负载全肿瘤抗原及SOCS1siRNA转染的树突状细胞杀伤儿童恶性肿瘤细胞的研究

发布时间：2018-05-11 02:23

本文选题：全肿瘤抗原 + 树突状细胞　；参考：《第四军医大学》2010年博士论文

【摘要】： 研究背景儿童恶性肿瘤现已成为危害儿童生命的常见疾病。与成人恶性肿瘤一样,目前对儿童恶性肿瘤的治疗多采用手术、化疗、放疗三大传统治疗。由于儿童的药代动力学和药效动力学不同于成人,常用的放化疗措施均伴随着明确的近、远期毒副作用,影响患者的生长发育及长期生存者的生活质量,且伴有转移率和复发率高的风险。因此,亟需一种高效、安全的治疗儿童恶性肿瘤的方法。基于树突状细胞(dendritic cells,DCs)的免疫治疗正逐渐成为治疗肿瘤患者的重要方法之一。正常情况下,DC以不成熟的形式存在。未成熟的DC激活T淋巴细胞的能力较弱,只有成熟的DC能刺激初始T淋巴细胞,使T细胞活化,刺激机体免疫应答。如何大量高效率制备体外诱导的DC,并增强DC诱导的抗肿瘤免疫能力,已成为研究DC疫苗的重点和难点问题。机体针对自身肿瘤相关抗原的免疫杀伤受到内源性抑制机制的制约,常导致杀伤效力减低,对许多肿瘤的免疫治疗因此难以取得预期效果。近年研究发现,细胞因子信号传导抑制蛋白(SOCS1)是存在于DC内调节T细胞激活能力及获得性免疫的内源性抑制分子。SOCS1可抑制T细胞及其它免疫细胞内的JAK活性,对多种细胞因子(如IFN-γ等)的信号转导均起抑制作用,是调节DC抗原提呈以及获得性免疫幅度的关键分子之一。在DC内抑制SOCS1的表达是否可增强DC诱导的特异性杀伤儿童恶性肿瘤细胞的能力,迄今鲜有报道。因此,本研究的目的是: 1)探索从外周血单核细胞(PBMC)获取大量成熟DC的可行性及刺激DC成熟的策略。 2)观察负载全肿瘤抗原的DC刺激T细胞对其增殖及杀伤自体及异体实体瘤鼻咽瘤细胞能力的影响。 3)探讨用siRNA技术沉默SOCS1表达,观察其对DC抗肿瘤免疫能力的作用,为提高DC临床免疫治疗儿童非实体瘤白血病的疗效提供实验基础和理论依据。实验方法 1)肿瘤细胞培养:活检组织采用组织块法培养。CNE-2Z及K562细胞按常规方法培养。 2)全肿瘤抗原制备:采用反复冻融法,并用60Coγ射线照射(剂量25Gy)。 3) DC的体外诱导及扩增:采用PBMC分离法,加入细胞因子GM-CSF、IL-4培养,培养第3天加入全肿瘤抗原、第7天加入TNF-α刺激DC成熟。 4) DC形态学鉴定:分别采用倒置显微镜、扫描电镜和投射电镜。 5) DC表面抗原及成熟度检测:采用流式细胞仪法。 6)肿瘤DC刺激T细胞增殖能力检测:采用四氮唑蓝试验(MTT法)。 7)肿瘤DC活化的CTL体外杀伤活性检测:采用MTT法。 8)肿瘤DC活化的CTL分泌IFN-γ能力检测:采用ELISPOT实验。 9) DC内SOCS1的表达:分别用RT-PCR和Western blot测定其mRNA和蛋白表达。实验结果 1) DC形态学鉴定:倒置显微镜及电镜下可见细胞表面典型的树突样突起。 2)培养5天负载全肿瘤抗原的DC(未成熟DC),其细胞表面标志性分子HLA-DR、CD1a、CD80、CD83和CD86经流式细胞检测阳性率分别为:(60.86±5.42)%、(21.84±2.52)%、(5.49±6.32)%、(6.82±1.24)%和(1.24±0.90)%,而经过TNF-α刺激的负载全肿瘤抗原的DC(成熟DC),上述细胞表面分子的阳性率分别为(86.14±8.32)%、(78.28±11.42)%、(78.24±12.64)%、(67.25±14.24)%和(85.26±9.14)%,各表面分子的表达明显高于未成熟DC(P 0.01~0.05)。 3)随着DC:T细胞比例由1:100增至1:5,各组T细胞增殖的刺激指数均呈增加趋势。负载全肿瘤抗原的DC刺激T细胞增殖的刺激指数高于未负载全肿瘤抗原的DC。 4)经过负载全肿瘤抗原的DC活化的CTL对自体或异体鼻咽癌细胞均具有杀伤活性,其对鼻咽癌细胞的杀伤率与效靶比成正比。 5)用负载全肿瘤抗原的DC孵育的T细胞中分泌IFN-γ的细胞数目显著多于未负载全肿瘤抗原的DC组和单独T细胞组(P㩳0.05)。 6)在培养第7天加入TNF-α(1000 U/mL)刺激DC成熟后,K562-DC中SOCS1 mRNA和蛋白表达增加。