本文關(guān)鍵詞：吲哚胺2，3-雙加氧酶在非霍奇金淋巴瘤免疫耐受中的作用及機制探討　出處：《山東大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 非霍奇金淋巴瘤 免疫耐受 調(diào)節(jié)性T細(xì)胞 吲哚胺-2 3雙加氧酶

【摘要】：研究背景 非霍奇金淋巴瘤(NHL)是一組起源于淋巴結(jié)或其他淋巴組織的常見惡性腫瘤,病因及發(fā)病機制至今仍不甚清,免疫耐受可能在腫瘤發(fā)生、發(fā)展和轉(zhuǎn)移中起關(guān)鍵作用。以利妥昔單抗(抗CD20單抗)為代表的針對淋巴瘤的免疫靶向治療已取得良好療效。但仍存在一個重要問題就是無法克服腫瘤細(xì)胞形成的免疫耐受,機體免疫系統(tǒng)無法對腫瘤細(xì)胞進行有效的識別和殺傷。免疫耐受形成過程中涉及到的主要細(xì)胞包括：樹突狀細(xì)胞(dendritic cells, DCs)、調(diào)節(jié)性T細(xì)胞(regulatory T cells, Tregs)、肥大細(xì)胞、甚至是睹酸性粒細(xì)胞,腫瘤細(xì)胞亦可直接或間接通過樹突狀細(xì)胞等誘導(dǎo)腫瘤內(nèi)部免疫耐受微環(huán)境。因此,研究NHL腫瘤細(xì)胞如何直接或間接通過抗原呈遞細(xì)胞形成免疫耐受的腫瘤微環(huán)境,從而針對性地阻斷腫瘤免疫耐受形成的關(guān)鍵通路,將對NHL治療起到關(guān)鍵性的輔助作用。 作為近年來的研究熱點,色氨酸代謝在誘導(dǎo)免疫耐受形成中的作用不斷見諸報道。吲哚胺2,3-雙加氧酶(Indoleamine 2,3-dioxygenase, IDO)是色氨酸代謝中肝臟以外唯一可催化色氨酸分子中吲哚環(huán)氧化裂解,從而沿犬尿酸途徑分解代謝的限速酶；主要表達(dá)于一些免疫耐受或免疫特赦組織中,如胸腺、胃腸道粘膜、附睪、胎盤及眼前房等,并且特異性地表達(dá)在巨噬細(xì)胞和樹突狀細(xì)胞上。通過對小鼠和人DCs的大量研究發(fā)現(xiàn),高表達(dá)IDO的DCs亞群與機體的免疫耐受密切相關(guān),如參與誘導(dǎo)T細(xì)胞凋亡、T細(xì)胞失能及活化調(diào)節(jié)性T細(xì)胞。研究表明,IDO在腫瘤和腫瘤轉(zhuǎn)移淋巴結(jié)中高表達(dá),從而被證實參與了腫瘤的免疫耐受機制,并在其中發(fā)揮重要作用。以1-甲基色氨酸(1-MT)為代表的IDO抑制劑已在國外用于Ⅰ期臨床試驗,并取得了良好的療效。 研究者發(fā)現(xiàn),凡是IDO高表達(dá)的部位,如胎盤、腫瘤及轉(zhuǎn)移淋巴結(jié)中,都存在大量調(diào)節(jié)性T細(xì)胞浸潤的現(xiàn)象。調(diào)節(jié)性T細(xì)胞于1995年首次報道,其中轉(zhuǎn)錄因子FoxP3是Tregs的特異性標(biāo)志,是發(fā)育的關(guān)鍵調(diào)節(jié)基因。Tregs同樣在維持自身免疫耐受中起重要作用,其作用已在多種自身免疫、腫瘤模型中得到證實,而且最新研究顯示Tregs的增殖及活化可能與IDO+DC亞群密切相關(guān)。已有研究證實：在多種腫瘤、腫瘤細(xì)胞系、急性白血病及成人T細(xì)胞白血病／淋巴瘤中存在著IDO的過表達(dá),并通過對小鼠淋巴瘤模型的研究證實了IDO的過表達(dá)與Tregs增殖存在相關(guān)性。然而IDO上調(diào)Tregs的具體機制尚待進一步的研究。 本課題旨在研究IDO在NHL患者組織水平上的表達(dá)情況,探討其與Tregs的相關(guān)性及其在NHL發(fā)生、發(fā)展和預(yù)后中的作用。進而通過體外實驗和建立NHL動物模型,驗證IDO在NHL腫瘤誘導(dǎo)的免疫耐受中的作用,并對其作用機制進行細(xì)致的研究和探討。為臨床發(fā)掘新的判斷NHL預(yù)后的指標(biāo),并為IDO抑制劑1-MT的臨床應(yīng)用提供有力的理論支持。 第Ⅰ部分IDO和FoxP3在人非霍奇金淋巴瘤中的表達(dá)及意義 研究目的 以人NHL標(biāo)本為研究對象,探討IDO在NHL中的表達(dá)情況,分析其與臨床特征以及與Tregs的相關(guān)性 研究方法 1.收集NHL空白石蠟切片(n=57),以反應(yīng)性增生(n=18)為對照,通過免疫組織化學(xué)技術(shù)研究IDO和FoxP3在組織中的表達(dá)及部位。 2.收集NHL新鮮標(biāo)本(n=20),以實時定量RT-PCR技術(shù)比較IDO和FoxP3在mRNA水平的表達(dá)情況。 3.收集NHL新鮮標(biāo)本(n=20),利用western blot技術(shù)比較IDO和FoxP3在蛋白水平的表達(dá)情況。 