本文選題：全腫瘤抗原 + 樹突狀細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2010年博士論文

【摘要】： 研究背景 兒童惡性腫瘤現(xiàn)已成為危害兒童生命的常見疾病。與成人惡性腫瘤一樣,目前對兒童惡性腫瘤的治療多采用手術(shù)、化療、放療三大傳統(tǒng)治療。由于兒童的藥代動力學(xué)和藥效動力學(xué)不同于成人,常用的放化療措施均伴隨著明確的近、遠(yuǎn)期毒副作用,影響患者的生長發(fā)育及長期生存者的生活質(zhì)量,且伴有轉(zhuǎn)移率和復(fù)發(fā)率高的風(fēng)險。因此,亟需一種高效、安全的治療兒童惡性腫瘤的方法。 基于樹突狀細(xì)胞(dendritic cells,DCs)的免疫治療正逐漸成為治療腫瘤患者的重要方法之一。正常情況下,DC以不成熟的形式存在。未成熟的DC激活T淋巴細(xì)胞的能力較弱,只有成熟的DC能刺激初始T淋巴細(xì)胞,使T細(xì)胞活化,刺激機(jī)體免疫應(yīng)答。如何大量高效率制備體外誘導(dǎo)的DC,并增強(qiáng)DC誘導(dǎo)的抗腫瘤免疫能力,已成為研究DC疫苗的重點(diǎn)和難點(diǎn)問題。機(jī)體針對自身腫瘤相關(guān)抗原的免疫殺傷受到內(nèi)源性抑制機(jī)制的制約,常導(dǎo)致殺傷效力減低,對許多腫瘤的免疫治療因此難以取得預(yù)期效果。近年研究發(fā)現(xiàn),細(xì)胞因子信號傳導(dǎo)抑制蛋白(SOCS1)是存在于DC內(nèi)調(diào)節(jié)T細(xì)胞激活能力及獲得性免疫的內(nèi)源性抑制分子。SOCS1可抑制T細(xì)胞及其它免疫細(xì)胞內(nèi)的JAK活性,對多種細(xì)胞因子(如IFN-γ等)的信號轉(zhuǎn)導(dǎo)均起抑制作用,是調(diào)節(jié)DC抗原提呈以及獲得性免疫幅度的關(guān)鍵分子之一。在DC內(nèi)抑制SOCS1的表達(dá)是否可增強(qiáng)DC誘導(dǎo)的特異性殺傷兒童惡性腫瘤細(xì)胞的能力,迄今鮮有報道。 因此,本研究的目的是: 1)探索從外周血單核細(xì)胞(PBMC)獲取大量成熟DC的可行性及刺激DC成熟的策略。 2)觀察負(fù)載全腫瘤抗原的DC刺激T細(xì)胞對其增殖及殺傷自體及異體實(shí)體瘤鼻咽瘤細(xì)胞能力的影響。 3)探討用siRNA技術(shù)沉默SOCS1表達(dá),觀察其對DC抗腫瘤免疫能力的作用,為提高DC臨床免疫治療兒童非實(shí)體瘤白血病的療效提供實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。 實(shí)驗(yàn)方法 1)腫瘤細(xì)胞培養(yǎng):活檢組織采用組織塊法培養(yǎng)。CNE-2Z及K562細(xì)胞按常規(guī)方法培養(yǎng)。 2)全腫瘤抗原制備:采用反復(fù)凍融法,并用60Coγ射線照射(劑量25Gy)。 3) DC的體外誘導(dǎo)及擴(kuò)增:采用PBMC分離法,加入細(xì)胞因子GM-CSF、IL-4培養(yǎng),培養(yǎng)第3天加入全腫瘤抗原、第7天加入TNF-α刺激DC成熟。 4) DC形態(tài)學(xué)鑒定:分別采用倒置顯微鏡、掃描電鏡和投射電鏡。 5) DC表面抗原及成熟度檢測:采用流式細(xì)胞儀法。 6)腫瘤DC刺激T細(xì)胞增殖能力檢測:采用四氮唑藍(lán)試驗(yàn)(MTT法)。 7)腫瘤DC活化的CTL體外殺傷活性檢測:采用MTT法。 8)腫瘤DC活化的CTL分泌IFN-γ能力檢測:采用ELISPOT實(shí)驗(yàn)。 9) DC內(nèi)SOCS1的表達(dá):分別用RT-PCR和Western blot測定其mRNA和蛋白表達(dá)。 實(shí)驗(yàn)結(jié)果 1) DC形態(tài)學(xué)鑒定:倒置顯微鏡及電鏡下可見細(xì)胞表面典型的樹突樣突起。 2)培養(yǎng)5天負(fù)載全腫瘤抗原的DC(未成熟DC),其細(xì)胞表面標(biāo)志性分子HLA-DR、CD1a、CD80、CD83和CD86經(jīng)流式細(xì)胞檢測陽性率分別為:(60.86±5.42)%、(21.84±2.52)%、(5.49±6.32)%、(6.82±1.24)%和(1.24±0.90)%,而經(jīng)過TNF-α刺激的負(fù)載全腫瘤抗原的DC(成熟DC),上述細(xì)胞表面分子的陽性率分別為(86.14±8.32)%、(78.28±11.42)%、(78.24±12.64)%、(67.25±14.24)%和(85.26±9.14)%,各表面分子的表達(dá)明顯高于未成熟DC(P 0.01~0.05)。 