重组人p53转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应

发布时间：2018-02-03 01:39

  本文关键词： 重组人p53腺病毒(rAd-p53) 树突状细胞 弥漫性大B细胞淋巴瘤　出处：《兰州大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:树突状细胞(dendritic cell,DC)是目前已知唯一能激活初始T细胞(naive Tcells)功能最强的抗原递呈细胞(antigen presenting cells,APC),因其强大的抗原递呈功能和激活抗原后特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocytes CTL)反应,成为肿瘤生物免疫治疗的重要载体,而淋巴瘤源性DC(L-DC)既带有淋巴瘤瘤细胞抗原,同时又是抗原提呈细胞,来源于患者本人,不存在MHC限制性,能有效地激活特异的抗肿瘤免疫,在淋巴瘤的生物免疫治疗中发挥重要作用。但L-DC与正常人CD14~+或CD34~+前体细胞来源的CD83~+的DC相比,其迁移、吞噬作用及抗原加工递呈功能减弱,使抗肿瘤免疫应答能力下降,同时由于肿瘤细胞释放细胞免疫抑制因子,引起DC的免疫耐受,这样就限制了DC在淋巴瘤免疫治疗中的作用,如何获得更具抗原递呈功能的DC,打破免疫耐受,成为目前研究的热点。有研究表明,p53基因突变和过度表达与NHL的发生发展有关[1],在弥漫性大B细胞淋巴瘤中,其异常表达率可高达50%[2]。目前,体内和体外试验,应用重组人p53腺病毒(rAd-p53),把wt-p53基因导入实体瘤中或与实体瘤细胞株共培养,可产生瘤细胞的生长抑制和诱导凋亡,同时增强了放疗的敏感性,从而使rAd-p53成为治疗实体瘤另外一种途径,为肿瘤的治疗提供了一种新的“武器”[3-10]。并且发现腺病毒p53修饰DC后可使其表达内源性抗原,上调DC共刺激分子表达,产生特异性CTL效应。因此,如何获得临床上需要的有功能的L-DC及纠正瘤细胞内的突变的p53基因的功能,是急需解决的问题。目前,虽然分子靶向治疗药物的问世,如CD20单抗(美罗华)等,使得DLBCL的治愈率提高了很多,但在淋巴瘤的治疗中如何解决微小残留病灶(MRD)、DC免疫耐受等问题,仍然是目前临床治疗淋巴瘤遇到的难题,因此,我们利用rAd-p53能否诱导淋巴瘤瘤细胞为成熟DC,作为肿瘤疫苗治疗淋巴瘤及解决MRD、DC免疫耐受问题是本实验目的所在。 方法:采集经病理确诊的弥漫性大B细胞淋巴瘤的淋巴结和外周血的单个核细胞(MNC)进行培养。分为3组:实验组A(淋巴结源性DC转染rAd-p53,L-rAd-p53-DC),实验组B(外周血源性DC转染rAd-p53,P-rAd-p53-DC);对照组A(淋巴结源性DC转染rAd,L-rAd-DC),对照组B(外周血源性DC转染rAd,P-rAd-DC);空白对照组A(淋巴结源性DC未转染,L-N-DC),空白对照组B(外周血源性DC未转染,P-N-DC),以上各组均用GM-CSF+IL-4培养,至第7天,实验组加rAd-p53;对照组加rAd;空白对照组未加任何物质;各组于培养第8天加入TNF-a+CD40mAb继续培养48小时后,流式细胞仪检测DC免疫表型、酶联免疫吸附法(ELISA)进行上清夜IL-12水平测定,四甲基偶氮唑盐比色(MTT)法观察各组刺激T淋巴细胞增殖能力(MLR)及乳酸脱氢酶(LDH)法针对淋巴瘤瘤细胞的细胞毒作用(CTL效应)。在分离外周血过程中,贴壁的进行DC培养,非贴壁的培养成效应细胞。 结果:淋巴结源性和外周血源性的单个核细胞,经培养后均成功获得具有树突状突起的细胞,但以实验组树突状突起明显。结果显示:DC的表型(CD_(1a)除外)CD83、CD80、CD86和HLA-DR实验组均较对照组及空白对照组明显增高(p＜0.05)。上清液中IL-12分泌水平实验组均较对照组及空白对照组明显增高(p＜0.05)。实验组具有明显的刺激自体淋巴细胞增殖的能力,且刺激能力随rAd-p53-DC与淋巴细胞比例的增加而升高。对照组也能刺激同种自体淋巴细胞增殖的能力,但较实验组组差(p＜0.05)。对靶细胞的细胞毒作用(CTL效应)结果显示,实验组介导的细胞杀伤率显著高于对照组及空白对照组(P＜0.05)。且实验组A的CTL效应明显高于实验组B,两者之间有显著性差异(P＜0.05)。 结论:利用rAd-p53可以成功地诱导弥漫性大B细胞淋巴瘤瘤细胞和外周血来源单个核细胞为树突状细胞,形态学观察及细胞表型检测证实均为成熟DC,其IL-12分泌水平及介导的MLR和CTL效应明显强于非rAd-p53组所诱导的DC组。提示以rAd-p53-DC为基础的肿瘤疫苗有可能在解决淋巴瘤的MRD、DC免疫耐受等问题上发挥强大的治疗作用,为淋巴瘤的治疗研究提供了一个新的思路和策略。