4.統(tǒng)計學(xué)分析IDO在NHL和反應(yīng)性增生中的表達(dá)差異及其與FoxP3的相關(guān)性。探討IDO與非霍奇金淋巴瘤臨床病例資料如年齡、性別、臨床分期、病理類型、LDH及預(yù)后等因素之間的關(guān)系。 研究結(jié)果 1.免疫組化結(jié)果顯示,對照組IDO陽性細(xì)胞率均在30%以下,而57例NHL石蠟切片中,IDO+NHL(陽性細(xì)胞率在30%以上者)為18例(31.58%)。18例IDO+NHL石蠟切片的FoxP3陽性率顯著高于對照組及39例IDO-NHL切片。IDO-NHL切片的FoxP3陽性率與對照組無顯著差異。結(jié)合NHL患者臨床病例資料,18例IDO+NHL患者的臨床分期多為Ⅲ或Ⅳ期,而39例IDO-NHL患者臨床分期相對較早,兩者差異有統(tǒng)計學(xué)意義。IDO+NHL患者LDH值以及腫瘤直徑同樣顯著大于IDO-NHL患者。以上數(shù)據(jù)均提示IDO與NHL預(yù)后密切相關(guān)。 2.20例新鮮NHL標(biāo)本經(jīng)實時定量RT-PCR測定,IDOmRNA表達(dá)量顯著高于對照組(2-dCt：0.00582～0.546 v.0～0.0103,P0.05)。對于NHL組內(nèi)進行IDO與FoxP3mRNA表達(dá)的相關(guān)性分析,兩者呈正相關(guān)(r=0.447)。 3.20例新鮮NHL標(biāo)本經(jīng)WB測定,IDO蛋白表達(dá)量顯著高于對照組(0.77±0.25 vs.0.18±0.18,P0.05)。NHL組內(nèi),IDO與FoxP3在蛋白水平上的表達(dá)呈正相關(guān)(r=0.613)。 結(jié)論 1.部分NHL組織內(nèi)存在IDOmRNA和蛋白高表達(dá)。 2.IDO的高表達(dá)與NHL臨床分期、LDH值等密切相關(guān),提示IDO可能指示了NHL患者預(yù)后不良。 3.IDO的表達(dá)與FoxP3呈正相關(guān),推測IDO+NHL腫瘤細(xì)胞可能與Tregs存在功能上的密切聯(lián)系。 第Ⅱ部分高表達(dá)IDO的淋巴瘤細(xì)胞株對Tregs調(diào)控作用的研究 研究目的 以高表達(dá)IDO的小鼠B系淋巴瘤細(xì)胞株A20及小鼠T細(xì)胞為研究對象,體外混合細(xì)胞培養(yǎng),證實高表達(dá)IDO的淋巴瘤細(xì)胞株可上調(diào)Tregs的比例。 研究方法 1.免疫磁珠技術(shù)(MACS)分離BALB/c小鼠CD4+CD25- T細(xì)胞,并通過流式細(xì)胞術(shù)驗證其純度。 2.選取高表達(dá)IDO的A20細(xì)胞系(源于BALB/c小鼠),將IDO+A20細(xì)胞系與BALB/c小鼠CD4+CD25-T細(xì)胞分為以下三組共同培養(yǎng)：①CD4+CD25- T細(xì)胞組；②CD4+CD25- T細(xì)胞+A20細(xì)胞組；③CD4+CD25- T細(xì)胞+A20細(xì)胞+1-甲基色氨酸組。 3.培養(yǎng)24小時后,流式細(xì)胞術(shù)測定CD4+CD25+T細(xì)胞的比例。觀察IDO+腫瘤細(xì)胞以及IDO抑制劑1-MT對Tregs增殖和功能的影響。 4.MACS技術(shù)收集第②組中培養(yǎng)后的CD4+CD25+T細(xì)胞；MACS技術(shù)分離BALB/c小鼠脾臟中的DCs; Ficoll分離BALB/c小鼠脾臟中的單個核細(xì)胞(MNC)。分為以下4組進行混合淋巴細(xì)胞反應(yīng)：①CD4+CD25+Tc+DCs+MNC；②MNC+DCs;③MNC組；④CD4+CD25+Tc組。培養(yǎng)72h后,MTT法測定其在570nm吸光度值(A值),計算CD4+CD25+T細(xì)胞的抑制率(supressive rate, SR)。SR=[1-(A1-A3-A4)/(A2-A3)]×100% 研究結(jié)果 1.流式細(xì)胞術(shù)驗證MACS技術(shù)分離得到的CD4+CD25- T細(xì)胞純度在98%以上。 2.混合細(xì)胞培養(yǎng)24小時后,流式細(xì)胞術(shù)測定CD4+CD25+ T細(xì)胞比例：第①組為0；第②組為7.87±1.65%；第③組為3.32±1.19%,證實高表達(dá)IDO的A20細(xì)胞系可誘導(dǎo)CD4+CD25-T細(xì)胞向CD4+CD25+表型轉(zhuǎn)化,而且1-MT可明顯抑制該過程。 