本文選題：人參皂苷Ro + 白血病��； 參考：《濟南大學》2017年碩士論文

【摘要】：白血病是一種特殊的血液系統(tǒng)惡性腫瘤。白血病患者體內(nèi)的白血病細胞增殖失去控制,從而在骨髓和其他造血組織中大量積累,抑制了正常造血功能。傳統(tǒng)的骨髓干細胞移植及化療存在供源受限、微小殘留病灶復發(fā)等問題。腫瘤生物治療是目前研究的熱點,其中誘導白血病細胞向免疫細胞定向分化呈現(xiàn)良好的應用前景。樹突狀細胞(DC)是體內(nèi)功能最強大的抗原提呈細胞(APC),是T淋巴細胞免疫應答的啟動者,在識別腫瘤抗原、活化T細胞殺傷腫瘤細胞中發(fā)揮著關鍵作用。然而白血病患者體內(nèi)DC數(shù)量明顯減少并且存在功能缺陷,這是特異性的腫瘤抗原不能有效激發(fā)機體特異性抗腫瘤細胞免疫應答的主要因素之一。DC在免疫應答中發(fā)揮重要的作用,已經(jīng)被用來作為治療腫瘤及免疫相關疾病,但DC在體內(nèi)含量少并且難分離純化限制了DC在臨床上的應用。因此,如何提高白血病患者DC數(shù)量及功能成為腫瘤領域研究的重點與難點。直接誘導白血病細胞分化為DC是潛在的有效方法,這種白血病來源的DC不僅具有了抗原遞呈細胞的特性,同時攜帶有白血病抗原,是腫瘤免疫治療的一個重要研究方向。目前體外誘導白血病源DC主要采用粒細胞巨噬細胞-集落刺激因子(GM-CSF)聯(lián)合白介素4(IL-4)的方法,但存在誘導及以體內(nèi)轉(zhuǎn)輸后效果不穩(wěn)定及價格昂貴的缺點。中藥治療腫瘤具有療效好、副作用小等優(yōu)點,成為目前白血病治療領域研究熱點。人參皂甙Ro是人參的主要有效單體成分之一,研究顯示其可以通過誘導腫瘤細胞凋亡、抑制腫瘤細胞浸潤和轉(zhuǎn)移、抑制血管形成等多個方面發(fā)揮抗腫瘤作用;但其是否具有誘導腫瘤細胞向DC分化的作用尚未見報道。JAK/STAT是調(diào)控DC分化及功能的關鍵信號通路,可啟動下游靶基因的轉(zhuǎn)錄與表達,調(diào)控DC的發(fā)育分化與功能,是目前DC發(fā)育分化領域研究的熱點。STAT5是STAT家族的重要成員,已證實STAT5通過活化DC特異性和轉(zhuǎn)錄因子Id2誘導DC的發(fā)育分化。但目前調(diào)節(jié)STAT信號通路調(diào)控白血病來源DC分化的關鍵機制尚不清楚,更缺乏藥物靶向研究。本實驗目的為探討人參皂苷Ro聯(lián)合細胞因子(GM-CSF與IL-4)促進白血病源樹突狀細胞(DC)分化的作用。篩選細胞因子誘導白血病源DC敏感細胞株;采用細胞因子聯(lián)合人參皂苷Ro誘導篩選的敏感細胞株人單核白血病細胞(THP-1)向DC分化。培養(yǎng)體系中分別加入小(5μmol/L)、中(10μmol/L)、大(20μmol/L)劑量人參皂苷Ro,流式細胞術檢測白血病來源DC表面標志CD1a、MHC-II、CD80、CD83、CD86及CCR7的表達;逆轉(zhuǎn)錄-聚合酶鏈式反應(RT-PCR)檢測白血病來源DC MHC-II、CD83、CD86及CCR7 mRNA轉(zhuǎn)錄水平;酶聯(lián)免疫吸附法(ELISA)檢測培養(yǎng)上清TNFa、IL-6蛋白水平。檢測結(jié)果顯示,THP-1是細胞因子誘導DC分化的敏感白血病細胞株,STAT5調(diào)控Id2在THP-1向DC分化中具有重要作用。與單純細胞因子誘導比較,人參皂苷Ro聯(lián)合細胞因子作用后白血病來源DC表面標志CD1a、MHC-II、CD80、CD83、CD86及CCR7的比例顯著升高(P0.05),MHC-II、CD83、CD86及CCR7的mRNA轉(zhuǎn)錄水平顯著升高(P0.05),培養(yǎng)上清TNFa、IL-6蛋白水平顯著升高(P0.05),提示人參皂苷Ro聯(lián)合細胞因子具有明顯促進白血病細胞向DC分化的作用;同時發(fā)現(xiàn)人參皂苷Ro明顯升高白血病來源DC STAT5、Id2的mRNA水平,并且呈劑量依賴性(P0.05),提示靶向調(diào)控STAT5信號通路可能是人參皂苷Ro誘導白血病細胞向DC的關鍵機制。以上結(jié)果提示,THP-1為GM-CSF與IL-4細胞因子誘導白血病源DC的敏感細胞株;人參皂苷Ro具有明顯促進白血病源DC誘導的作用,靶向調(diào)控STAT5信號通路可能是人參皂苷Ro誘導白血病細胞向DC的關鍵機制。
[Abstract]:Leukemia is a special malignant tumor of the blood system. The proliferation of leukemic cells in the leukemia patients is not controlled, thus accumulating in the bone marrow and other hematopoietic tissues and inhibiting the normal hematopoiesis. The traditional bone marrow stem cell transplantation and chemotherapy have the problem of limited source of donor and the recurrence of small residual focus. Therapy is a hot spot of current research, which induces a good prospect of leukemic cells to be directed to Immunocytic differentiation. Dendritic cells (DC) are the most powerful antigen presenting cells (APC) in the body. It is the promoter of the immune response of T lymphocytes. It plays a key role in identifying tumor antigenic and activating T cells to kill tumor cells. However, there is a significant decrease in the number of DC in the patients with leukemia and the existence of functional defects. This is one of the main factors that the specific tumor antigen can not effectively stimulate the immune response of the body..DC plays an important role in the immune response and has been used as a treatment for cancer and immune related diseases, but DC is contained in the body. Less and difficult separation and purification limit the clinical application of DC. Therefore, how to improve the number and function of DC in leukemia patients is the key and difficult point in the field of cancer research. Direct induction of leukemia cells to differentiate into DC is a potential effective method. The DC derived from this leukemia is not only characterized by antigen presenting cells, but also carried by