【摘要】： 目的：CD4+CD25+調(diào)節(jié)性T細(xì)胞(Regulatory T cells,Treg)占正常人體外周CD4+T淋巴細(xì)胞的5%-10%,參與體內(nèi)免疫抑制過程,具有免疫抑制和免疫無能性。在多種腫瘤患者外周血及腫瘤組織局部的數(shù)量增多,誘導(dǎo)維持外周免疫耐受。Treg可分為天然性Treg和獲得性Treg。在一定的環(huán)境條件下由于抗原提呈細(xì)胞(antigen-presenting cell,APC)表面共刺激分子表達(dá)的改變或細(xì)胞因子種類的變化,使CD4+T細(xì)胞分化為獲得性Treg。Treg通過降低對(duì)腫瘤相關(guān)抗原的初始T細(xì)胞免疫,導(dǎo)致腫瘤免疫逃逸,從而在腫瘤患者的免疫病理中發(fā)揮重要作用。有報(bào)道表明,減少Treg的數(shù)量在腫瘤生物治療中至關(guān)重要。減少Treg的數(shù)量就可以減少Treg介導(dǎo)的免疫抑制作用,從而增強(qiáng)T細(xì)胞的效能。另有研究證實(shí),未成熟樹突狀細(xì)胞可促使CD4+T細(xì)胞向獲得性Treg分化,從而促進(jìn)Treg的產(chǎn)生。目前腫瘤免疫治療策略的研究重點(diǎn)集中在打破免疫耐受和提高宿主T細(xì)胞反應(yīng)。銅綠假單胞菌注射液(Vaccine of Pseudomonas aeruginosa with Mannose Sensitive Hemagglutination pili,PA-MSHA)是從帶甘露糖敏感血凝菌毛的銅綠假單胞菌中制備而來,因其產(chǎn)生免疫調(diào)節(jié)作用及低毒無副作用而倍受青睞,目前已廣泛應(yīng)用于抗感染和抗炎治療,甚至作為免疫調(diào)節(jié)劑用于抗腫瘤治療。據(jù)報(bào)道,PA-MSHA作為白血病、惡性淋巴瘤、肺癌、乳腺癌、胃癌、肝癌等惡性腫瘤的輔助治療可提高臨床有效率,其抗腫瘤的可能機(jī)制在于通過增加樹突狀細(xì)胞的抗原遞呈能力,提高Th1/Th2的比例,調(diào)節(jié)細(xì)胞免疫和體液免疫的不平衡狀態(tài),達(dá)到有效的治療腫瘤的目的,但其具體作用機(jī)制尚不十分明確。急性髓細(xì)胞白血病(Acutemyeloid leukemia,AML)是造血干細(xì)胞異�？寺≡錾鷮�(dǎo)致的一種惡性腫瘤性疾病,伴有嚴(yán)重的免疫系統(tǒng)損傷。與正常人相比,AML患者外周血和骨髓血中均含有更高比例Treg。孫文平報(bào)道了PA-MSHA疫苗能明顯提高急性白血病患者的免疫功能,增強(qiáng)其自身的免疫殺傷能力。PA-MSHA疫苗治療組患者其IL-2水平、NK細(xì)胞活性、CD4+/CD8+比值均明顯高于對(duì)照組。但是PA-MSHA疫苗是否可以通過降低AML患者體內(nèi)的Treg細(xì)胞的數(shù)量和功能,從而發(fā)揮其抗腫瘤作用,還尚未有報(bào)道。本研究通過檢測(cè)經(jīng)銅綠假單胞菌注射液刺激的急性髓細(xì)胞白血病源性樹突狀細(xì)胞(dendritic cells derived from aute myeloid leukemia,AML-DCs)對(duì)Treg作用的影響,探討PA-MSHA對(duì)AML的免疫調(diào)節(jié)作用。 方法：用來源于初診未治急性髓細(xì)胞白血病患者的經(jīng)由外周血細(xì)胞單采術(shù)得到的富集白細(xì)胞層與PBS液1：1稀釋,采用淋巴細(xì)胞分離液進(jìn)行淋巴細(xì)胞分離,3h后棄懸浮細(xì)胞,加入重組人粒細(xì)胞巨噬細(xì)胞集落刺激因子(rhGM-CSF),重組人白細(xì)胞介素-4(rhIL-4)和RPMI1640完全培養(yǎng)基體外培養(yǎng)至第7天,分為三組：第一組為對(duì)照組,不給予任何處理；第二組加入PA-MSHA;第三組加入TNF-a。繼續(xù)培養(yǎng)24小時(shí)。每天取三組DC在倒置顯微鏡下觀察細(xì)胞形態(tài)。第8天用流式細(xì)胞儀檢測(cè)其細(xì)胞表型,并行混合淋巴細(xì)胞反應(yīng)檢測(cè)AML-DCs對(duì)淋巴細(xì)胞的增殖作用。將第8天的三組AML-DCs分別與MACS法分離得到的正常人外周血CD4+T細(xì)胞共培養(yǎng)7天,然后ELISA法分別測(cè)量三組上清液IL-10、TGF-β水平,流式細(xì)胞儀檢測(cè)CD4、CD25的表達(dá),RT-PCR測(cè)量Foxp3mRNA水平。 結(jié)果：PA-MSHA組和TNF-a組的DC呈現(xiàn)明顯的樹突狀形態(tài),這兩組DC的CD1a、CD80、CD83、CD86和HLA-DR表達(dá)較對(duì)照組明顯升高(P0.05),且這兩組DC誘導(dǎo)的T細(xì)胞增殖指數(shù)隨刺激細(xì)胞數(shù)量的增加而增加,并明顯高于對(duì)照組。此外,PA-MSHA組和TNF-a組DC誘導(dǎo)Treg產(chǎn)生后所測(cè)的IL-10、TGF-β水平,CD4、CD25的表達(dá)及Foxp3 mRNA水平均較對(duì)照組明顯降低(P0.05)。 結(jié)論：PA注射液可以促進(jìn)AML-DC的進(jìn)一步成熟,并抑制初始T細(xì)胞向Treg方向分化,從而增強(qiáng)對(duì)急性髓細(xì)胞白血病細(xì)胞的抗腫瘤活性。
[Abstract]:Objective: CD4 + CD25 + regulatory T cells (Treg) account for 5% - 10% of the normal human peripheral CD4 + T lymphocytes, participate in the process of immunosuppression in vivo, with immunosuppression and immune anergy. CD4 + T cells differentiate into acquired Treg. Treg induces tumor immune escape by reducing the initial T cell immunity to tumor-associated antigens under certain environmental conditions due to changes in the expression of costimulatory molecules on the surface of antigen-presenting cells (APC) or changes in the types of cytokines. It has been reported that reducing the number of Treg is essential in tumor biotherapy. Reducing the number of Treg can reduce Treg-mediated immunosuppressive effects and thus enhance T cell potency. Current studies on tumor immunotherapy strategies have focused on breaking immune tolerance and enhancing