3.MTT法分析測定轉(zhuǎn)化后CD4+CD25+T細(xì)胞的抑制功能：陽性對照組A值明顯高于陰性對照組(0.8657±0.0353 vs 0.3434±0.0319,P0.05),說明T細(xì)胞在DCs的作用下增殖、活化；而實驗組A值明顯小于陽性對照組(0.4331±0.0280 vs0.8657±0.0353,P0.05),證實轉(zhuǎn)化后的Tregs具有抑制T細(xì)胞增殖的作用(抑制率為82.54±7.31%)。 結(jié)論 1.A20細(xì)胞系在體外可穩(wěn)定高表達(dá)IDO。 2.免疫磁珠技術(shù)是一種簡單有效的細(xì)胞分離手段,可被廣泛應(yīng)用于臨床和基礎(chǔ)研究中。 3.IDO+腫瘤細(xì)胞可在體外將CD4+CD25-T細(xì)胞向CD4+CD25+ T細(xì)胞轉(zhuǎn)化,轉(zhuǎn)化后的CD4+CD25-T細(xì)胞具有免疫抑制功能。 4.IDO抑制劑1-MT可逆轉(zhuǎn)體外IDO+腫瘤細(xì)胞將CD4+CD25-T細(xì)胞轉(zhuǎn)化為CD4+CD25+T細(xì)胞的過程,說明這一轉(zhuǎn)化具有IDO依賴性。 第Ⅲ部分動物模型的建立及IDO對Tregs調(diào)控作用的探討 研究目的 通過NHL動物模型,研究IDO+NHL小鼠Tregs在腫瘤、引流淋巴結(jié)及脾臟中的含量以及使用IDO抑制劑l-MT對Tregs的影響 研究方法 1.建立NHL動物模型：選6-8周齡左右BALB/c小鼠,皮下注射A20細(xì)胞懸液(5×106/只)。 2.1周后選取腫瘤大小相近的小鼠入組實驗,以同周齡的正常BALB/c小鼠為對照,分為以下三組：正常對照組(n=6),腫瘤組(n=6),1-MT干預(yù)組(n=6)。 3.對于1-MT干預(yù)組,每日瘤內(nèi)注射1-MT溶液,正常組和腫瘤組注射同體積PBS。 3.2周后處死小鼠,收集腫瘤、脾臟、淋巴結(jié),通過流式細(xì)胞術(shù)檢測各組Tregs在腫瘤組織、淋巴結(jié)及脾臟中的含量；通過Western Blot檢測IDO和FoxP3在組織中的表達(dá)。收集數(shù)據(jù)進行統(tǒng)計學(xué)分析,比較各組差異。 研究結(jié)果 1.A20 B細(xì)胞淋巴瘤模型成瘤率為95%,成瘤時間為7天左右。 2.流式細(xì)胞術(shù)檢測結(jié)果 就CD+CD25+T細(xì)胞在腫瘤組織、脾臟、淋巴結(jié)中占淋巴細(xì)胞的比例而言,腫瘤組均明顯高于1-MT干預(yù)組,也明顯高于正常對照組。 CD4+T細(xì)胞在腫瘤組織、脾臟內(nèi)占淋巴細(xì)胞的比例,腫瘤組、1-MT組和對照組之間無差異。在腫瘤引流淋巴結(jié)和對照淋巴結(jié)內(nèi),CD4+ T細(xì)胞占淋巴細(xì)胞的比例腫瘤組明顯低于正常對照組,但與1-MT干預(yù)組之間無明顯差異,1-MT干預(yù)組同樣明顯低于正常對照組。 腫瘤組織內(nèi),CD4+CD25+T細(xì)胞/CD4+ T細(xì)胞腫瘤組明顯高于1-MT干預(yù)組。脾臟內(nèi),CD4+CD25+T細(xì)胞/CD4+T細(xì)胞在腫瘤組中的比例為明顯高于正常組,也明顯高于1-MT組。正常對照組和1-MT組之間差異無統(tǒng)計學(xué)意義。腫瘤引流淋巴結(jié)和對照淋巴結(jié)內(nèi),CD4+CD25+T細(xì)胞/CD4+T細(xì)胞在腫瘤組中的比例明顯高于正常組(P0.001),也明顯高于1-MT組。1-MT組仍高于正常對照組。 3. Western Blot 結(jié)果顯示,IDO在腫瘤組和1-MT組腫瘤、脾臟、淋巴結(jié)中表達(dá)均明顯高于對照組。 FoxP3在腫瘤組腫瘤、脾臟和淋巴結(jié)中的表達(dá)明顯高于常對照組,也明顯高于1-MT組。l-MT組與正常對照組之間無明顯差異。 結(jié)論 1.A20 B細(xì)胞淋巴瘤是一種良好的NHL動物模型。 2.IDO在動物模型的腫瘤、脾臟、腫瘤引流淋巴結(jié)內(nèi)高表達(dá),通過上調(diào)Tregs誘導(dǎo)免疫耐受。 3.1-MT通過減弱動物模型中IDO對Tregs的上調(diào)作用,從而對淋巴瘤的化學(xué)和免疫治療提供有效的協(xié)同作用,在抗腫瘤治療中極具應(yīng)用前景。
[Abstract]:Research background
