HLAⅠ类分子的缺失与肿瘤逃逸

人类白细胞抗原（ＨＬＡ）Ⅰ类分子在肿瘤细胞上的缺失呈异质性,现已发现至少有５种之多,并有证据显示这一改变与肿瘤细胞逃避免疫细胞的杀伤、促进肿瘤进展和转移密切相关,此外,还有可能影响肿瘤的特异性免疫或生物治疗。     

食管鳞癌组织HLA-Ⅰ和TAP1及LMP2的表达相关性研究

目的探讨食管鳞癌组织中发生肿瘤免疫逃逸机制的相关指标人类白细胞抗原-Ⅰ(human leukocyte antigen-Ⅰ,HLA-Ⅰ)、抗原相关转运蛋白1(transporter associated with antigen proeessing1,TAP1)和低分子量多肽2(low molecular weight protein2,LMP2)的表达,分析三者表达的差异性和相关性。方法应用免疫组化技术,检测济宁医学院附属医院胸外科2012-01-01-2013-12-31收治的临床资料完整的手术切除87例食管癌和配对正常食管组织中HLA-Ⅰ、TAP1和LMP2的表达,并分析其在食管癌组织中表达变化与临床病理特征的关系。结果正常黏膜组织样本中HLA-Ⅰ阳性表达率为100.0%(87/87),TAP1为98.9%(86/87),LMP2为98.9%(86/87)。在食管癌组织中HLA-Ⅰ阳性表达率为42.5%(37/87),TAP1为41.4%(36/87),LMP2为46.0%(40/87)。与病理因素的相关分析显示,癌组织的分化程度、是否有淋巴结转移和肿瘤的TNM临床分期均与HLA-Ⅰ、TAP1和LMP2的表达相关,P值均<0.05。HLA-Ⅰ类分子的表达与TAP1(P=0.05)和LMP2(P=0.04)的表达呈正相关,即随着TAP1和LMP2的低表达,HLA-Ⅰ也出现低表达。结论在肿瘤发生免疫逃逸机制中,食管鳞癌组织HLA-Ⅰ、TAP1和LMP2出现低表达与组织分化程度、淋巴结转移和TNM临床分期相关。提示HLA-Ⅰ、TAP1和LMP2可能与食管鳞癌的发生和转移有关,三者可同成为食管癌诊断和预警转移有价值的生物标志物。     

肿瘤细胞HLAⅠ类分子表达对NK抗性的影响及IFN-γ的调节作用

目的 探讨肿瘤细胞表面HLAⅠ类分子的表达与NK杀伤的关系及IFN－γ的调节作用。方法 用间接免疫荧光法检测7种肿瘤细胞表面HLA－ABC分子的表达;用鼠抗人HLA－ABC单抗封闭靶细胞后,观察NK杀伤的变化;用IFN-γ处理靶细胞后,观察瘤细胞表面HLA分子表达的变化及NK杀伤的变化。结果 不同肿瘤细胞株表达HLA－ABC分子的比例及荧光强度均不相同,除Karpas外,均表现为不同程度的下降。HLA－ABC表达阴性的K562细胞对NK杀伤最敏感,其他细胞的敏感性均有下降。肿瘤细胞表达HLA－ABC分子的表达多有不同程度的下降。用抗HLA单抗封闭相应位 点后,可使NK杀伤明显增强。用IFN－γ500U／ml处理48h后,白血病细胞K562、黑色素瘤细胞M21和高转移肺癌细胞PG表面HLAⅠ类分子表达明显增加,对NK杀伤的敏感性降低;而人T细胞淋巴瘤Karpas、髓样白血病细胞HL60和结直肠癌细胞HT29经处理后,HLAⅠ类分子表达无明显变化,对NK杀伤的敏感性反而明显增强。IFN－γ促进HL60和HT29细胞的凋亡。结论 NK细胞能识别HLA－ABC分子表达下降或缺陷的肿瘤细胞并发挥杀伤作用;INF－γ能恢复部分肿瘤细胞HLA－ABC分子的丢失,并能促进部分肿瘤细胞的凋亡,提高肿瘤对机体抗瘤机制的敏感性。     

