關(guān)研究進(jìn)展比較緩慢,目前已成為淋巴瘤治療中最具挑戰(zhàn)性的研究領(lǐng)域之一。目前尚無有效的標(biāo)準(zhǔn)一線治療方案,在既往的治療中CHOP或CHOP樣的化療仍是患者最常用的方案,但相比彌漫大B細(xì)胞淋巴瘤(DLBCL),T細(xì)胞淋巴瘤緩解率低,緩解持續(xù)時(shí)間短。雖然增強(qiáng)的聯(lián)合化療及干細(xì)胞移植在一定程度上可提高療效,但難治復(fù)發(fā)仍是突出難題,5年生存率僅30%左右,亟待新的更有效的治療策略。T細(xì)胞淋巴瘤病因和發(fā)病機(jī)制還不完全明確,一般認(rèn)為是多種因素相互作用的結(jié)果,如感染因素(包括EB病毒、人類T淋巴細(xì)胞病毒等)、理化刺激、免疫缺陷、遺傳變異等。眾所周知,正常的信號(hào)傳導(dǎo)是人體各項(xiàng)生命活動(dòng)的基礎(chǔ)。許多信號(hào)傳導(dǎo)通路失調(diào)參與細(xì)胞惡性轉(zhuǎn)化、腫瘤血管生成、侵襲、遷移等重要過程。國內(nèi)外研究發(fā)現(xiàn)T細(xì)胞淋巴瘤中許多信號(hào)傳導(dǎo)通路(如Notch、Wnt、PI3K/AKT、 P53等)調(diào)節(jié)異常,并且某些關(guān)鍵靶標(biāo)或小分子標(biāo)志物與淋巴瘤的復(fù)發(fā)、耐藥等密切相關(guān)。研究信號(hào)傳導(dǎo)通路在T細(xì)胞淋巴瘤中的調(diào)控作用,將為探尋新的治療靶點(diǎn)及改善疾病預(yù)后提供依據(jù)。Hedgehog(Hh)信號(hào)通路是一個(gè)高度保守的細(xì)胞間信號(hào)傳導(dǎo)通路,該信號(hào)通路的異常活化已見于許多實(shí)體腫瘤及血液腫瘤,尤其是該通路上關(guān)鍵轉(zhuǎn)錄因子GLI1(glioma-associated oncogene-1)在多種腫瘤中顯著上調(diào),表明Hh通路在腫瘤發(fā)生中起重要調(diào)控作用。STAT3 (Signal transducer and activator of transcription 3)可將上游細(xì)胞因子及生長(zhǎng)因子受體信號(hào)傳遞到胞內(nèi),調(diào)節(jié)靶基因轉(zhuǎn)錄,STAT3持續(xù)激活可進(jìn)而參與腫瘤形成。越來越多的研究表明調(diào)節(jié)異常的Hedgehog與STAT3信號(hào)通路可能通過促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡、誘發(fā)腫瘤血管形成及放化療抵抗等促進(jìn)腫瘤形成與生存,目前已成為國內(nèi)外研究的熱點(diǎn)領(lǐng)域。然而上述兩大信號(hào)傳導(dǎo)通路在T細(xì)胞淋巴瘤中的作用機(jī)制尚不明確亟待研究。本研究探討了GANT61 (Hh/GLI1抑制劑)及WP1066 (STAT3抑制劑)在T細(xì)胞淋巴瘤中的抑制作用及潛在的作用機(jī)制,將為該疾病的診斷、預(yù)后評(píng)估及靶向治療等提供新的思路與實(shí)驗(yàn)依據(jù)。第一部分：GANT61在T細(xì)胞淋巴瘤細(xì)胞中的抗腫瘤作用及其機(jī)制研究目的：T細(xì)胞淋巴瘤是一類侵襲性高、預(yù)后差的淋巴系統(tǒng)惡性腫瘤。由于確切病因不明,目前尚無統(tǒng)一有效的治療策略。Hh信號(hào)通路的異常活化及其與多種癌通路的相互作用已在許多腫瘤中報(bào)道。此外研究發(fā)現(xiàn)信號(hào)傳導(dǎo)與轉(zhuǎn)錄激活因子3(STAT3)和細(xì)胞因子信號(hào)轉(zhuǎn)導(dǎo)抑制分子3(SOCS3)表達(dá)異常多種實(shí)體瘤及血液腫瘤的發(fā)病相關(guān),Hh信號(hào)通路的促腫瘤作用是否可通過STAT3信號(hào)通路介導(dǎo)亦不明確。