Non Hodgkin's lymphoma (NHL) is a common malignant tumor originated from lymph node or other lymphoid tissues, the etiology and pathogenesis of the disease is still not very clear, immune tolerance may play a key role in tumorigenesis, development and metastasis. With rituximab (anti CD20 antibody) as the representative of the target for immune lymphoma the treatment has achieved good curative effect. But there is still an important problem is to overcome the formation of tumor cell immune tolerance, immune system is unable to effectively recognize tumor cells and kill. The formation of immune tolerance cells mainly involved in the process include: dendritic cells (dendritic cells, DCs), regulatory T cells (regulatory T cells, Tregs), mast cells, and even see the eosinophil, tumor cells can directly or indirectly through dendritic cells induced tumor immune tolerance by micro environment. Therefore, research To investigate how NHL tumor cells directly or indirectly form immune tolerance tumor microenvironment through antigen presenting cells, so as to block the key pathway of tumor immune tolerance, it will play a key role in NHL treatment.
As a research hotspot in recent years, the role of tryptophan metabolism in inducing immune tolerance in constantly reported. Indoleamine 2,3- dioxygenase (Indoleamine 2,3-dioxygenase IDO) is a liver tryptophan metabolism only can catalyze tryptophan indole ring oxidation cracking, thus limiting enzyme along the kynurenine catabolism pathway; mainly expressed in some immune tolerance or immune amnesty, such as thymus, gastrointestinal mucosa, epididymis, placenta and anterior chamber, and is specifically expressed in macrophages and dendritic cells. Through a lot of research on mouse and human DCs found that the high expression of IDO and DCs subsets the immune tolerance is closely related, such as participation in inducing T cell apoptosis, T cell anergy and activation of regulatory T cells. The results show that IDO high expression in tumor and tumor metastasis in lymph nodes, which proved the argument and tumor free Immune tolerance mechanism, and play an important role in it. The 1- methyltryptophan (1-MT) as the representative of the IDO inhibitor has been used in phase I clinical trials in foreign countries, and achieved good results.
