本文選題：CD137mAb + CD3~+CD56~+細(xì)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(dá)在活化的T細(xì)胞、自然殺傷細(xì)胞(natural killer,NK cells)和樹(shù)突狀細(xì)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(hào)可促進(jìn)T細(xì)胞和NK細(xì)胞增殖、誘導(dǎo)細(xì)胞因子的分泌以及減少活化誘導(dǎo)的細(xì)胞死亡(activation-induced cell death,AICD),增加細(xì)胞存活能力。細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(cytokineinduced killer cells,CIK細(xì)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(xì)胞。但目前CIK細(xì)胞的擴(kuò)增效率和治療實(shí)體瘤的療效仍有待進(jìn)一步提高。CIK細(xì)胞為一群異質(zhì)細(xì)胞群,包括CD3~+CD56~+細(xì)胞和CD3~+CD8~+細(xì)胞,其中CD3~+CD56~+細(xì)胞是CIK細(xì)胞發(fā)揮直接和間接抗腫瘤作用最主要的效應(yīng)細(xì)胞。因此通過(guò)提高CIK細(xì)胞中CD3~+CD56~+細(xì)胞的數(shù)量和抗腫瘤的活性可有效提高CIK細(xì)胞過(guò)繼免疫治療的療效。然而目前CD137信號(hào)對(duì)CD3~+CD56~+細(xì)胞亞群的功能調(diào)節(jié)國(guó)內(nèi)外未見(jiàn)報(bào)道。在本研究中,通過(guò)CD137mAb激活CD137共刺激信號(hào)通路,可以顯著提高CIK細(xì)胞的增殖能力和殺傷活性。在此基礎(chǔ)上,探討CD137信號(hào)對(duì)CIK細(xì)胞功能的調(diào)節(jié)機(jī)制。本實(shí)驗(yàn)結(jié)果為提高CIK細(xì)胞治療實(shí)體瘤的療效提供新的理論依據(jù)。 目的:探討CD137信號(hào)對(duì)健康人和肺癌患者外周血來(lái)源的CIK細(xì)胞的擴(kuò)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(gè)核細(xì)胞(peripheral bloodmononuclear cells,PBMCs),實(shí)驗(yàn)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(duì)照組在常規(guī)CIK培養(yǎng)體系中加入鼠IgG1同型對(duì)照,即IgG1-CIK組。 健康人CIK細(xì)胞:(1)苔盼藍(lán)拒染法細(xì)胞計(jì)數(shù)分析細(xì)胞增殖;(2)LDH酶釋放法檢測(cè)CIK細(xì)胞對(duì)A549細(xì)胞殺傷活性;(3)AnnexinV-FITC/PI試劑盒檢測(cè)CIK細(xì)胞凋亡率;(4)流式細(xì)胞術(shù)檢測(cè)CIK細(xì)胞表型分布及CD3~+CD56~+細(xì)胞內(nèi)細(xì)胞因子及其表面NK細(xì)胞受體(NK cell receptor,NKR)的表達(dá);(5)CIK細(xì)胞和CD4~+Th0細(xì)胞共培養(yǎng)后,流式細(xì)胞儀分析CD4~+T細(xì)胞內(nèi)IFN-γ和IL-4的表達(dá)水平。 肺癌患者CIK細(xì)胞:(1)流式細(xì)胞術(shù)分析CIK細(xì)胞表型;(2)LDH酶釋放法檢測(cè)CIK細(xì)胞對(duì)A549細(xì)胞殺傷活性。 結(jié)果:健康人:(1)CD137-CIK組細(xì)胞體外擴(kuò)增效率顯著高于IgG1-CIK組,CD137-CIK組細(xì)胞濃度最高達(dá)(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(xì)胞中CD3~+CD56~+細(xì)胞比例分別達(dá)到(39.86±4.69)%和(29.14±5.12)%(p＜0.05);(3)培養(yǎng)至14天,CD137-CIK組細(xì)胞凋亡壞死率明顯低于IgG1-CIK組;(4)CD137-CIK組細(xì)胞體外殺傷肺癌細(xì)胞株A549活性高于對(duì)照組(20:1,P＞0.05;10:1和5:1時(shí),P＜0.05);(5)培養(yǎng)至14天,CD137mAb上調(diào)CD3~+CD56~+細(xì)胞內(nèi)IL-2,IFN-γ,TNF-α表達(dá),同時(shí)下調(diào)IL-4,TGF-β_1,IL-10表達(dá);(6)與IgG1-CIK組相比,CD137-CIK組中CD3~+CD56~+細(xì)胞表面NKG2D的表達(dá)較IgG1-CIK組明顯增強(qiáng),而NKG2A的表達(dá)則減弱;(7)結(jié)果顯示,CD137-CIK組細(xì)胞可顯著提高CD4~+T細(xì)胞IFN-γ表達(dá)水平,同時(shí)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(xì)胞比例都顯著提高,分別達(dá)到(30.1±7.3)%和(21.7±5.7)%(p＜0.01);(2)CD137-CIK組細(xì)胞體外殺傷肺癌細(xì)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(hào)可有效的提高健康人和肺癌患者外周血中CIK細(xì)胞的擴(kuò)增效率并增強(qiáng)CIK細(xì)胞體外非MHC限制性殺傷腫瘤細(xì)胞的能力;同時(shí)CD137信號(hào)通過(guò)提高CD3~+CD56~+細(xì)胞分泌Th1類(lèi)細(xì)胞因子的能力,促使CIK細(xì)胞誘導(dǎo)CD4~+Th0細(xì)胞向Th1方向分化,從而實(shí)現(xiàn)輔助激活體內(nèi)細(xì)胞毒性T淋巴細(xì)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(xì)胞在實(shí)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(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R392;R734.2

