肺癌患者外周血树突状细胞的生物学特性研究

目的：探讨肺癌患者外周血来源的树突状细胞（dendritic cells, DC）生物学特性。方法：从肺癌患者外周血分离获得单个核细胞，用细胞因子GMCSF (100 μg/L)，IL4 (50 μg/L)体外培养诱导。凋亡肿瘤细胞负载24 h后加入TNFα（10 μg/L）或CD40激发型单抗于培养DC中，继续诱导3～4 d。分别测定DC的表型、DC摄取抗原能力；混合淋巴细胞反应（MLR）对T细胞增殖能力的测定；细胞计数和3HTdR掺入观察DC对T细胞的激发和扩增效应，并与健康志愿者外周血来源的DC进行比较。结果： 患者外周血单个核细胞和正常人外周血单个核细胞来源的DC均高表达CD1α，CD83，CD80，CD86和HLADR等DC的相关抗原和共刺激分子；患者的未成熟DC能有效摄取FITCDextran，经TNFα或CD40激发型单抗激发诱导后，成为成熟和有功能的DC,几乎完全失去对抗原的摄取能力，与健康人外周血来源DC组相比无明显差别(P>0.05)；患者单个核细胞来源的DC在体外具有激发自体和同种异体外周血T细胞增殖的能力。结论：肺癌患者的外周血来源单个核细胞可以诱导成为具有功能的成熟DC。     

急性B淋巴细胞白血病免疫球蛋白重链可变区的细胞毒T淋巴细胞识别表位

目的分析儿童急性B淋巴细胞白血病(B-ALL)细胞免疫球蛋白重链可变区(IgHV)的基因特征,确定IgHV的细胞毒T淋巴细胞(CTL)识别表位。方法PCR扩增37例儿童B-ALL的7个：IgHV基因家族,PCR产物直接测序,利用生物信息资源,分析所得序列的特征并预测B-ALL细胞IgHV上与HLA-A*0201分子结合的九肽。合成预测九肽QLNQSGAEV,进行T-B杂交瘤细胞系(T2)结合实验,用荷肽抗原呈递细胞,反复刺激HLA-A*0201正常供者外周血中肽特异性CD8^ T细胞的扩增。结果37例B-ALL均检测到IgHV基因,经PCR产物测序,得到40份IgHV基因序列,它们优先利用基因片段VH4．34(12．5％)和VH4．59(10．0％)。V-ALL细胞的IgHV基因利用D7．27片段的频率(15．4％)和在DJH结合区缺乏非编码核苷酸的频率(20．0％),均明显高于正常成人外周血淋巴细胞的IgHV基因。17．5％的序列含有不到2％的替代突变。从40份B-ALL细胞的IgHV序列,预测出12条与HLA-A*0201分子有高亲和力的九肽,10条(83．0％)位于框架1和3区。合成九肽QLNQSGAEV使T2细胞表面HLA-A*0201分子的表达强度提高1．6倍。HLA-A*0201正常供者外周血中的QLVQSGAEV特异性CD8^ T细胞,在荷肽抗原呈递细胞的重复刺激下,由2轮刺激后的1．6％增至3轮刺激后的82．6％。结论儿童B-ALL的IgHV基因为胚系基因,重链框架区有与HLA-A*0201结合的抗原九肽,这些九肽能够诱导特异性CD8^ T细胞的活化和增殖。     

修饰的肿瘤抗原肽CEA-610D疫苗的体外抗肿瘤作用

目的: 评价修饰的CEA610D抗原肽疫苗体外抗肿瘤免疫的有效性,为其临床应用提供依据。方法： 多肽固相合成法合成HLAA2 CEA修饰肽(CEA610D)、HLAA2天然肽CEA605613（天然肽）和HLAA2无关肽MAGE3（无关肽）,采用同种异体混合淋巴细胞与肽共孵育的方法,诱导肽特异性细胞毒性T淋巴细胞(CTL),利用MTT法检测CTL的增殖和杀伤活性;流式细胞术观察细胞表型;RTPCR检测细胞穿孔素的表达;ELISA法检测CTL上清中IFNγ的水平。结果：CEA610D疫苗产生肽特异性CTL的能力最强（P<0.05）,其CD8+T细胞数也高于天然肽组和无关肽组;在效靶比为40〖DK〗∶1时,CEA610D和天然肽所诱导的CTL对CEA+HLAA2限制性人结肠癌细胞T84的杀伤活性可达(56.7±3.73)%和(51.2±1.86)%,而3种肽特异性CTL细胞对CEA+HLAA2人结肠癌lovo细胞杀伤活性维持在本底水平;CEA610D组的CTL所释放的杀伤相关介质穿孔素和上清中IFNγ的水平也显著高于其余两组（P<0.01）。结论：与天然肽相比,CEA610D疫苗可打破自身表位的免疫耐受,在体外抗肿瘤免疫中更具优势。     