host T cell responses. Vaccine of Pseudomonas aeruginosa with Mannose Sensitive Hemagglutination pili (PA-MSHA) is a mannose-sensitive Pseudomonas aeruginosa strain. PA-MSHA has been widely used in anti-infection and anti-inflammation therapy, even as an immunomodulator in anti-tumor therapy. It is reported that PA-MSHA as an adjuvant therapy for leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and other malignant tumors can be improved. The possible mechanism of its anti-tumor effect is to increase the antigen presenting ability of dendritic cells, increase the ratio of Th1/Th2, regulate the imbalance of cellular immunity and humoral immunity, so as to achieve the goal of effective treatment of tumor, but the specific mechanism is not very clear. AML is a malignant disease caused by abnormal clonal proliferation of hematopoietic stem cells, accompanied by severe immune system damage. Compared with normal people, the peripheral blood and bone marrow blood of AML patients contain a higher proportion of Treg. Sun Wenping reported that PA-MSHA vaccine can significantly improve the immune function of patients with acute leukemia and enhance their own immunity. The levels of IL-2, NK cell activity and CD4 +/CD8 + ratio in PA-MSHA vaccine group were significantly higher than those in control group. However, whether PA-MSHA vaccine can exert its anti-tumor effect by reducing the number and function of Treg cells in AML patients has not been reported. The effect of dendritic cells derived from aute myeloid leukemia (AML-DCs) on Treg was studied to investigate the immunomodulatory effect of PA-MSHA on AML.
Methods: The enriched leukocyte layer obtained from untreated patients with acute myeloid leukemia was diluted with PBS solution 1:1 by peripheral blood mono-collection. Lymphocyte isolation was carried out with lymphocyte isolation solution. The suspension cells were discarded after 3 hours and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was added to recombinant human leukocyte fine. Interleukin-4 (rhIL-4) and RPMI 1640 were cultured in vitro for 7 days and divided into three groups: the first group was the control group without any treatment; the second group was added PA-MSHA; the third group was added TNF-a. Mixed lymphocyte reaction was used to detect the proliferation of AML-DCs. On the 8th day, three groups of AML-DCs were co-cultured with normal human peripheral blood CD4+T cells separated by MACS for 7 days, then the levels of IL-10 and TGF-beta in the supernatants of three groups were measured by ELISA, the expressions of CD4 and CD25 were detected by flow cytometry, and Foxp3 mRNA water was measured by RT-PCR. Flat.
Results: DCs in PA-MSHA group and TNF-a group showed obvious dendritic morphology. The expressions of CD1a, CD80, CD83, CD86 and HLA-DR in the two groups were significantly higher than those in the control group (P The levels of IL-10, TGF-beta, CD4, CD25 and Foxp3 mRNA were significantly lower than those in the control group (P 0.05).
CONCLUSION: PA injection can promote the maturation of AML-DC and inhibit the differentiation of initial T cells toward Treg, thus enhancing the antitumor activity of AML-DC cells.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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