有研究表明抑制Hh信號(hào)通路活性對(duì)T細(xì)胞淋巴瘤有抑制作用,GLI1抑制劑GANT61是一種很有前景的Hh抑制劑。本研究旨在探討GANT61對(duì)T細(xì)胞淋巴瘤細(xì)胞的抑制作用及其潛在的作用機(jī)制。材料與方法：1.病例標(biāo)本收集；2.免疫組織化學(xué)染色；3.外周血T細(xì)胞的分離提��；4.細(xì)胞培養(yǎng)；5.慢病毒轉(zhuǎn)染介導(dǎo)的RNAi靶向沉默淋巴瘤細(xì)胞株的GLI1基因；6.采用CCK-8法檢測(cè)藥物對(duì)細(xì)胞增殖的影響；7.采用AnnexinV/7-AAD雙染法進(jìn)行細(xì)胞調(diào)亡分析；8.細(xì)胞蛋白提取、蛋白印跡分析；9.免疫共沉淀；10.統(tǒng)計(jì)學(xué)分析。結(jié)果：1.通過對(duì)35例T細(xì)胞淋巴瘤患者的石蠟組織切片進(jìn)行免疫組化染色(以反應(yīng)性增生的淋巴結(jié)組織作為對(duì)照)發(fā)現(xiàn)：GLI1、p-STAT3、STAT3及SOCS3蛋白在淋巴瘤組織中表達(dá)的陽性率分別為28/35(80.0%),26/35(74.2%),32/35(91.4%)；24/35(68.60%)；且p-STAT3及SOCS3的蛋白表達(dá)與GLI1呈正相關(guān)(P0.05)。2.蛋白質(zhì)印跡方法分析表明T細(xì)胞淋巴瘤細(xì)胞株(Jurkat,Karpass299及Myla3676)中GLI1,p-STAT3及SOCS3的蛋白表達(dá)顯著高于健康志愿者的外周血T細(xì)胞,且免疫共沉淀證實(shí)上述淋巴瘤細(xì)胞內(nèi)存在Gli1與p-STAT3的物理性結(jié)合。3.不同濃度的GANT61分別處理三種T細(xì)胞淋巴瘤細(xì)胞株48小時(shí)后通過CCK8方法檢測(cè)細(xì)胞增殖活性,結(jié)果顯示該藥物對(duì)細(xì)胞增殖的抑制作用呈現(xiàn)藥物濃度依賴性增強(qiáng),IC50值分別為Jurkat 13.761±0.81μM;Karpass299 6.81±0.91μM; Myla3676 10.23±0.94μM。4.不同濃度的GANT61分別處理三種T細(xì)胞淋巴瘤細(xì)胞24小時(shí)后應(yīng)用AnexinV-PE/7AAD流式試劑盒檢測(cè)細(xì)胞凋亡率,結(jié)果表明三種細(xì)胞凋亡率呈藥物濃度依賴性增加,同時(shí)蛋白印跡分析顯示細(xì)胞中GLI1、p-STAT3及SOCS3蛋白隨藥物作用濃度增高而表達(dá)下降。5. GLI1-RNAi慢病毒和陰性對(duì)照慢病毒分別轉(zhuǎn)染上述三種細(xì)胞,與未轉(zhuǎn)染的細(xì)胞株相比,轉(zhuǎn)染了GLI1-RNAi慢病毒的細(xì)胞GLI11、p-STAT3及SOCS3的蛋白表達(dá)明顯下調(diào),細(xì)胞增殖活性下降,細(xì)胞凋亡增加；而轉(zhuǎn)染陰性對(duì)照病毒的細(xì)胞較未轉(zhuǎn)染組無明顯變化。結(jié)論：上述研究結(jié)果發(fā)現(xiàn)T細(xì)胞淋巴瘤組織及細(xì)胞株中GLI1、p-STAT3、STAT3及SOCS3蛋白表達(dá)是顯著上調(diào)的。應(yīng)用抑制劑或者RNA干擾方法抑制T淋巴瘤細(xì)胞株GLI1表達(dá),可以抑制細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡,同時(shí)引起p-STAT3和SOCS3蛋白表達(dá)的下調(diào)。