[Abstract]:Objective: dendritic cells (dendritic, cell, DC) is currently the only known to activate naive T cells (naive Tcells) the most potent antigen-presenting cells (antigen presenting cells, APC), because of its powerful antigen-presenting function and activation of antigen specific cytotoxic T lymphocytes (cytotoxic T lymphocytes CTL) the reaction, become an important carrier for tumor immunotherapy, and lymphoma derived DC (L-DC) is a tumor cell antigen, and antigen presenting cells from the patients themselves, without limitation of MHC, can effectively activate specific antitumor immunity and play an important role in the biological treatment of lymphoma. But compared with normal CD14~+, L-DC or CD34~+ precursor cells derived CD83~+ DC migration, phagocytosis and antigen processing and presentation function, make the antitumor immune response ability, at the same time due to the tumor Cellular immune cells release inhibiting factor, DC induced immune tolerance, thus limiting the role of DC in lymphoma treatment, how to get more antigen-presenting function of DC, to break the immune tolerance and become the hotspot of research. Studies have shown that p53 gene mutation and overexpression is related to carcinogenesis and development of [1] NHL and in diffuse large B cell lymphoma, the abnormal expression rate can be as high as 50%[2]. at present, in vivo and in vitro experiments using recombinant human p53 adenovirus (rAd-p53), wt-p53 gene was transfected into solid tumors or tumor cell lines were cultured, can produce tumor cell growth inhibition and apoptosis induction, at the same time to enhance the sensitivity of radiotherapy, so rAd-p53 has become the treatment of solid tumors another way for tumor therapy provides a new "weapon" [3-10]. and adenovirus p53 DC modified by the expression of endogenous antigen, DC up-regulated the expression of costimulatory molecules to produce specific CTL effect. Therefore, how to obtain the p53 gene mutations in tumor cell L-DC and correct the clinical need of function within the function, is an urgent problem. At present, although the molecular targeted therapy drugs, such as CD20 (Mei Luohua), the monoclonal antibody the cure rate of DLBCL has improved a lot, but in the treatment of lymphoma in how to solve the minimal residual disease (MRD), DC immune tolerance and other issues, is still the current clinical treatment of lymphoma problem, therefore, we use rAd-p53 can induce lymphoma tumor cells into mature DC, as a tumor vaccine for the treatment of lymphoma and MRD. The immune tolerance of DC problem is the purpose of this study lies.
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