the leukemia cells. Leukemic antigen is an important research direction in tumor immunotherapy. At present, the main method of inducing leukemia source DC in vitro is the method of granulocyte macrophage colony stimulating factor (GM-CSF) combined with interleukin 4 (IL-4), but there is a shortcoming of instability and high price after induction and transfer in vivo. The therapeutic effect of traditional Chinese medicine on tumor is effective. Good, small side effects and so on, it has become a hot spot in the field of leukemia treatment. Ginsenoside Ro is one of the main effective monomer components of ginseng. The study shows that it can induce tumor cell apoptosis, inhibit tumor cell infiltration and metastasis, inhibit vascular formation and other sides to play anti-tumor effect, but it can induce swelling. The role of tumor cell differentiation to DC has not yet been reported that.JAK/STAT is the key signaling pathway to regulate the differentiation and function of DC, which can activate the transcription and expression of the target genes of the downstream target, regulate the development and differentiation and function of the DC, which is a key member of the STAT family in the field of DC development and differentiation, and has confirmed that STAT5 is specific and turned through the activation of DC. The transcriptional factor Id2 induces the development and differentiation of DC. However, the key mechanism of regulating the differentiation of leukemia source DC by the STAT signaling pathway is still unclear, and it lacks the drug targeting study. The purpose of this experiment is to explore the role of ginsenoside Ro combined cytokine (GM-CSF and IL-4) in promoting the differentiation of leukemia derived dendritic cells (DC). DC sensitive cell line of the source of white blood disease; the human mononuclear leukemic cells (THP-1), which were induced by cytokines and ginsenoside Ro, were differentiated into DC. In the culture system, small (5 mu mol/L), medium (10 mu), and large (20 mu mol/L) dose ginsenoside Ro were used, and the flow cytometry was used to detect the DC surface marker CD1a, MHC-II, CD. 80, CD83, CD86 and CCR7 expression; reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transcriptional level of DC MHC-II, CD83, CD86 and CCR7 mRNA, and enzyme linked immunosorbent assay (ELISA) was used to detect and cultivate the supernatant TNFa and protein level. THP-1 has an important role in the differentiation of DC. Compared with simple cytokine induction, ginsenoside Ro combined with cytokines, the proportion of leukemia source DC surface markers CD1a, MHC-II, CD80, CD83, CD86 and CCR7 increased significantly (P0.05). Significantly increased (P0.05), suggesting that ginsenoside Ro combined with cytokines can significantly promote the differentiation of leukemic cells to DC, and that ginsenoside Ro significantly increases the mRNA level of leukemia source DC STAT5, Id2, and is dose-dependent (P0.05), suggesting that the target regulation of STAT5 signaling pathway may be the Ro induction of Ginsenoside induced leukemia. The key mechanism of cell to DC. The results suggest that THP-1 is a sensitive cell line of leukemia source DC induced by GM-CSF and IL-4 cytokine, and ginsenoside Ro can obviously promote the DC induction of leukemia source, and the target regulation of STAT5 signaling pathway may be the key mechanism of ginsenoside Ro induced leukemic cell to DC.

【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.7

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