肝癌MAGE-n抗原HLA-A2限制性细胞毒性T细胞表位的预测

目的 预测肝癌MAGE—n抗原HLA—A2限制性细胞毒性T淋巴细胞(CTL)表位。方法 应用SYFPEITHI超基序多项式法远程预测系统和量化基序多项式法结合的预测方法，筛选MAGE—n抗原HLA—A2限制性CTL表位。结果 筛选到MAGE—n抗原5个HLA—A2限制性CTL表位(九肽)。结论 SYFPEITHI超基序法和量化基序多项式法结合的预测方法是一种高效和准确的表位预测方法。     

食管癌患者外周血MAGE-C2抗原特异性CD8+T细胞的功能及临床意义

目的:评价食管癌患者外周血恶性黑色素瘤相关抗原(melanoma-associated antigen,MAGE)-C2抗原特异性CD8+T细胞的功能,并分析其与临床病理参数的相关性.方法:密度梯度离心法获取130例食管癌初治患者(收集自2014年11月至2015年11月郑州大学第一附属医院胸外科)的PBMC,流式细胞术、RT-PCR方法筛选HLA-A2* 0201和MAGE-C2共阳性的患者,其PBMC培养7、14 d后加MAGE-C2(336-344)抗原肽1μg/m1、IL-2 100 μg/ml和CD3/CD28 Dynabeads 2μl处理,诱导扩增抗原特异性CD8+T细胞,流式细胞术检测CD8+T细胞表面CD107a和胞内IFN-γ的水平,并分析其与临床参数的相关性.结果:筛选出52例HLA-A2* 0201和MAGE-C2共阳性的患者,MAGE-C2(336-344)处理其PBMC后,MAGE-C2(336-344)处理组CD8+T细胞中CD107a+ IFN-γ+,CD107a+ IFN-γ-和CD107a IFN-γ+细胞的比例明显升高(P＜0.05);CDI07a的表达在早期、未发生淋巴结转移和高分化的食管癌患者明显高于晚期、发生淋巴结转移和低分化的癌症患者(P＜0.05);IFN-γ的表达在高分化的食管癌患者明显高于低分化的食管癌患者(P＜0.05).结论:成功诱导扩增MAGE-C2抗原特异性CD8+T细胞,并发现其功能与食管癌的分期和淋巴结转移相关,似可作为一种理想的免疫治疗靶点.     

肿瘤相关抗原OVA66单克隆抗体的制备和鉴定

目的：制备和鉴定人卵巢癌cDNA文库筛选到的肿瘤相关抗原OVA66的单克隆抗体,为研究其生物学功能提供手段。方法：采用基因重组方法构建pET32bOVA66重组表达质粒,经大肠杆菌诱导表达OVA66融合蛋白,利用NiTED亲和层析技术分离纯化HisOVA66融合蛋白;采用经典的杂交瘤技术制备OVA66单克隆抗体;ELISA和Western blotting鉴定单克隆抗体的生物学和免疫学特性,并对OVA66单克隆抗体在细胞免疫荧光法、流式细胞术和免疫组化等方法中进行了初步应用。结果：成功构建重组表达载体pET32bOVA66,并诱导表达和纯化OVA66融合蛋白。制备获得两株小鼠OVA66单克隆抗体杂交廇细胞株5F4和4G9。两株细胞分泌的抗体亚类均为IgG1,轻链为κ型,亲和力常数Ka分别为2.96×1010和0.4×1010L/mol,初步表位分析结果显示两者针对不同的抗原表位;两株抗体均可以采用细胞免疫荧光和流式细胞术检测OVA66的表达,5F4还可通过免疫组织化学检测肿瘤组织中OVA66的表达。结论：成功制备两株特异性的小鼠OVA66单克隆抗体杂交瘤细胞株,为进一步研究OVA66的生物学特性和临床应用奠定了基础。     