乳腺癌组织中C-erbB-2蛋白和MHCⅠ类分子表达与预后的关系

目的了解乳腺癌组织中C-erbB-2蛋白和MHCⅠ类分子表达与预后的关系。方法58例乳腺癌根据有无淋巴结转移分为淋巴结转移组和淋巴结未转移组;根据组织学分级分为Ⅰ级、Ⅱ级和Ⅲ级。应用免疫组织化学S-P法检测乳腺癌组织C-erbB-2蛋白表达;应用流式细胞仪检测MHCⅠ类分子表达。结果乳腺癌淋巴结转移组C-erbB-2蛋白的阳性表达率(84.2%)明显高于淋巴结未转移组(41.0%);C-erbB-2蛋白在Ⅰ级、Ⅱ级和Ⅲ级的阳性表达率分别为42.1%、55.5%和75.0%;MHCⅠ类分子在Ⅰ级、Ⅱ级和Ⅲ级的阳性表达率分别为50.21%±15.70%、30.17%±16.23%和14.27%±9.36%。结论C-erbB-2蛋白和MHCⅠ类分子的表达可作为判断乳腺癌预后的重要指标。     

苯乙酸钠增强肿瘤细胞表面HLA分子的表达

以分化绣导剂苯乙酸钠处理肿瘤细胞,以ELISA检测肿瘤细胞表面HLA Ⅰ、Ⅱ类分子表达的变化。结果发现MCF-7、MDA-453、MKN-45以及Hela细胞表面HLA Ⅰ类分子表达较低,而MKN-28和3AO细胞表面HLA Ⅰ类分子高表达。MDA-453、MKN-28、MKN-45、Hela以及3AO细胞表面亦表达HLA Ⅱ类分子,而MCF-7细胞表面缺乏HLA Ⅱ类分子。苯乙酸钠能够诱导MCF-7细胞表面表达HLA Ⅱ类分子;增强MCF-7细胞表面HLA Ⅰ类分子,MDA-453、MKN-28、MKN-45、Hela以及3AO细胞表面HLA Ⅰ、Ⅱ类分子的表达。并且肿瘤细胞表面HLA Ⅰ类分子的表达与苯乙酸钠的作用时间和剂量呈正相关。     

肝癌细胞系中DNA甲基转移酶和HLAⅠ类分子的表达及意义

目的:探讨甲基转移酶(DNMT)3a及HLA Ⅰ类分子在肝癌细胞系中表达及意义.方法:提取HepG2、MHCC97L、MHCC97M3、SMMC7421和BEL7402的mRNA,应用RT-PCR及实时定量PCR检测DNMT及HLA的表达情况.结果:RT-PCR显示.在5个肝癌细胞系中DN-MT1和DNMT3b均高表达.而DNMT3a在高转移的MHCC97M3中的表达低于其他细胞.另外,HLA-A、HLA-B和HLA-C则普遍低表达.定量PCR示,DNMT3a在MHCC97L中的相对表达量为1,在MHCC97M3中为0.33.同时,前者的HLA-A、HLA-B和HLA-C的相对表达量分别为4、4.199和3.9,而在后者中分别是2.129、2.621和2.55.结论:在相同的遗传背景下,DNMT3a和HLAⅠ类分子的高表达与肝癌细胞系的低转移能力相关.     