這些結(jié)果提示GANT61對(duì)T細(xì)胞淋巴瘤細(xì)胞株的抑制作用可能部分是通過下調(diào)p-STAT3和SOCS3的表達(dá)介導(dǎo)的。GANT61是很有前景的靶向抗腫瘤藥物,Hh與STAT3信號(hào)通路的相互作用仍需進(jìn)一步研究。目的：鼻型NK/T細(xì)胞淋巴瘤是一類侵襲性高、預(yù)后差的淋巴系統(tǒng)惡性腫瘤,我國發(fā)病率遠(yuǎn)高于西方國家。雖然三分之二病例發(fā)病時(shí)僅局限于局部病灶,但由于該疾病病因不明、侵襲性高、病程進(jìn)展快伴放化療抵抗等原因,目前尚缺乏有效治愈方案,預(yù)后差。大量研究表明STAT3信號(hào)通路過度激活與許多腫瘤的發(fā)生及進(jìn)展有關(guān)。WP1066是一種小分子STAT3抑制劑,可抑制STAT3信號(hào)通路的激活,在腎細(xì)胞癌、惡性膠質(zhì)瘤等多種腫瘤細(xì)胞的體外實(shí)驗(yàn)及動(dòng)物模型中已經(jīng)表現(xiàn)出顯著的抗腫瘤作用。本研究旨在評(píng)估WP1066對(duì)鼻型NK/T細(xì)胞淋巴瘤細(xì)胞的抑制作用,并進(jìn)一步揭示潛在相關(guān)的分子生物學(xué)機(jī)制。材料與方法：1.病例標(biāo)本收集；2.免疫組化；3.細(xì)胞培養(yǎng)；4.免疫熒光；5.采用CCK-8法檢測(cè)藥物對(duì)細(xì)胞增殖的影響；6.采用AnnexinV/7-AAD雙染法進(jìn)行細(xì)胞調(diào)亡分析；7.蛋白提取和western-blot分析；8.RNA提取、逆轉(zhuǎn)錄和實(shí)時(shí)定量PCR；9.統(tǒng)計(jì)學(xué)分析。結(jié)果：1.通過對(duì)28例鼻型NK/T細(xì)胞淋巴瘤患者的石蠟組織切片進(jìn)行免疫組化染色分析發(fā)現(xiàn),有21例患者(75.0%)p-STAT3蛋白表達(dá)呈陽性;同時(shí)通過對(duì)免疫組化結(jié)果分析發(fā)現(xiàn)p-STAT3蛋白表達(dá)與Ki-67水平呈正相關(guān)(P0.05)。2.CCK-8方法及AnexinV-PE/7AAD流式雙染法檢測(cè)表明STAT3抑制劑WP1066對(duì)SNK6細(xì)胞具有顯著的生長(zhǎng)抑制和凋亡促進(jìn)作用。3.蛋白質(zhì)印跡及免疫熒光實(shí)驗(yàn)均表明STAT3抑制劑WP1066可以降低SNK6細(xì)胞中STAT3的磷酸化水平。4.不同濃度的WP1066處理SNK6細(xì)胞24小時(shí)后,蛋白印跡及RT-PCR分析顯示W(wǎng)P1066可抑制SNK6細(xì)胞中c-Myc,cyclinD1,及Bcl-2的蛋白及mRNA的表達(dá),且該抑制作用具有藥物濃度依賴性。結(jié)論：本研究證實(shí)STAT3在鼻型NK/T細(xì)胞淋巴瘤組織及細(xì)胞株中存在組成性活化。STAT3抑制劑WP1066對(duì)鼻型NK/T細(xì)胞淋巴瘤細(xì)胞株SNK6具有抑制增殖、誘導(dǎo)凋亡的作用；其潛在的分子機(jī)制可能與WP1066抑制STAT3激活,下調(diào)c-Myc, cyclinD1及Bcl-2的mRNA及蛋白表達(dá)相關(guān),這將為進(jìn)一步提高鼻型NK/T細(xì)胞淋巴瘤的療效及改善預(yù)后提供新的治療策略。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.1
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本文編號(hào)：2084891