3)隨著DC:T細(xì)胞比例由1:100增至1:5,各組T細(xì)胞增殖的刺激指數(shù)均呈增加趨勢。負(fù)載全腫瘤抗原的DC刺激T細(xì)胞增殖的刺激指數(shù)高于未負(fù)載全腫瘤抗原的DC。 4)經(jīng)過負(fù)載全腫瘤抗原的DC活化的CTL對自體或異體鼻咽癌細(xì)胞均具有殺傷活性,其對鼻咽癌細(xì)胞的殺傷率與效靶比成正比。 5)用負(fù)載全腫瘤抗原的DC孵育的T細(xì)胞中分泌IFN-γ的細(xì)胞數(shù)目顯著多于未負(fù)載全腫瘤抗原的DC組和單獨(dú)T細(xì)胞組(P㩳0.05)。 6)在培養(yǎng)第7天加入TNF-α(1000 U/mL)刺激DC成熟后,K562-DC中SOCS1 mRNA和蛋白表達(dá)增加。經(jīng)SOCS1 siRNA(SOCS1 siRNA1和siRNA2)轉(zhuǎn)染成熟DC 24 h可明顯降低K562-DC中SOCS1 mRNA及蛋白的表達(dá)。 7)培養(yǎng)5天負(fù)載全腫瘤抗原的DC(未成熟DC),其細(xì)胞表面標(biāo)志性分子HLA-DR,CD1a、CD80、CD86和CD83經(jīng)流式細(xì)胞檢測陽性率分別為:(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誘導(dǎo)K562-DC成熟,各表面分子的陽性率分別為(69.52±8.68)%、(58.97±4.25)%、(63.84±7.32)%、(72.69±6.23)%和(71.46±4.96)%,均顯著高于未成熟DC(P 0.01~0.05)。經(jīng)SOCS1 siRNA轉(zhuǎn)染成熟DC 24 h后,上述分子的陽性率分別為(67.17±9.53)%、(59.46±5.17)%、(65.72±6.58)%、(71.47±5.68)%和(87.92±3.94)%。其中CD83的陽性率顯著高于未轉(zhuǎn)染DC組(P 0.05)。 8) TNF-α刺激成熟的K562-DCs其刺激T細(xì)胞增殖的能力明顯高于未成熟DC,且隨著DC: T細(xì)胞比例的增加,刺激能力增強(qiáng),以效靶比1: 5時刺激作用最為顯著(效靶比1:20及1:5時均有P 0.01)。SOCS1 siRNA轉(zhuǎn)染后K562-DCs刺激T細(xì)胞增殖的能力較之scramble siRNA后的K562-DCs強(qiáng),二者比較有顯著性差異(P 0.05)。 9)經(jīng)TNF-α刺激成熟的K562-DCs其刺激外周血T細(xì)胞對K562細(xì)胞的殺傷率明顯增加,在效靶比為5~20:1時殺傷率增加尤為明顯(成熟DC vs.未成熟DC, P 0.01)。采用SOCS1 siRNA轉(zhuǎn)染的K562-DCs刺激外周血T細(xì)胞可使T細(xì)胞對K562細(xì)胞的殺傷率進(jìn)一步增加(P 0.05),其對K562細(xì)胞的殺傷率的影響強(qiáng)度與效靶比成正比。 10)成熟的K562-DCs刺激后,外周血T細(xì)胞分泌IFN-γ的數(shù)目增加(成熟DC vs.未成熟DC,P 0.01)。而采用SOCS1 siRNA轉(zhuǎn)染的K562-DCs刺激外周血T細(xì)胞可進(jìn)一步增加IFN-γ的T細(xì)胞數(shù)目(P 0.05)。 結(jié)論 1)從PBMC來源的、負(fù)載全腫瘤抗原的DC經(jīng)細(xì)胞因子誘導(dǎo)成熟后,可刺激T細(xì)胞增殖,并增強(qiáng)其特異性殺傷腫瘤細(xì)胞的能力。 2) SOCS1 siRNA轉(zhuǎn)染可有效抑制DC內(nèi)SOCS1表達(dá),可突破內(nèi)源性抑制機(jī)制的制約,使成熟DC誘導(dǎo)出更強(qiáng)的反應(yīng),表現(xiàn)為T細(xì)胞增殖反應(yīng)增加及其對K562細(xì)胞的殺傷能力增強(qiáng)。 綜上所述,我們的結(jié)果提示,負(fù)載全腫瘤抗原的DC可能是治療兒童惡性腫瘤的一種有效方法,具有潛在的臨床應(yīng)用價值。而采用SOCS1 siRNA抑制SOCS1,增強(qiáng)CTL對腫瘤細(xì)胞的殺傷效應(yīng),可能是一種增強(qiáng)獲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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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