制备抗原肽所用宫颈癌细胞培养方法的建立及HLA表达的检测

目的 探讨制备抗原肽所用宫颈癌细胞的培养方法及检测其HLA的表达。方法 先利用CO2孵箱和普通培养瓶进行培养，测定其生长状况，然后流式细胞仪检测HLA-Ⅰ、Ⅱ类分子的表达，再用免疫荧光法检测不同浓度IFN-γ对Hela细胞HLA-Ⅰ、Ⅱ类分子的表达和IFN-γ对不同培养时间Hela细胞HLA分子的表达。结果 IFN-γ能够明显提高对数生长期的Hela细胞HLA-Ⅰ类分子的表达，而IFN-γ刺激前后Hela细胞HLA-Ⅱ类分子的表达均为阴性。结论 本方法可缩短细胞培养时间，利用IFN-γ诱导细胞提高HLA-Ⅰ类分子的表达，为提供制备抗原肽所用宫颈癌细胞奠定了基础。     

重组hDCN对肝癌细胞株HepG2表面HLA分子表达的影响

目的研究peDNA3．1（＋）-核心蛋白聚糖（DCN）重组质粒对体外培养的HepG2细胞株表面HLAⅠ、Ⅱ类分子表达的影响,探讨DCN增强抗肿瘤免疫应答的作用。方法重组质粒pcDNA3．1（＋）-DCN转染HepG2细胞,用流式细胞术（FCM）检测转染前后HepG2细胞表面HLAⅠ、Ⅱ类分子的表达。结果HepG2细胞低表达HLAⅠ、Ⅱ类分子,经转染重组质粒作用后,HLAⅠ、Ⅱ类分子的荧光强度明显增强。结论rhDCN核心蛋白能上调肿瘤细胞表面HLAⅠ、Ⅱ类分子的表达,这一作用可能参与其抗肿瘤效应机制。     

鼻咽癌中EB病毒LMP1基因N端XhoⅠ酶切位点的丢失

背景与目的:众所周知, EB病毒 LMP1基因在鼻咽癌变过程起着一定的作用.本研究通过检测广东地区鼻咽癌组织 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失,探讨 LMP1基因变异在鼻咽癌发生发展中的作用.方法 :收集中山大学肿瘤防治中心鼻咽癌患者鼻咽新鲜活检标本 63例.收集 EB病毒健康携带者外周血单个核细胞 (PBMCs) 10例作为对照.采用 QIAamp DNA Mini Kit和 QIAamp DNA Blood Mini Kit分别抽取组织和外周血单个核细胞的 DNA,应用巢式 PCR扩增 EB病毒 LMP1基因的 N-末端区,并用 XhoⅠ对扩增产物进行酶切.采用四色荧光 末端终止法对扩增产物进行序列分析.结果: 10例健康携带者外周血单个核细胞的 EB病毒 LMP1基因 N-末端区均未见 XhoⅠ酶切位点的丢失.63例鼻咽癌组织中有 50例(79.37%)出现 XhoⅠ酶切位点的丢失(XhoⅠ-loss),还有 4例(6.34%)为 XhoⅠ酶切位点部分丢失,只有 9例(14.29%)未见 XhoⅠ酶切位点的丢失(wt-XhoⅠ ).除了 XhoⅠ酶切位点的丢失 (nt: 169423～169428; GAGCTC→ GATCTC)外,还发现四个错义点突变.结论:本研究所检测的广东地区 EB病毒健康携带者外周血单个核细胞所携带的 EB病毒 LMP1基因为 wt-XhoⅠ,而在鼻咽癌组织中主要为 XhoⅠ-loss.因此,我们认为 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的.     

冷冻影响肝癌细胞HLA和B7分子表达的实验研究

目的：探讨冷冻治疗后机体免疫功能提高的机制。方法：应用流式细胞仪检测人肝癌细胞经0℃或－20℃冷冻后，HLA和B7分子表达率和平均荧光强度的变化。结果：0℃组与对照组比较，HLA－Ⅰ分子和B7－2分子的表达率和平均荧光强度均无明显变化；HLA－Ⅱ分子和B7－1分子的表达率增高，平均荧光强度增强。－20℃组与对照组比较，HLA－Ⅰ、HLA-Ⅱ、B7-1和B7－2分子的表达率均增高，平均荧光强度均增强。－20℃组和0℃组比较，HLA－Ⅱ和B7－1分子的表达率和平均荧光强度均无显著性差异；HLA－Ⅰ和B7－2分子表达率均增高，平均荧光强度均增强。结论：0℃和－20℃冷冻可增强人肝癌细胞HLA和B7分子的表达，这可能是冷冻治疗提高机体免疫功能的机制之一。     