The researchers found that high expression of all IDO parts, such as the placenta, tumors and lymph node metastases, there are a lot of T cell infiltration phenomenon. Regulation of regulatory T cells was first reported in 1995, the transcription factor FoxP3 is a specific marker of Tregs, is the development of key regulatory gene.Tregs plays an important role in the same maintaining self tolerance, its role has been confirmed in a variety of immune, tumor models, and the latest research shows that Tregs may be closely related to the proliferation and activation of IDO+DC subsets. Studies have confirmed: in a variety of tumors, tumor cell lines, acute leukemia and adult T cell leukemia / lymphoma there too the expression of IDO, and through the research of the murine lymphoma model confirmed the correlation between IDO expression and Tregs proliferation. However the specific mechanism of IDO regulation of Tregs remains to be further studied.
The purpose of this study is to investigate the expression of IDO in patients with NHL in the tissues, and explore the correlation between Tregs and NHL in the occurrence, development and prognosis effect. Then through experiments in vitro and animal model of NHL, IDO in NHL verification of tumor induced immune tolerance, and the mechanism of detailed study for the clinical prognosis of NHL. To explore new targets, and provide strong theoretical support for the clinical application of IDO inhibitor 1-MT.
Expression and significance of part I IDO and FoxP3 in human non Hodgkin's lymphoma
research objective
Taking human NHL as a study object, the expression of IDO in NHL was discussed, and the correlation with the clinical features and the correlation with Tregs was analyzed.
research method
1. the NHL blank paraffin section (n=57) was collected and the reactive hyperplasia (n=18) was used as the control. The expression and location of IDO and FoxP3 in the tissues were studied by immunohistochemistry.
2. NHL fresh specimens (n=20) were collected, and the expression of IDO and FoxP3 at mRNA level was compared by real-time quantitative RT-PCR technique.
3. the fresh specimens of NHL (n=20) were collected, and the expression of IDO and FoxP3 at protein level was compared by Western blot technique.
4. statistical analysis of IDO expression difference in NHL and reactive hyperplasia and its correlation with FoxP3. To explore the relationship between IDO and clinical data of non Hodgkin lymphoma, such as age, gender, clinical stage, pathological type, LDH and prognosis.
Research results
1. immunohistochemistry results showed that IDO positive cells in control group were below 30%, and 57 cases of NHL paraffin sections, IDO+NHL (positive rate in more than 30%) 18 cases (31.58%) the positive rate of FoxP3.18 cases of IDO+NHL paraffin section was significantly higher than the control group and 39 cases of.IDO-NHL positive IDO-NHL slice slice the rate of FoxP3 had no significant difference with control group. The clinical data of patients with NHL, 18 patients with clinical IDO+NHL staging for stage III or IV, and 39 cases of IDO-NHL patients with clinical stage relatively early, the difference was statistically significant in patients with.IDO+NHL LDH and the same diameter of the tumor was significantly higher than IDO-NHL patients. The above data indicate that IDO and NHL is closely related to the prognosis.
The expression of IDOmRNA in 2.20 fresh NHL specimens was significantly higher than that in the control group (2-dCt:0.00582 ~ 0.546 v.0 ~ 0.0103, P0.05). The expression of IDO and FoxP3mRNA in NHL group was positively correlated (r=0.447). The expression level of IDOmRNA was significantly higher in the NHL group than that in the control group.
The expression of IDO protein in 3.20 fresh NHL specimens was significantly higher than that in the control group (WB, 0.77 + 0.25 vs.0.18 + 0.18, P0.05). The expression of IDO and FoxP3 on protein level was positively correlated (r=0.613) in the.NHL group.
conclusion
There is a high expression of IDOmRNA and protein in the 1. part of NHL.
The high expression of 2.IDO is closely related to the clinical staging of NHL and the value of LDH, suggesting that IDO may indicate poor prognosis in patients with NHL.
