青蒿琥酯对MDS来源的髓样抑制细胞增殖和凋亡的体外研究

发布时间：2018-07-09 20:12

本文选题：骨髓增生异常综合征 + 髓样抑制细胞　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:骨髓增生异常综合征(Myelodysplastic syndrome,MDS)是起源于造血干细胞的克隆性疾病,以一系或多系外周血细胞减少、骨髓无效和/或病态造血,并易向急性白血病(Acute leucemia,AL)转化为特点。MDS的骨髓微环境是MDS形成和发展的关键机制,肿瘤细胞通过利用和改变微环境来逃避免疫杀伤而优势于正常细胞生长。髓样抑制细胞(Myeloid-derived suppressor cells,MDSCs)是一群具有相似生物学功能的异质性细胞,包括骨髓祖细胞、未成熟髓系细胞(Immature myeloid cells,IMCs)、未成熟的巨噬细胞和未成熟的树突状细胞。MDSCs可通过调节固有免疫及适应性免疫应答促进肿瘤发生免疫逃逸,如抑制CD4+T细胞、CD8+T细胞和自然杀伤细胞(Natural kill cells,NK)等的活化及免疫应答。肿瘤微环境和MDSCs诱导的免疫耐受相互作用来促进MDS的发展。因MDS多见于老年人,所以不能承受强烈化疗及行异基因造血干细胞移植(Allogeneic hematopoietic stem cell transplantation,Allo-HSCT),尤其对于高危的MDS患者,极易向白血病转化,故预后极差。对于不适用行Allo-HSCT的患者,仍以药物治疗为主。MDS的药物治疗包括去甲基化药物、组蛋白去乙酰化酶抑制剂、铁螯合剂及抗血管增殖药物等。尽管这些药物已经应用于临床治疗MDS,但这些药物都没有获得持久疗效。为中高危患者寻找低毒、有效、经济的药物治疗是十分需要解决的临床问题。青蒿琥酯(Artesunate,ART)是一种从中药青蒿中提取出来的青蒿素的半合成衍生物,是治疗恶性疟疾最有效的低毒的药物之一,研究发现其除具有抗疟作用外,在体外研究中还发现其具有抗肿瘤作用,ART可诱导MDS细胞系SKM-1细胞凋亡,并且ART与传统化疗药物的杀伤机制不同,可能与其他MDS治疗药物存在协同作用。并且ART在治疗肿瘤方面具有显著的优势,如价格低廉,高效低毒、肿瘤靶向性等。本实验通过研究MDS患者外周血MDSCs、CD34+细胞、NK细胞数目以及与MDS疾病恶性程度之间的关系。通过体外细胞培养实验,检测ART对MDS来源的MDSCs、NK细胞、CD34+细胞的各细胞群的变化,对进一步研究MDS的发病机制,ART抗MDS机制有十分重要的临床意义,对寻找新的药物治疗MDS具有重要意义。方法:选择2016年4月至2016年12月于河北医科大学第二医院血液内科初治的MDS患者20例以及健康体检者10例为实验对象。同时将MDS患者行IPSS评分对疾病恶性程度进行分层分析。取实验对象外周血各2ml,采用流式细胞术(Flow cytometry,FCM)检测其外周血中MDSCs(CD33+CD11b+CD14-HLA-DR-)、NK细胞(CD3-CD56+)和CD34+细胞比例。抽取3例MDS患者外周血各20ml,提取外周血单个核细胞(Peripheral blood mononuclear cells,PBMCs),并接种于96孔板,用不同浓度的ART(终浓度分别为1、2.5、5、10umol/L),处理相应各孔48小时,用FCM检测MDSCs、NK细胞、CD34+各群细胞的增殖和凋亡变化。结果:1健康者外周血MDSCs比例(0.65%±0.28%)明显低于MDS患者(2.39%±0.74%)(P0.05),健康者外周血MDSCs比例(0.65%±0.28%)低于低危及中危-ⅠMDS患者(1.46%±0.17%)(P0.05);低危及中危-ⅠMDS患者外周血MDSCs比例(1.46%±0.17%)低于中危-Ⅱ及高危MDS患者(2.69%±0.56%)(P0.05)。2健康者外周血NK细胞占淋巴细胞百分比(12.88%±4.51%)明显高于MDS患者(9.08%±3.59%)(P0.05);健康者外周血NK细胞占淋巴百分比(12.88%±4.51%)与低危及中危-ⅠMDS患者(13.71%±2.46%)相比,差异无统计学意义(P0.05);低危及中危-ⅠMDS患者外周血NK细胞占淋巴细胞百分比(13.71%±2.46%)高于中危-Ⅱ及高危MDS患者(7.53%±2.36%)(P0.05)。3健康者外周血CD34+细胞百分数(0.03%±0.01%)明显低于MDS患者(3.75%±3.12%)(P0.05);健康者外周血CD34+细胞百分数(0.03%±0.01%)低于低危及中危-ⅠMDS患者(0.34%±0.36%)(P0.05);低危及中危-ⅠMDS患者(0.34%±0.36%)低于中危-Ⅱ及高危MDS患者(4.88%±2.77%)(P0.05)。4不同浓度的ART对MDS外周血来源的MDSCs、NK细胞、CD34+细胞的增殖和凋亡均未见明显差异(P0.05)。结论:1 MDS患者外周血循环中MDSCs和CD34+细胞比例均是升高的,且随着疾病危险程度增加而增高;其NK细胞比例是降低的,并随着疾病危险程度增加而降低。2 ART对MDS外周血来源的MDSCs、NK细胞、CD34+细胞的增殖和凋亡暂未见明显影响。
[Abstract]:Objective: Myelodysplastic syndrome (MDS) is a clonogenic disease originating from hematopoietic stem cells. It is a key mechanism for the formation and development of MDS to reduce the blood cells of one or more peripheral peripheral blood, bone marrow ineffective and / or morbid hematopoiesis, and the transformation of Acute leucemia (AL) to the characteristic.MDS in the bone marrow microenvironment. Myeloid-derived suppressor cells (MDSCs) is a group of heterogeneous cells with similar biological functions, including bone marrow progenitor cells, immature myeloid cells (Immature myeloid cells, IMCs), immature macrophages, by using and changing microenvironment to escape immune killing. Cells and immature dendritic cells.MDSCs can promote immune escape by regulating inherent immunity and adaptive immune response, such as inhibition of CD4+T cells, CD8+T cells and natural killer cells (Natural kill cells, NK) and other activation and immune responses. The interaction of tumor microenvironment and MDSCs induced immune tolerance promotes MDS Development. Because MDS is more common in the elderly, it can not bear strong chemotherapy and transgene hematopoietic stem cell transplantation (Allogeneic hematopoietic stem cell transplantation, Allo-HSCT), especially for high-risk MDS patients, it is very easy to convert to leukemia, so the prognosis is extremely poor. For patients who do not apply Allo-HSCT, they are still treated with drug therapy as the main.MDS. Drug treatment includes demethylation drugs, histone deacetylase inhibitors, iron chelating agents and antiproliferative drugs. Although these drugs have been used in clinical treatment of MDS, these drugs have not achieved a lasting effect. It is a very important clinical question to find low toxicity, effective and economic treatment