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 任金榮,孫麗霞,楊淑琴,何蘭欣,艾軍;食管癌及其癌前病變患者血清補(bǔ)體總活性與紅細(xì)胞CR1表達(dá)及臨床意義分析[J];癌變.畸變.突變;2004年02期

2 孫麗霞;任金榮;單保恩;李元振;何蘭欣;艾軍;;大黃制劑對(duì)小鼠的急性毒性和自然免疫調(diào)節(jié)作用[J];癌變.畸變.突變;2006年01期

3 徐偉剛,胡梅齊,陸群;血清TSGF、CEA、CA242、CA19-9聯(lián)合檢測(cè)對(duì)消化道惡性腫瘤的診斷價(jià)值[J];蚌埠醫(yī)學(xué)院學(xué)報(bào);2004年02期

4 張葛;花寶金;;晚期大腸癌中醫(yī)藥治療研究進(jìn)展[J];北京中醫(yī)藥;2012年06期

5 ;4-1BB (CD137) Ligand Enhanced Anti-Tumor Immune Response against Mouse Forestomach Carcinoma In Vivo[J];Cellular & Molecular Immunology;2008年05期

6 顧敏;范原銘;王強(qiáng);;乳腺癌患者外周血CD4~+、CD8~+以及CD4~+CD25~+調(diào)節(jié)性T細(xì)胞變化及其意義[J];重慶醫(yī)學(xué);2009年07期

7 杜文峰;楊道貴;;術(shù)后應(yīng)用吲哚美辛栓對(duì)胃腸道腫瘤病人免疫功能的影響[J];腸外與腸內(nèi)營(yíng)養(yǎng);2012年01期

8 申鄭堂,海健,毛杰;烏體林斯注射液對(duì)乳腺癌患者輔助化療后免疫功能的影響[J];中國(guó)醫(yī)師雜志;2003年01期

9 李建華,李琳;惡性腫瘤患者胸腺移植前后IL-10檢測(cè)及其臨床意義[J];中國(guó)醫(yī)師雜志;2005年05期

10 邵俊;陳穎;楊大明;;進(jìn)展期胃癌圍手術(shù)期細(xì)胞免疫水平變化及臨床意義[J];中國(guó)醫(yī)藥導(dǎo)刊;2012年01期