经SOCS1 siRNA(SOCS1 siRNA1和siRNA2)转染成熟DC 24 h可明显降低K562-DC中SOCS1 mRNA及蛋白的表达。 7)培养5天负载全肿瘤抗原的DC(未成熟DC),其细胞表面标志性分子HLA-DR,CD1a、CD80、CD86和CD83经流式细胞检测阳性率分别为:(54.65±3.28)%、(17.42±6.78)%、(6.27±5.29)%、(3.02±2.47)%和(3.28±2.79)%,而用TNF-α诱导K562-DC成熟,各表面分子的阳性率分别为(69.52±8.68)%、(58.97±4.25)%、(63.84±7.32)%、(72.69±6.23)%和(71.46±4.96)%,均显著高于未成熟DC(P 0.01~0.05)。经SOCS1 siRNA转染成熟DC 24 h后,上述分子的阳性率分别为(67.17±9.53)%、(59.46±5.17)%、(65.72±6.58)%、(71.47±5.68)%和(87.92±3.94)%。其中CD83的阳性率显著高于未转染DC组(P 0.05)。 8) TNF-α刺激成熟的K562-DCs其刺激T细胞增殖的能力明显高于未成熟DC,且随着DC: T细胞比例的增加,刺激能力增强,以效靶比1: 5时刺激作用最为显著(效靶比1:20及1:5时均有P 0.01)。SOCS1 siRNA转染后K562-DCs刺激T细胞增殖的能力较之scramble siRNA后的K562-DCs强,二者比较有显著性差异(P 0.05)。 9)经TNF-α刺激成熟的K562-DCs其刺激外周血T细胞对K562细胞的杀伤率明显增加,在效靶比为5~20:1时杀伤率增加尤为明显(成熟DC vs.未成熟DC, P 0.01)。采用SOCS1 siRNA转染的K562-DCs刺激外周血T细胞可使T细胞对K562细胞的杀伤率进一步增加(P 0.05),其对K562细胞的杀伤率的影响强度与效靶比成正比。 10)成熟的K562-DCs刺激后,外周血T细胞分泌IFN-γ的数目增加(成熟DC vs.未成熟DC,P 0.01)。而采用SOCS1 siRNA转染的K562-DCs刺激外周血T细胞可进一步增加IFN-γ的T细胞数目(P 0.05)。结论 1)从PBMC来源的、负载全肿瘤抗原的DC经细胞因子诱导成熟后,可刺激T细胞增殖,并增强其特异性杀伤肿瘤细胞的能力。 2) SOCS1 siRNA转染可有效抑制DC内SOCS1表达,可突破内源性抑制机制的制约,使成熟DC诱导出更强的反应,表现为T细胞增殖反应增加及其对K562细胞的杀伤能力增强。综上所述,我们的结果提示,负载全肿瘤抗原的DC可能是治疗儿童恶性肿瘤的一种有效方法,具有潜在的临床应用价值。而采用SOCS1 siRNA抑制SOCS1,增强CTL对肿瘤细胞的杀伤效应,可能是一种增强获得性免疫的重要策略。
[Abstract]:Research background
Malignant tumor in children is now a common disease which is harmful to children's life. Like adult malignant tumors, the treatment of malignant tumor in children is currently treated with three traditional treatments, such as surgery, chemotherapy and radiotherapy. The pharmacokinetics and pharmacodynamics of children are different from those of adults. Side effects, which affect the growth and development of the patients and the quality of life of the long-term survivors, are associated with a high risk of high metastasis and recurrence. Therefore, there is an urgent need for a efficient and safe method for the treatment of malignant tumors in children.