杂色曲霉素对人外周血单个核细胞TAP1及LM P2基因表达的影响

目的:探讨杂色曲霉素(ST)对体外培养的人外周血单个核细胞(HPBMc)抗原加工递呈相关基因TAP1、LMP2表达的影响.方法:采用RT-PCR和FCM方法,分析5种浓度ST处理对体外培养的HPBMc TAP1、LMP2基因在mRNA和蛋白水平表达的影响.结果:RT-PCR检测结果表明,ST处理24h后,低浓度ST组(0.125mg/L、0.25mg/L)HPBMc TAP1 mRNA相对表达量明显高于对照组,而较高浓度组(0.5mg/L、1mg/L和2mg/L)TAP1 mRNA则明显低于对照组,尤以1mg/L和2mg/L组为著.FCM定量分析表明,低浓度ST组HPBMc TAP1蛋白平均表达量略低于对照组,但无统计学意义,而较高浓度组TAP1表达则明显低于对照组(P＜0.05).在0.125mg/L到1mg/L浓度范围内,各ST处理组HPBMc LMP2蛋白表达量和mRNA的相对表达量均高于对照组.结论:ST对体外培养的HPBMc TAP1mRNA表达和蛋白表达的影响按ST剂量不同而变化,在0.125～1mg/L浓度范围内ST在mRNA及蛋白水平均可剂量依赖地提高体外培养的HPBMc LMP2的表达.     

c-Myc and EBV-LMP1: two opposing regulators of the HLA class I antigen presentation machinery in epithelial cells

Background:

Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) up-regulates the human leukocyte antigen (HLA) class I antigen presentation machinery (APM). This appears counterintuitive with immune evasion in EBV-associated tumours like nasopharyngeal carcinoma (NPC).

Methods:

Latent membrane protein 1-transfected epithelial cell lines were used as a model system to study the impact of LMP1 and c-Myc on HLA class I components. The expression of components of the HLA class I APM, c-Myc and Ki-67 was analysed in LMP1+ and LMP1− NPC by immunohistochemistry.

Results:

In epithelial cells, LMP1 up-regulated HLA class I APM. This effect could be counteracted by c-Myc, which itself was up-regulated by LMP1 apparently through IL6 induction and Jak3/STAT3 activation. Studies of NPC biopsies revealed down-regulation of HLA class I APM expression. No difference was observed between LMP1+ and LMP1− NPC. However, expression of Ki-67 and c-Myc were up-regulated in LMP1+ tumours.

Conclusion:

These findings raise the possibility that c-Myc activation in NPC might antagonise the effect of LMP1 on HLA class I expression thus contributing to immune escape of tumour cells.     

Human leukocyte antigen class I expression is an independent prognostic factor in advanced ovarian cancer resistant to first-line platinum chemotherapy

Background:

Loss of HLA class I is important in ovarian cancer prognosis but its role as a prognostic indicator in relation to therapy remains unproven. We studied the prognostic potential of this antigen and its significance in relation to platinum therapy.

Methods:

A total of 157 primary ovarian cancers were assessed for HLA class I immunohistochemically and linked to a comprehensive database of clinicopathological variables, treatment details, and platinum sensitivity.

Results:

Tumours expressing high levels of HLA class I had significantly improved survival (P=0.044). There was a 19-month difference in the median overall survival between tumours with high and low antigen expression. HLA class I antigen expression, stage, and platinum sensitivity were independently predictive of prognosis on multivariate analysis. HLA class I antigen was shown to be expressed at higher levels in patients with good overall survival in platinum-resistant patients (P=0.042). HLA class I significantly correlated with overall survival on multivariate analyses (P=0.034).

