發(fā)布時間：2018-06-28 05:40

  本文選題：CD137mAb + CD3~+CD56~+細胞�。� 參考：《南京醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 共刺激分子CD137是近年新發(fā)現(xiàn)的TNFR超家族的新成員,其主要表達在活化的T細胞、自然殺傷細胞(natural killer,NK cells)和樹突狀細胞(dendritic cells,DCs)表面。研究發(fā)現(xiàn)CD137與其配體結(jié)合后,介導(dǎo)的共刺激信號可促進T細胞和NK細胞增殖、誘導(dǎo)細胞因子的分泌以及減少活化誘導(dǎo)的細胞死亡(activation-induced cell death,AICD),增加細胞存活能力。細胞因子誘導(dǎo)的殺傷細胞(cytokineinduced killer cells,CIK細胞)是一種新型的具有廣譜殺瘤活性的非主要組織相容性復(fù)合物(non-major histocompatibility complex,MHC)限制性的免疫效應(yīng)細胞。但目前CIK細胞的擴增效率和治療實體瘤的療效仍有待進一步提高。CIK細胞為一群異質(zhì)細胞群,包括CD3~+CD56~+細胞和CD3~+CD8~+細胞,其中CD3~+CD56~+細胞是CIK細胞發(fā)揮直接和間接抗腫瘤作用最主要的效應(yīng)細胞。因此通過提高CIK細胞中CD3~+CD56~+細胞的數(shù)量和抗腫瘤的活性可有效提高CIK細胞過繼免疫治療的療效。然而目前CD137信號對CD3~+CD56~+細胞亞群的功能調(diào)節(jié)國內(nèi)外未見報道。在本研究中,通過CD137mAb激活CD137共刺激信號通路,可以顯著提高CIK細胞的增殖能力和殺傷活性。在此基礎(chǔ)上,探討CD137信號對CIK細胞功能的調(diào)節(jié)機制。本實驗結(jié)果為提高CIK細胞治療實體瘤的療效提供新的理論依據(jù)。 目的:探討CD137信號對健康人和肺癌患者外周血來源的CIK細胞的擴增和功能調(diào)節(jié)作用。 方法:分離健康人和肺癌患者外周血單個核細胞(peripheral bloodmononuclear cells,PBMCs),實驗組在常規(guī)CIK培養(yǎng)體系中加入cD137單克隆抗體(monoclonal antibody,mAb),即CD137-CIK組;對照組在常規(guī)CIK培養(yǎng)體系中加入鼠IgG1同型對照,即IgG1-CIK組。 健康人CIK細胞:(1)苔盼藍拒染法細胞計數(shù)分析細胞增殖;(2)LDH酶釋放法檢測CIK細胞對A549細胞殺傷活性;(3)AnnexinV-FITC/PI試劑盒檢測CIK細胞凋亡率;(4)流式細胞術(shù)檢測CIK細胞表型分布及CD3~+CD56~+細胞內(nèi)細胞因子及其表面NK細胞受體(NK cell receptor,NKR)的表達;(5)CIK細胞和CD4~+Th0細胞共培養(yǎng)后,流式細胞儀分析CD4~+T細胞內(nèi)IFN-γ和IL-4的表達水平。 肺癌患者CIK細胞:(1)流式細胞術(shù)分析CIK細胞表型;(2)LDH酶釋放法檢測CIK細胞對A549細胞殺傷活性。 結(jié)果:健康人:(1)CD137-CIK組細胞體外擴增效率顯著高于IgG1-CIK組,CD137-CIK組細胞濃度最高達(9.87±0.57)×10~6/ml;IgG1-CIK組濃度最高為(7.02±0.68)×10~6/ml(p＜0.05);(2)培養(yǎng)至28天,CD137-CIK組和IgG1-CIK組細胞中CD3~+CD56~+細胞比例分別達到(39.86±4.69)%和(29.14±5.12)%(p＜0.05);(3)培養(yǎng)至14天,CD137-CIK組細胞凋亡壞死率明顯低于IgG1-CIK組;(4)CD137-CIK組細胞體外殺傷肺癌細胞株A549活性高于對照組(20:1,P＞0.05;10:1和5:1時,P＜0.05);(5)培養(yǎng)至14天,CD137mAb上調(diào)CD3~+CD56~+細胞內(nèi)IL-2,IFN-γ,TNF-α表達,同時下調(diào)IL-4,TGF-β_1,IL-10表達;(6)與IgG1-CIK組相比,CD137-CIK組中CD3~+CD56~+細胞表面NKG2D的表達較IgG1-CIK組明顯增強,而NKG2A的表達則減弱;(7)結(jié)果顯示,CD137-CIK組細胞可顯著提高CD4~+T細胞IFN-γ表達水平,同時降低IL-4的產(chǎn)生。 肺癌患者:(1)第28天,CD137-CIK組和IgG1-CIK組中CD3~+CD56~+細胞比例都顯著提高,分別達到(30.1±7.3)%和(21.7±5.7)%(p＜0.01);(2)CD137-CIK組細胞體外殺傷肺癌細胞株A549活性明顯高于IgG1-CIK組(10:1和5:1,P＜0.01;20:1,P＜0.05)。 結(jié)論:CD137共刺激信號可有效的提高健康人和肺癌患者外周血中CIK細胞的擴增效率并增強CIK細胞體外非MHC限制性殺傷腫瘤細胞的能力;同時CD137信號通過提高CD3~+CD56~+細胞分泌Th1類細胞因子的能力,促使CIK細胞誘導(dǎo)CD4~+Th0細胞向Th1方向分化,從而實現(xiàn)輔助激活體內(nèi)細胞毒性T淋巴細胞(cytotoxic T lymphocyte,CTL)免疫反應(yīng),發(fā)揮MHC限制性的抗腫瘤作用。上述研究結(jié)果為提高CIK細胞在實體瘤中的治療效果提供更多的可行性依據(jù)。
[Abstract]:Co stimulator CD137 is a new member of the newly discovered TNFR superfamily in recent years. It is mainly expressed in activated T cells, natural killer cells (natural killer, NK cells) and dendritic cells (dendritic cells, DCs). The study found that the co stimulatory signal mediated by CD137 and its ligand can promote the proliferation of T cells and cells and induce fine induction. Cytokine secretion and reduced activation induced cell death (activation-induced cell death, AICD), increasing cell viability. Cytokine induced killer cells (cytokineinduced killer cells, CIK cells) are a new type of non major histocompatibility complex (non-major histocompatibility) with broad-spectrum antitumor activity (non-major histocompatibility) Complex, MHC) restrictive immune effector cells. However, the amplification efficiency of CIK cells and the efficacy of the treatment of solid tumors still need to be further enhanced by.CIK cells as a group of heterogeneous cells, including CD3~+CD56~+ and CD3~+CD8~+ cells, in which CD3~+CD56~+ cells are the most important effects of CIK cells in direct and indirect antitumor effects. So by increasing the number of CD3~+CD56~+ cells in CIK cells and antitumor activity, the effect of the adoptive immunotherapy of CIK cells can be effectively improved. However, the function regulation of the CD137 signal on the CD3~+CD56~+ cell subgroup has not been reported at home and abroad. In this study, the activation of the CD137 co stimulatory signal pathway by CD137mAb can be significantly improved. On the basis of the proliferation and killing activity of high CIK cells, the regulation mechanism of CD137 signal on the function of CIK cells is discussed. The results of this experiment provide a new theoretical basis for improving the curative effect of CIK cells in the treatment of solid tumors.