for middle risk patients. Artesunate (ART) is a semi synthetic derivative of artemisinin extracted from the Chinese herb Artemisia Artemisia. It is one of the most effective and low toxic drugs for the treatment of malarial malaria. It is found that it has anti swelling effect in vitro, and ART can induce apoptosis of SKM-1 cells in MDS cell line in vitro. And ART is different from the traditional chemotherapeutic drugs, and may have synergistic effects with other MDS drugs. And ART has significant advantages in the treatment of tumor, such as low price, high efficiency and low toxicity, and tumor targeting. In this study, the number of MDSCs, CD34+ cells, the number of NK cells and the malignancy of MDS disease in the peripheral blood of MDS patients were studied. Through in vitro cell culture experiment, it is important to detect the changes in the cell groups of MDSCs, NK and CD34+ cells derived from MDS by ART. It is of great significance to further study the pathogenesis of MDS and the mechanism of ART anti MDS, and is of great significance for finding new drugs to treat MDS. Method: choose from April 2016 to December 2016. In the second hospital of the second hospital of Northern Medical University, 20 cases of MDS and 10 healthy subjects were treated as subjects. At the same time, the malignant degree of the disease was stratified by IPSS score of the MDS patients. The peripheral blood of the experimental subjects was taken 2ml, and MDSCs (CD33+CD11b+CD14-HLA-DR-) in peripheral blood was detected by flow cytometry (FCM), and the flow cytometry (FCM) was used to detect MDSCs (CD33+CD11b+CD14-HLA-DR-) in peripheral blood. The proportion of NK cells (CD3-CD56+) and CD34+ cells. The peripheral blood of 3 MDS patients was extracted from the peripheral blood of each 20ml, and the peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMCs) were extracted and inoculated on the 96 orifice. Results: in 1 healthy subjects, the proportion of MDSCs in peripheral blood (0.65% + 0.28%) was significantly lower than that in MDS (2.39% + 0.74%) (2.39% + 0.74%), and the proportion of MDSCs in peripheral blood (0.65% + 0.28%) in healthy subjects was lower than that of low risk - I MDS (1.46% + 0.17%) (P0.05), and the proportion of MDSCs in peripheral blood (1.46% +) in patients with low and middle risk MDS was lower than that of middle risk - II. The percentage of peripheral blood NK cells in peripheral blood (12.88% + 4.51%) of peripheral blood (12.88% + 4.51%) in patients with high risk MDS (P0.05).2 health was significantly higher than that of MDS (9.08% + 3.59%) (P0.05), and the percentage of NK cells in peripheral blood (12.88% + 4.51%) in healthy subjects was not statistically significant (P0.05) compared with those of low risk - I MDS patients (13.71% + 2.46%), and low endanger. The percentage of NK cells in peripheral blood (13.71% + 2.46%) of peripheral blood (13.71% + 2.46%) in patients with middle risk I was higher than that in middle risk - II and high risk MDS patients (7.53% + 2.36%) (P0.05).3 healthy persons (0.03% + 0.01%) in peripheral blood (0.03% + 0.01%) were significantly lower than those of MDS patients (3.75% + 3.12%) (P0.05), and the percentage of peripheral blood CD34+ cells (0.03% + 0.01%) in healthy subjects was lower than that of low endanger. Risk I MDS patients (0.34% + 0.36%) (P0.05); low risk middle risk - I MDS patients (0.34% + 0.36%) were lower than middle risk - II and high risk MDS patients (4.88% + 2.77%) (4.88% + 2.77%) (P0.05).4 different concentrations of MDSCs, NK cells, CD34+ cells of the proliferation and apoptosis of MDS (P0.05). Conclusion: 1 patients of peripheral blood circulation in the peripheral circulation The proportion of 4+ cells increased and increased with the increase of the risk of disease; the proportion of NK cells was reduced, and with the increase of the risk of disease,.2 ART had no significant effect on the proliferation and apoptosis of MDS peripheral blood of MDSCs, NK cells, CD34+ cells.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R551.3

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