The expression of 3.IDO is positively related to FoxP3, and it is suggested that the IDO+NHL tumor cells may be closely related to the function of Tregs.
Study on the regulation of Tregs in part II of the lymphoma cell line with high expression of IDO
research objective
The B lymphoma cell line A20 and mouse T cells with high expression of IDO were selected as the research objects. In vitro mixed cell culture, it was confirmed that the high expression of IDO lymphoma cell line could increase the proportion of Tregs.
research method
1. immunomagnetic bead technique (MACS) was used to isolate CD4+CD25- T cells from BALB/c mice and the purity of the cells was verified by flow cytometry.
The selection of 2. A20 high expression cell line IDO (from BALB/c mice), IDO+A20 cells and BALB/c mice CD4+CD25-T cells co culture into the following three groups: CD4+CD25- group, CD4+CD25- T T cells; +A20 cells; the CD4+CD25- T +A20 cell +1- methyltryptophan group.
After 24 hours of culture, the proportion of CD4+CD25+T cells was measured by flow cytometry. The effects of IDO+ tumor cells and IDO inhibitor 1-MT on the proliferation and function of Tregs were observed.
4.MACS collection of cultured CD4+CD25+T cells of the second group; separation of BALB/c mice spleen DCs MACS; mononuclear cells were isolated from mouse spleen BALB/c in Ficoll (MNC). Divided into the following 4 groups of mixed lymphocyte reaction: CD4+CD25+Tc+DCs+MNC; MNC+DCs; MNC group; CD4+CD25+Tc group. After 72h of culture determination of the absorbance at 570nm, MTT (A), calculate the inhibition rate of CD4+CD25+T cells (supressive, rate, SR).SR=[1- (A1-A3-A4) / (A2-A3)] x 100%
Research results
1. flow cytometry proved that the purity of CD4+CD25- T cells isolated by MACS technology was more than 98%.
2. mixed cell culture after 24 hours, determination of the proportion of T cells CD4+CD25+ by flow cytometry: the first group was 0; the second group is 7.87 + 1.65%; the third group is 3.32 + 1.19%, confirmed that the high expression of A20 in IDO cell line can be transformed to the CD4+CD25+ phenotype of CD4+CD25-T cells induced by 1-MT, and can inhibit the process of.
Determination of inhibitory function of CD4+CD25+T cells transformed by 3.MTT: positive control group A was significantly higher than the negative control group (0.8657 + 0.0353 vs 0.3434 + 0.0319, P0.05), the proliferation of T cells that, under the effect of DCs activation; and A value of experimental group was significantly less than the positive control group (0.4331 + 0.0280 vs0.8657 + 0.0353. P0.05), confirmed after the conversion of Tregs can inhibit the proliferation of T cells (the inhibition rate was 82.54 + 7.31%).
conclusion
1.A20 cell line is stable and high expression of IDO. in vitro
2. immunomagnetic bead technology is a simple and effective method of cell separation, which can be widely used in clinical and basic research.
The 3.IDO+ tumor cells can transform the CD4+CD25-T cells into CD4+CD25+ T cells in vitro, and the transformed CD4+CD25-T cells have the immunosuppressive function.
4.IDO inhibitor 1-MT can reverse the transformation of CD4+CD25-T cells into CD4+CD25+T cells by IDO+ tumor cells in vitro, indicating that this transformation is IDO dependent.
The establishment of Part III animal model and the study of the effect of IDO on the regulation of Tregs
research objective
The content of Tregs in the tumor, drainage lymph node and spleen of IDO+NHL mice and the effect of IDO inhibitor l-MT on Tregs were studied by NHL animal model.
research method
1. the animal model of NHL was established: 6-8 weeks old BALB/c mice were selected and A20 cell suspension was injected subcutaneously (5 x 106/).
After 2.1 weeks, the mice with similar tumor size were enrolled in the experiment. The normal BALB/c mice of the same age were divided into the following three groups: the normal control group (n=6), the tumor group (n=6) and the 1-MT intervention group (n=6).
3. for the 1-MT intervention group, 1-MT solution was injected daily in the tumor, and the normal group and the tumor group were injected with the same volume of PBS..
After 3.2 weeks, the mice were killed, and the tumor, spleen and lymph nodes were collected. The content of Tregs in tumor tissues, lymph nodes and spleen was detected by flow cytometry. The expression of IDO and FoxP3 in tissues was detected by Western Blot. Data were collected and analyzed statistically.