Conclusion:

Low-level HLA class I expression is an independent prognostic indicator of poor clinical outcome in ovarian cancer. The survival advantage of patients with platinum-resistant tumours expressing high levels of HLA class I suggests that immunotherapy may be of use in these ovarian cancers resistant to standard chemotherapy.     

SIRT7 Exhibits Oncogenic Potential in Human Ovarian Cancer Cells

Background: Sirtuin7 (SIRT7) is a type of nicotinamide adenine dinucleotide oxidized form (NAD+)-dependentdeacetylase and the least understood member of the sirtuins family; it is implicated in various processes, such asaging, DNA damage repair and cell signaling transduction. There is some evidence that SIRT7 may function asa tumor trigger for human malignancy. Here, we aimed to explore the biological function of SIRT7 in ovariancarcinoma cells and its potential mechanism. Materials and Methods: Expression of SIRT7 in ovarian cancercell lines was detected by western blotting. Transduced cell lines with SIRT7 knockdown or overexpression wereconstructed. Cell viability, cologenic, apoptosis-associated and motility assays were performed to elucidate thebiological function of SIRT7 in ovarian cancer cells. Results: SIRT7 demonstrated a higher level in ovariancancer cell lines compared with normal cells. On the one hand, down-regulation of SIRT7 significantly reducedovarian cancer cell growth, repressed colony formation and increased cancer cell apoptosis; on the other hand,up-regulation promoted the migration of cancer cells. Additionally, repression of SIRT7 also induced changein apoptosis-related molecules and subunits of the NF-κB family. Conclusions: In the present study, our dataindicated that SIRT7 might play a role of oncogene in ovarian malignancy and be a potential therapeutic target.     

Anti-viral cytotoxic T cells inhibit the growth of cancer cells with antibody targeted HLA class I/peptide complexes in SCID mice

A number of experimental antibody mediated cancer therapies aim to redirect cytotoxic T cells (CTLs) of non-tumour specificity to cancer cells. It has been previously demonstrated that cancer cells targeted with recombinant HLA-class I/viral peptide complexes via antibody delivery systems can be killed by virus specific CTLs. This novel therapeutic system has been developed with a simple pre-clinical model using the recombinant anti-CD20 B9E9 scFvSA fusion protein to target HLA-A2/peptide complexes to CD20 +ve Daudi lymphoma cells. In vitro data confirmed that, although binding of the B9E9 scFvSA fusion protein alone to Daudi cells had no effect on their growth, effective CTL mediated killing of Daudi cells could be achieved by targeting with B9E9 sfvScSA and recombinant HLA-A2/MI complexes at dilutions as low as 100 pg/ml. In contrast the free HLA-A2/MI complexes only significantly inhibited CTL activity at concentrations in excess of 100 ng/ml. The in vivo tumour protection assays in SCID mice demonstrated that only 1 of the 4 mice that received anti-HLA-A2/M1 CTLs and Daudi cells targeted with the B9E9 scFvSA fusion protein and HLA-A2/M1 complexes developed a tumour. In contrast in the control mice that received CTL and native Daudi cells all 4 developed tumours, as did all 4 that received targeted Daudi cells but no CTLs. Similar results were obtained in a parallel experiment using Daudi cells targeted with B9E9 scFvSA and HLA-A2/BMLF1 complexes and a CTL line to HLA-A2/BMLF1. The demonstration of in vivo activity for targeted HLA class I/peptide complexes combined with anti-viral T cells, supports the further clinical development of the system where it may be combined with autologous CTLs produced by vaccination or ex vivo expansion.     