HLA-A*0201的基因克隆及逆转录病毒载体构建

CTL对肿瘤的特异性杀伤作用受到HLA的限制.在中国人群中,HLA-A2的频率最高,所以,克隆HLA-A*0201基因,构建逆转录病毒载体可以用于研究肿瘤抗原排斥基因及HLA-A*0201限制性的肿瘤抗原肽.为了克隆HLA-A*0201全长基因,我们首先抽提B淋巴母细胞(HLA-A*0201,A19)的总RNA,在HLA-A座位cDNA特异性引物两端分别加上HindⅢ,SalⅠ的酶切序列,作RT-PCR.用HindⅢ,SalⅠ同时酶切PCR产物及测序载体pBlueScript SK( /-),低熔点琼脂糖凝胶分离,切胶、纯化、连接,用TSS转化法转入XL1-Blue细菌中.接种至涂布X-gal,IPTG的含氯苄青霉素的LB平板上,挑选白色菌落,摇菌,抽     

造血干细胞移植后淋巴细胞增殖症患者单个核细胞来源DC负载EBV抗原肽制备DC-CIK

目的：以造血干细胞移植后并发淋巴细胞增殖症（posttransplant lympholiferative disorder, PTLD)患者外周血单核细胞培养诱导DC,负载抗原肽后制备DCCIK（cytokineinduced killer cell）,为探索新的PTLD治疗方法奠定基础。方法: 分离造血干细胞移植后EBV（EpsteinBarr virus)感染致PLTD患者外周血单个核细胞,贴壁细胞培养诱导DC,悬浮细胞诱导CIK;负载EBV抗原肽LMP2后建立DCCIK共培养体系。流式细胞仪分析共培养前后细胞的免疫表型,ELISA检测共培养前后细胞上清IFNγ的分泌水平,基因扫描仪分析T细胞受体(T cell receptor, TCR)β家族基因谱。结果: 成功制备负载EBV抗原肽的DCCIK,HLADR+CD86+DC细胞从诱导前的12.5%增加到91.17%;DCCIK共培养14 d后,两例患者的CIK数量分别增加了5.3和6.8倍;CD3+、CD8+、CD3+CD8+以及CD3+CD56+ 细胞比例在DCCIK共培养后均明显升高(均P<005)。抗原肽负载的DCCIK共培养体系中IFNγ的分泌水平明显高于未经抗原肽负载的DC组\[（1 332.6±92.38）pg/ml vs (693.42±62.41) pg/ml,P<0.01）\]。DCCIK培养后细胞的TCRβ家族基因在5.2家族出现单克隆表达峰。结论：EBV抗原肽负载后DC可诱导DCCIK共培养体系中CD3+CD8+以及CD3+CD56+ 细胞扩增,并分泌高水平IFNγ,为临床应用DCCIK对移植后EBV感染致PTLD患者进行过继性细胞免疫治疗提供实验基础。     

肿瘤抗原MAGE-12新的HLA-A2限制性细胞毒性T细胞表位的预测

目的预测黑色素瘤抗原MAGE-12的HLA—A2限制性CTL表位。方法采用SYFPEITHI超基序远程预测系统与量化基序多项式法、延展基序联合应用，筛选MAGE-12抗原HLA—A0201限制性CTL表位。结果共预测出5个MAGE-12抗原HLA—A2限制性CTL表位。结论超基序法与量化基序，延展基序联合应用可以提高预测效率，为实验方法探索MAGE-12的表位提供有用线索。     

肿瘤的HLA相关生物治疗

人类白细胞抗原(HLA)作为人类最复杂、最具多态性的遗传系统，其功能涉及到机体免疫的各个方面，肿瘤的发生发展和HLA有非常重要的关系，因此HLA相关的肿瘤生物治疗成为近年来肿瘤研究的热点，包括发现和鉴定某HLA基因型匹配的、CD8^ T细胞所识别的肿瘤特异性抗原肽，寻找CD4^ T细胞所识别的抗原肽，针对HLA表型缺失的不同原因而采取的各种HLA基因治疗，以及对HLA分子表达异质性和动态性等的研究，各项研究的进展将为肿瘤的免疫基因治疗提供更科学的理论依据。     

CD44 and Hyaluronan Binding by Human Myeloid Cells

The CD44 cell surface molecule has been shown to be the principal cell surface receptor for hyaluronan (or hyaluronic acid), a glycosaminoglycan component of marrow extracellular matrix. However, its affinity for hyaluronan is not constitutive, since it depends on the cell type, the stage of differentiation and on activation by external stimuli including certain anti-CD44 antibodies and phorbol esters. Except for a few lymphoid cell lines, hematopoietic cells do not spontaneously bind hyaluronan and initial studies reported that, contrary to lymphocytes, myeloid cells could not be activated to bind hyaluronan.