Immunotherapy based on dendritic cells (DCs) is becoming one of the most important methods for the treatment of cancer patients. Under normal conditions, DC exists in the form of immature DC. The ability of immature DC to activate T lymphocytes is weak. Only mature DC can stimulate the initial T drenched cells to activate the T cells and stimulate the immune response. It has become a key and difficult problem for the study of the high efficient preparation of DC induced by high efficiency and the enhancement of DC induced anti-tumor immunity. The immune killing of the body against its tumor related antigen is restricted by endogenous inhibition mechanism, which often leads to reduced killing effect. Therefore, the immunotherapy for many tumors is difficult to obtain. In recent years, it has been found that cytokine signaling inhibitory protein (SOCS1) is an endogenous inhibitory molecule that regulates T cell activation and acquired immunity in DC, and.SOCS1 inhibits JAK activity in T cells and other immune cells, and inhibits the signal transduction of a variety of cytokines, such as IFN- gamma, and is the regulation of DC resistance. It is rarely reported that suppressing the expression of SOCS1 in DC can enhance the ability of DC to kill malignant tumor cells in children.
Therefore, the purpose of this study is to:
1) explore the feasibility of obtaining a large number of mature DC from peripheral blood mononuclear cells (PBMC) and the strategy to stimulate DC maturation.
2) to observe the effect of DC loaded with whole tumor antigen on T cells proliferation and killing the ability of autologous and xenogenic solid tumor nasopharynx cells.
3) to explore the effect of SOCS1 expression by siRNA technique to observe the anti tumor immunity of DC, and to provide experimental basis and theoretical basis for improving the curative effect of DC clinical immunotherapy for non solid tumor leukemia in children.
Experimental method
1) tumor cell culture: tissue biopsy was used to culture.CNE-2Z and K562 cells.
2) preparation of whole tumor antigen: repeated freezing and thawing and irradiation with 60Co gamma rays (dose 25Gy).
3) in vitro induction and amplification of DC: using PBMC separation method, adding cytokine GM-CSF, IL-4 culture, culture and adding all tumor antigen for third days, and adding TNF- to TNF- to stimulate the maturation of DC in seventh days.
4) morphological identification of DC: inverted microscope, scanning electron microscope and projection electron microscope respectively.
5) DC surface antigen and maturity detection: flow cytometry.
6) the proliferation ability of T cells stimulated by tumor DC: the tetrazolium blue test (MTT).
7) in vitro killing activity of CTL activated by tumor DC: MTT method.
8) the ability of CTL activated IFN- gamma in tumor DC activation: ELISPOT test.
9) SOCS1 expression in DC: mRNA and protein expression was measured by RT-PCR and Western blot respectively.
experimental result
1) morphological identification of DC: typical dendritic dendrites on cell surface can be seen under inverted microscope and electron microscope.
2) DC (immature DC) loaded with total tumor antigen for 5 days, the positive rates of cell surface marker molecules HLA-DR, CD1a, CD80, CD83 and CD86 were respectively (60.86 + 5.42)%, (21.84 + 2.52)%, (5.49 + 6.32)%, (6.82 + 1.24)% and (1.24 + 0.90)%, while DC (mature DC) loaded with TNF- alpha stimulated total tumor antigen (mature DC), the above cells The positive rates of surface molecules were (86.14 + 8.32)%, (78.28 + 11.42)%, (78.24 + 12.64)%, (67.25 + 14.24)% and (85.26 + 9.14)%, and the expression of each surface molecule was significantly higher than that of unmature DC (P 0.01~0.05).