卵巢癌组织和细胞株中miR-141表达水平与卡铂耐药性的关系

目的探讨上皮性卵巢癌中微小RNA-141(microRNAs,miRNAs)的表达情况,及其与卡铂治疗敏感性的相关性。方法上皮性卵巢癌68例,术后接受规范的多西他赛+卡铂化疗方案治疗。30例卵巢癌良性肿瘤标本作为正常对照组。采用real-time PCR方法检测标本中miR-141的表达水平,采用MTT和克隆形成实验方法,检测miR-141对浆液性卵巢癌细胞株SKOV-3和人卵巢透明细胞癌细胞ES-2卡铂敏感度的影响。结果 30例卵巢良性肿瘤标本中miR-141表达水平为(0.09±0.0032),68例卵巢上皮癌组织中miR-141表达水平为(5.03±0.17),两组间有显著性差异(P=0.0014)。68例卵巢上皮癌组织中58例miR-141表达水平高于良性肿瘤的平均表达水平,其相对表达水平为(5.54±0.11);有10例miR-141表达水平低于良性肿瘤的平均表达水平,其相对表达水平为(0.03±0.0093)。卡铂耐药患者19例(19/68,27.9%),卡铂敏感患者49例(49/68,72.1%)。miR-141表达水平与患者对卡铂的敏感性呈显著负相关性(γ=-0.7816,P=0.0032)。miR-141表达与患者5年生存率亦呈显著负相关性(γ=-0.5296,P=0.0128)。miR-141表达与卵巢上皮癌患者年龄、病理类型、组织学分级、FIGO分期、残留肿瘤大小均无相关性(P>0.05)。MTT和克隆形成实验结果显示,与未转染组和转染阴性miRNA模拟物(miRNA mimics)组比较,转染miR-141模拟物(miR-141 mimics)组SKOV-3和ES-2细胞对卡铂的敏感性显著降低。结论检测MiR-141的表达水平,对预测上皮性卵巢癌对卡铂敏感性、评估患者预后及其合理指导卵巢癌的综合治疗等具有重要的临床意义。     

Downregulation of HLA Class I molecules in the tumour is associated with a poor prognosis in patients with oesophageal squamous cell carcinoma

As antigenic peptides in the context of human leukocyte antigen (HLA) class I molecules are recognised by cytotoxic T lymphocytes (CTL), the downregulation of HLA class I molecules is one of the reasons why tumour cells can evade CTL-mediated anti-tumour immunity. In this study, we investigated HLA class I expression in oesophageal squamous cell carcinoma (ESCC) (n=70) and in their metastatic lesions (lymph nodes (n=40) and liver (n=3)), by immunohistochemistry with anti-HLA class I monoclonal antibody (EMR8-5). As a result, the downregulation of HLA class I expression in primary lesions of ESCC was observed in 43%, and that in metastatic lymph nodes was noted in 90%. Furthermore, patients with preserved HLA class I expression in primary tumours showed a better survival in comparison to those with downregulated HLA class I molecules (P<0.01). Furthermore, multivariate analysis using Cox's proportional hazards model revealed that the downregulated expression of HLA class I in primary lesions was an independent, unfavourable prognostic factor (P<0.01). In conclusion, the downregulation of HLA class I expression frequently occurred in primary tumour and, to a greater extent, in metastatic lesions of patients with ESCC and was associated with patient survival.     

逆转录病毒载体介导的γ-干扰素基因在人肝癌细胞中的表达研究

以逆转录病毒载体(pLXSN)将人源γ-干扰素(huIFN-γ)基因转导入4种不同的人肝癌细胞株,经G418抗性筛选均获得了阳性克隆.PCR和RT-PCR结果均表明IFN-γ基因已在基因组DNA中整合并表达.IFN-γ生物活性检测结果表明,在基因修饰的4种不同个体人肝癌细胞株及同一细胞株的5种不同克隆中,分泌的IFN-γ活性有较大差别.流式细胞仪检测细胞表面HLAⅠ类分子,结果表明基因修饰肿瘤细胞表面HLAⅠ类分子表达有显著提高.同时还首次对HLAⅠ类分子专一位点A2表达进行了分析,结果表明经IFN-γ基因修饰后,A2表达增加与HLAⅠ类分子总体增加相一致,本实验为进行基因工程修饰的肿瘤疫苗研究奠定了基础.