相關(guān)博士學(xué)位論文 前10條

1 王琳;新等位基因HLA-B~*15：133的鑒定、重鏈胞外區(qū)多肽的原核表達(dá)及特異性單克隆抗體的制備[D];北京市結(jié)核病胸部腫瘤研究所;2010年

2 王忠敏;影像引導(dǎo)下~125I放射性粒子組織間植入治療胰腺癌的基礎(chǔ)和臨床研究[D];蘇州大學(xué);2011年

3 楊傳標(biāo);連黛膠囊治療胃腸腫瘤臨床療效觀察與分子機(jī)理研究[D];廣州中醫(yī)藥大學(xué);2001年

4 韓鳳娟;海馬生髓丸對(duì)婦科惡性腫瘤減毒增效、抗腫瘤免疫機(jī)理的研究[D];黑龍江中醫(yī)藥大學(xué);2001年

5 韓秀婕;超聲引導(dǎo)瘤區(qū)注射高聚生協(xié)同微波治療肝癌局部細(xì)胞免疫變化與臨床療效的研究[D];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院;2003年

6 劉承利;轉(zhuǎn)4-1BBL基因的小鼠肝癌細(xì)胞疫苗抗腫瘤作用的研究[D];中國(guó)人民解放軍第四軍醫(yī)大學(xué);2003年

7 馬維東;肝癌組織中MAGE-3和NY-ESO-1的表達(dá)以及相應(yīng)抗原肽疫苗的基礎(chǔ)研究[D];天津醫(yī)科大學(xué);2003年

8 黃立中;益氣平懸飲對(duì)肺癌性胸水局部免疫功能影響及臨床療效之研究[D];湖南中醫(yī)學(xué)院;2003年

9 鄭大勇;完全人源化基因工程抗體anti-HBsAg Fab原核體系表達(dá)純化工藝路線放大可行性研究[D];第一軍醫(yī)大學(xué);2004年

10 蘇延軍;外周血單核細(xì)胞來(lái)源的樹(shù)突狀細(xì)胞體外培養(yǎng)及抗原負(fù)載策略性研究[D];天津醫(yī)科大學(xué);2004年

相關(guān)碩士學(xué)位論文 前10條

1 蘇紅權(quán);血清CA19-9在肝外惡性梗阻性黃疽的診斷價(jià)值[D];廣西醫(yī)科大學(xué);2011年

2 韓曉玉;肝門(mén)部膽管癌的診斷與手術(shù)治療[D];山西醫(yī)科大學(xué);2011年

3 苑曉娟;WT1-DNA疫苗聯(lián)合小劑量環(huán)磷酰胺抗腫瘤效應(yīng)的研究[D];山西醫(yī)科大學(xué);2011年

4 尹曦;參芪扶正注射液減輕肺癌化療后副反應(yīng)的臨床觀察[D];山東中醫(yī)藥大學(xué);2011年

5 唐鴻;消癌平注射液治療原發(fā)性肝癌的療效及對(duì)機(jī)體免疫功能的影響[D];南京中醫(yī)藥大學(xué);2011年

6 郭寧寧;肺癌患者TCR Vβ亞家族表達(dá)及T細(xì)胞功能的研究[D];廣東藥學(xué)院;2011年

7 石紅兵;CD8~+T細(xì)胞在胃癌組織中的表達(dá)及其臨床意義[D];蘇州大學(xué);2011年

8 匡志鵬;CD3AK細(xì)胞治療惡性腫瘤的實(shí)驗(yàn)和臨床初步應(yīng)用研究[D];廣西醫(yī)科大學(xué);2000年

9 曾剛;鏈球菌制劑抗瘤效應(yīng)與細(xì)胞因子關(guān)系的研究[D];蘇州大學(xué);2001年

10 胡沛臻;HSP70在人原發(fā)性肝細(xì)胞肝癌中的表達(dá)及其基因轉(zhuǎn)染對(duì)肝癌細(xì)胞系HepG2的影響[D];第四軍醫(yī)大學(xué);2001年

，

本文編號(hào)：2076953