Objective: To investigate the amplification and function regulation of CD137 signal in peripheral blood CIK cells from healthy and lung cancer patients.
Methods: the peripheral blood mononuclear cells (peripheral bloodmononuclear cells, PBMCs) were isolated from the healthy and lung cancer patients. The experimental group added the cD137 monoclonal antibody (monoclonal antibody, mAb) to the CD137-CIK group in the routine CIK culture system, and the control group added the rat IgG1 same type control in the routine CIK culture system, that is, the group.
Healthy human CIK cells: (1) trypan blue staining method cell count analysis and analysis of cell proliferation; (2) LDH enzyme release assay to detect the killing activity of CIK cells to A549 cells; (3) AnnexinV-FITC/PI kit to detect the apoptosis rate of CIK cells; (4) flow cytometry detection of CIK cell phenotype distribution and CD3~+CD56~+ cell cytokines and the surface NK cell receptor (NK cell R) Eceptor (NKR) expression; (5) CIK cells and CD4~+Th0 cells co cultured, and the expression levels of IFN- and IL-4 in CD4~+T cells were analyzed by flow cytometry.
CIK cells in lung cancer patients: (1) flow cytometry was used to analyze the phenotype of CIK cells; (2) LDH enzyme release assay was used to detect the killing activity of CIK cells against A549 cells.
Results: (1) the efficiency of cell amplification in CD137-CIK group was significantly higher than that in group IgG1-CIK, and the highest cell concentration in group CD137-CIK was (9.87 + 0.57) x 10~6/ml, and the highest concentration in IgG1-CIK group was (7.02 + 0.68) x 10~6/ml (P < 0.05); (2) culture to 28 days, the ratio of CD3~+CD56~+ cells in CD137-CIK and IgG1-CIK groups reached (39.86 + 4.69)% and (39.86 + 4.69)%, respectively. 29.14 + 5.12)% (P < 0.05); (3) the apoptosis and necrosis rate in group CD137-CIK was significantly lower than that in group IgG1-CIK; (4) the activity of A549 in group CD137-CIK was higher than that of the control group (20:1, P > 0.05; 10:1 and 5:1, P < 0.05); (5) cultured to 14 days, CD137mAb increased the expression of CD3~+CD56~+ cells. IL-4, TGF- beta _1, IL-10 expression; (6) compared with the IgG1-CIK group, the expression of NKG2D on the surface of CD3~+CD56~+ cells in the CD137-CIK group was significantly enhanced and the expression of NKG2A decreased. (7) the results showed that the CD137-CIK group cells could significantly increase the expression level of CD4~+T cells and reduce the production of the CD3~+CD56~+ cells.
Patients with lung cancer: (1) on the twenty-eighth day, the proportion of CD3~+CD56~+ cells in group CD137-CIK and group IgG1-CIK increased significantly (30.1 + 7.3)% and (21.7 + 5.7)% respectively (P < 0.01). (2) the cytotoxic activity of lung cancer cell line in CD137-CIK group was significantly higher than that of IgG1-CIK group (10:1 and 5:1, P < 0.01; 20:1, P < 0.05).
Conclusion: CD137 co stimulation signal can effectively improve the amplification efficiency of CIK cells in the peripheral blood of healthy and lung cancer patients and enhance the ability of non MHC restrictive killer cells in CIK cells in vitro, and CD137 signal can induce CD4~+Th0 cells to induce CD4~+Th0 cells to Th1 direction by enhancing the ability of CD3~+CD56~+ cells to secrete Th1 cell factors. Differentiation, so as to assist in activating the immune response of cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) in vivo, and exerting MHC restrictive antitumor effect, the above results provide more feasible basis for improving the therapeutic effect of CIK cells in solid tumor.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392;R734.2

【共引文獻】

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