Research results
The tumor formation rate of 1.A20 B cell lymphoma model is 95%, and the time of tumor formation is about 7 days.
Results of 2. flow cytometry
In terms of the proportion of CD+CD25+T cells in tumor tissue, spleen and lymph nodes in the proportion of lymphocytes, the tumor group was significantly higher than that in the 1-MT intervention group, which was also significantly higher than that in the normal control group.
CD4+T cells in the tumor, accounting for the proportion of lymphocytes in spleen, tumor group, no difference between 1-MT group and control group. The tumor draining lymph node and control lymph nodes, CD4+ T cell percentage of lymphocytes in tumor group was significantly lower than that in normal control group, but no significant difference between the intervention group and 1-MT intervention group, 1-MT the same was significantly lower than the normal control group.
In tumor tissue, CD4+CD25+T cells of /CD4+ T cell tumor group was significantly higher than that of 1-MT group. In the spleen, the proportion of CD4+CD25+T cells and /CD4+T cells in tumor group was significantly higher than the normal group, were significantly higher than those in 1-MT group. The difference between normal control group and 1-MT group had no statistical significance. The tumor draining lymph node and control lymph nodes. The proportion of CD4+CD25+T cells, /CD4+T cells in the tumor group was significantly higher than that in normal group (P0.001), was significantly higher than 1-MT group.1-MT group was still higher than the normal control group.
3. Western Blot
The results showed that the expression of IDO in tumor, spleen and lymph nodes in tumor group and 1-MT group was significantly higher than that in control group.
The expression of FoxP3 in tumor group, spleen and lymph node was significantly higher than that of normal control group, but it was obviously higher than that of group 1-MT and normal control group, no significant difference was found between.L-MT group and normal control group.
conclusion
1.A20 B cell lymphoma is a good animal model of NHL.
2.IDO is highly expressed in the tumor, spleen, and tumor drainage lymph nodes of animal models, and induces immune tolerance by up regulation of Tregs.
3.1-MT, by reducing the up regulation effect of IDO on Tregs in animal models, provides an effective synergistic effect on the chemical and immunotherapy of lymphoma, and has great potential in anti-tumor therapy.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 黃燕萍,郭秀嬋,何祖根,趙健,曾毅;EB病毒誘導(dǎo)胸腺惡性T細(xì)胞淋巴瘤的研究[J];病毒學(xué)報;2001年04期

2 蔡大川,李用國,任紅,梁增偉,羅云萍;人臍血來源的樹突狀細(xì)胞的體外培養(yǎng)擴增[J];免疫學(xué)雜志;2002年S1期

3 張寧,脫朝偉,劉秋珍,吳澤全;人脾原發(fā)性惡性淋巴瘤裸小鼠皮下及原位移植模型的建立[J];消化外科;2002年03期

4 張寧;脫帥;劉秋珍;楊波;王明耀;朱希偉;;人肝惡性淋巴瘤裸小鼠原位和皮下移植模型的建立及生物學(xué)特性的研究[J];消化外科;2006年01期

5 邵曉楓,楊純正,熊冬生,許元富,彭暉,賴增祖,朱禎平;人B淋巴瘤裸鼠腹腔內(nèi)移植模型[J];中國腫瘤臨床;2001年03期

6 高艷芳;彭瑞清;伍小軍;丁婭;萬德森;張曉實;;吲哚胺2,3-雙加氧酶在結(jié)腸癌及其區(qū)域淋巴結(jié)中的表達(dá)與臨床意義[J];中國腫瘤臨床;2007年14期

7 林蘋,張潔,陸燕蓉,周宏遠(yuǎn),寧其志,黃孝忠;體外誘導(dǎo)成熟樹突狀細(xì)胞的研究[J];中國免疫學(xué)雜志;2001年03期

8 尹志華,劉騰飛,唐運蓮,王麗江,甘潤良;環(huán)孢素A對穩(wěn)定EBV誘發(fā)淋巴瘤模型的作用和意義[J];中國免疫學(xué)雜志;2002年11期

9 王云甫,孫圣剛,楊敬寧,曹學(xué)兵,吳雄文;大鼠樹突狀細(xì)胞的體外擴增與初步鑒定(英文)[J];中國現(xiàn)代醫(yī)學(xué)雜志;2005年01期

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