Because CD44 plays an important role in the initial phases of hematopoiesis, as shown by experiments using blocking anti-CD44 monoclonal antibodies, its capacity to mediate adhesion of primitive myeloid cells has been investigated. It was found that CD44 could mediate spontaneous adhesion to hyaluronan of immature myeloid cell lines KG1, KG1a, and TF1, which serve as a model for hematopoietic progenitors. However, despite expressing high amounts of CD44, no more than 15% of bone marrow progenitors could adhere to hyaluronan. Recent experiments have shown that a very important feature of CD44 is its capacity to be rapidly activated by certain antibodies and cytokines (GM-CSF and KL) from a low affinity to a high affinity state for hyaluronan. These data shed light on striking similarities in the functional regulation of CD44 and of the two integrin receptors VLA-4 (a4b1), and VLA-5 (a5b1), which are also expressed on hematopoietic progenitors. The relevance of these data to the regulation of normal hematopoiesis and mobilization of CD34+ progenitors in the view of cell grafting is analyzed. In addition, we show that in idiopathic myelofibrosis, the amount of hyaluronan is markedly increased in the extracellular matrix from the myeloproliferative spleen. Considering that the production of cytokines is enhanced in this disease, we discuss whether CD44-hyaluronan interaction may have a role in the pathophysiology of this myeloproliferative syndrome.     

钙结合蛋白S100A4与肿瘤

钙结合蛋白家族成员其结构相似,都具有与钙离子结合的区域,但功能不尽相同,特别是与肿瘤的关系的研究显示出不同的功能作用。S100A4是一个与细胞分化及肿瘤的发生、转移、预后密切相关的蛋白。文章将对钙结合蛋白S100A4基因的定位、结构特点、组织分布及其与肿瘤的关系等加以回顾。     

Binding and interstitial penetration of liposomes within avascular tumor spheroids

The liposomal delivery of cancer therapeutics, including gene therapy vectors, is an area of intense study. Poor penetration of liposomes into interstitial tumor spaces remains a problem, however. In this work, the penetration of different liposomal formulations into prostate carcinoma spheroids was examined. Spheroid penetration was assessed by confocal microscopy of fluorescently labeled liposomes. The impact of liposomal surface charge, mean diameter, lipid bilayer fluidity and fusogenicity on spheroid penetration was examined. A variety of different liposome systems relevant to clinical or preclinical protocols have been studied, including classical zwitterionic (DMPC:chol) and sterically stabilized liposomes (DMPC:chol:DOPE-PEG2000), both used clinically, and cationic liposomes (DMPC:DOPE:DC-chol and DOTAP), forming the basis of the vast majority of nonviral gene transfer vectors tested in various cancer trials. Surface interactions between strongly cationic vesicles and the tumor cells led to an electrostatically derived binding-site barrier effect, inhibiting further association of the delivery systems with the tumor spheroids (DMPC:DC-chol). However, inclusion of the fusogenic lipid DOPE and use of a cationic lipid of lower surface charge density (DOTAP instead of DC-chol) led to improvements in the observed intratumoral distribution characteristics. Sterically stabilized liposomes did not interact with the tumor spheroids, whereas small unilamellar classical liposomes exhibit extensive distribution deeper into the tumor volume. Engineering liposomal delivery systems with a relatively low charge molar ratio and enhanced fusogenicity, or electrostatically neutral liposomes with fluid bilayers, offered enhanced intratumoral penetration. This study shows that a delicate balance exists between the strong affinity of delivery systems for the tumor cells and the efficient penetration and distribution within the tumor mass, similar to previous work studying targeted delivery by ligand-receptor interactions of monoclonal antibodies. Structure-function relationships from the interaction of different liposome systems with 3-dimensional tumor spheroids can lead to construction of delivery systems able to target efficiently and penetrate deeper within the tumor interstitium and act as a screening tool for a variety of therapeutics against cancer.     

Binding of soybean agglutinin lectin to prostatic hyperplasia and adenocarcinoma

The binding of soybean agglutinin (SBA) lectin to routinely processed surgical pathology material from cases of prostatic hyperplasia, atypical hyperplasia, and adenocarcinoma was studied using an immunoperoxidase technique. Positive staining was obtained in eight of eight cases of nodular hyperplasia, eight of eight cases of atypical hyperplasia, and ten of 11 cases of adenocarcinoma. This report indicates that this lectin is not useful in distinguishing prostatic hyperplasia from adenocarcinoma.