3) as the proportion of DC:T cells increased from 1:100 to 1:5, the stimulation index of the proliferation of T cells in each group increased. The stimulation index of DC stimulated T cells with full tumor antigen was higher than that of the unloaded total tumor antigen.
4) the DC activated CTL, which is loaded with all tumor antigen, has a killing activity to both autologous or allogenic nasopharyngeal carcinoma cells, and the killing rate of the nasopharyngeal carcinoma cells is proportional to the target ratio.
5) the number of cells secreting IFN- gamma in T cells incubated with DC loaded with whole tumor antigen was significantly higher than that in DC group and T cell group without P (P? 0.05).
6) after the addition of TNF- alpha (1000 U/mL) to the maturation of DC for seventh days, the expression of SOCS1 mRNA and protein in K562-DC increased. The expression of mature DC 24 could be significantly reduced via SOCS1 siRNA (SOCS1 siRNA1 and siRNA2) transfected with mature DC 24.
7) DC (immature DC) loaded with total tumor antigen for 5 days, and the positive rates of cell surface marker molecules HLA-DR, CD1a, CD80, CD86 and CD83 were respectively (54.65 + 3.28)%, (17.42 + 6.78)%, (6.27 + 5.29)%, (3.02 + 2.47)% and 3.28 + 2.79)%, and the positive rates of each surface molecule were respectively (69.52) using TNF- alpha. (+ 8.68)%, (58.97 + 4.25)%, (63.84 + 7.32)%, (72.69 + 6.23)% and (71.46 + 4.96)%, significantly higher than unmature DC (P 0.01~0.05). After SOCS1 siRNA transfected to mature DC 24 h, the positive rates of these molecules were (67.17 + 9.53)%, (59.46 +%)%, (72.69%)% and (72.69)%, respectively. The positive rate of CD83 was significantly higher than that of unconverted. DC group (P 0.05).
8) the ability of TNF- alpha to stimulate mature K562-DCs to stimulate T cell proliferation is significantly higher than that of immature DC, and with the increase of DC: T cell ratio, the stimulation ability is enhanced, and the stimulation effect is the most significant when the target target ratio 1: 5 is most significant (the target ratio at 1:20 and 1:5 is P 0.01).SOCS1 siRNA after the.SOCS1 siRNA transfection. After K562-DCs, there was a significant difference between the two groups (P 0.05).
9) the killing rate of T cells in peripheral blood stimulated by K562-DCs stimulated by TNF- alpha was significantly increased in K562 cells, especially when the target target ratio was 5~20:1 (mature DC vs. immature DC, P 0.01). SOCS1 siRNA transfected K562-DCs stimulated peripheral blood cells to further increase the killing rate of peripheral blood cells (0 5) the intensity of the killing effect on K562 cells is directly proportional to the target ratio.
10) after the mature K562-DCs stimulation, the number of IFN- gamma secreted in peripheral blood T cells increased (mature DC vs. immature DC, P 0.01), while SOCS1 siRNA transfected K562-DCs stimulated the number of IFN- gamma cells to increase the number of IFN- gamma cells (0.05).
conclusion
1) from PBMC, DC loaded with whole tumor antigen can induce T cells to proliferate and enhance their ability to specifically kill tumor cells after induced maturation by cytokines.
2) SOCS1 siRNA transfection can effectively inhibit the expression of SOCS1 in DC. It can break through the restriction of endogenous inhibition mechanism and induce the mature DC to induce a stronger reaction, which shows the increase of T cell proliferation and the enhancement of the killing ability to K562 cells.
To sum up, our results suggest that DC loaded with total tumor antigen may be an effective method for the treatment of malignant tumor in children, and it is of potential clinical value. The use of SOCS1 siRNA to inhibit SOCS1 and enhance the killing effect of CTL on tumor cells may be an important strategy for enhancing acquired immunity.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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