MUC1/Y基因真核表达载体构建及MUC1、MUC1/Y体外修饰DC诱导特异性抗肿瘤免疫应答

目的　构建MUC1 Y全长cDNA真核表达载体 ,并以MUC1及MUC1 Y真核表达载体修饰树突状细胞 (DC)诱导特异性抗肿瘤免疫。方法　构建MUC1 YDNA真核表达载体 ,选取 10例HLA A2乳腺癌患者 ,体外IL 4、GM CSF联合诱导DC ,并以pCDNA3.1 MUC1和pCDNA3.1 MUC1 Y转染DC ,同时以空载体为对照 ,以pIRES2 EGFP MUC1 Y观察转染效率 ,转染后DC与自体T细胞混合培养 ,诱导CTL。以MCF 7为特异性靶细胞 ,Raji为非特异性靶细胞 ,通过乳酸脱氢酶释放实验检测杀伤活性 ,以annexinⅤ FITC检测特异性CTL诱导靶细胞凋亡的情况。以ELISA法测定基因修饰后DC刺激自体T细胞分泌IFN γ的能力。结果　酶切鉴定及测序结果表明成功构建了MUC1 Y真核表达载体pIRES2 EGFP MUC1 Y和pCDNA3.1 MUC1 Y。pIRES2 EGFP MUC1 Y转染效率基本为 10 %左右 ,DC中MUC1表达率为 8%。杀伤实验表明T DC pCDNA3.1 MUC的杀伤活性为 6 4 % ,T DC pCDNA3.1 MUC1 Y为 4 6 %。这两组间差异有显著性 ,同时与对照组比较差异均有显著性。annexinⅤ FITC标记实验显示 ,T DC pCDNA3.1 MUC1对靶细胞的杀伤和诱导凋亡的能力显著高于T DC pCDNA3.1 MUC1 Y及对照组。基因修饰DC刺激自体T细胞分泌IFN γ的能力显著升高。结论　构建的真核表达载体pIRES2 EGFP MUC1 Y可用     

MUC1/Y基因在胃肠道肿瘤中的表达及诱导抗肿瘤免疫

目的 研究MUC1／Y基因异常表达在消化道肿瘤中的临床意义；构建MUC1／YcDNA全长载体，修饰树突状细胞(DC)诱导杀伤细胞治疗消化道肿瘤。方法 采用RT-PCR方法检测标本中MUC1／YmRNA表达。选取8例HLA-A2^ 消化道肿瘤患者，体外诱导DC，以构建的MUCl／YcDNA真核表达载体pcDNA3．1-MUC1／Y修饰DC诱导CTL。通过检测对靶细胞SW620和Raji的杀伤作用和诱导靶细胞凋亡的能力来评价抗肿瘤免疫反应。结果 93．88％肿瘤组织表达较高水平的MUC1／Y。分析表明：消化道肿瘤中MUC1／YmRNA表达与组织学分型、肿瘤生长方式、浸润程度及TNM临床分期有关；与性别、肿瘤发生部位及分化程度无关。pcDNA3．1-MUC1／Y修饰DC诱导的CTL对特异性靶细胞的杀伤活性显著高于对照组，并能有效诱导靶细胞凋亡。结论 MUC1／YmRNA表达在消化道肿瘤组织具有重要意义；经pcDNA3．1-MUC1／Y修饰的DC可有效诱导特异性抗肿瘤免疫应答。     

MUC1/Y基因修饰的树突状细胞体外抗卵巢癌效应的研究

目的研究人MUC1／Y基因转染人外周血树突状细胞(DC)后在体外诱导的特异性抗MUC1／Y^ 卵巢癌效应。方法通过人外周血单核细胞诱导DC，采用脂质体法将已构建好的含人MUC1／Y全长cDNA的真核表达载体pEGFP-N1／MUC1/Y转染到DC中，荧光显微镜下观察其表达的蛋白定位；与自体T细胞共培养，观察其致敏的细胞毒性T淋巴细胞(CTL)对MUC1／Y^ 的卵巢癌细胞A2780的杀伤活性，并与转染空载体的DC诱导的CTL进行比较。结果pEGFP-N1／MUC1/Y转染DC后，其绿色荧光蛋白主要表达于DC细胞膜上，可诱导出对卵巢癌细胞系A2780特异性的细胞毒性T细胞。结论MUC1／Y基因修饰的DC可诱导特异的抗MUC1／Y^ 卵巢癌效应。     

MUC1 mRNA体外负载树突状细胞诱导细胞毒性T细胞对非小细胞肺癌的杀伤作用

 目的：将体外扩增的黏蛋白1 （MUC1） mRNA转染入成熟的树突状细胞（DCs）,观察其体外诱导的特异性抗肿瘤效应。方法：将分离提纯的单核细胞培养诱导为DC并用流式细胞术鉴定。构建pcDNA3.1（+）-MUC1质粒,体外转录为mRNA,电穿孔法转染DCs。定量 PCR检测转染的DCs中MUC1的表达;MTT法检测T细胞增殖情况;流式细胞术检测CD8+ 在T细胞的表达;LDH释放法测定细胞毒性,ELISA检测IFN-γ分泌水平。结果：流式细胞术结果表明成熟DCs标志表型的表达明显高于对照组。定量PCR结果说明转染后的DCs MUC1 mRNA相对表达量增高。转染组DCs与T细胞按1∶10共培养时,刺激增殖能力明显高于未转染组,且CD8+  T细胞表达率高于未转染组,诱导产生特异性的细胞毒性T细胞杀伤表达MUC1蛋白的靶细胞,而未转染组的杀伤作用较弱。转染组DCs与T细胞共培养的上清中IFN-γ的分泌水平高于未转染组。结论：电穿孔法可以将MUC1 mRNA成功转染至DCs,产生特异性杀伤效应,为以MUC1为靶点的非小细胞肺癌的免疫治疗提供实验和理论依据。     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

肺癌细胞总RNA转染的DC疫苗体外诱导肺癌患者特异性抗肿瘤免疫的实验研究

目的：为了探讨肺癌细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力。方法：采用分离肺癌患者外周血单核细胞体外诱导DC细胞，Trizol法提取肺癌细胞系Calu-6总RNA，用脂质体包裹总RNA转染DC并诱导特异性CTL的扩增，LDH法和ELISA法检测CTL的杀伤活性和IFN-γ分泌。结果：经肺癌细胞总RNA转染的DC特异性表面标志及功能相关分子表达均上调，转染后的DC可显著刺激异体／自体T淋巴细胞增殖，诱导的特异性CTL对携带Calu-6抗原的靶细胞的杀伤率显著高于LAK细胞，再次接触相同抗原时其IFN-γ分泌量显著增高。结论：肺癌细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫。     

应用肺癌细胞系诱生肿瘤特异性T细胞的初步研究

目的 探讨通过构建表达人白细胞抗原(HLA)HLA-A2的肺癌细胞系以诱生肿瘤特异性细胞毒性T淋巴细胞(CTL).方法 将编码HLA-A2基因的质粒pcDNA3.1D/V5转染人肺腺癌A549细胞,得到能够稳定表达HLA-A2的肺腺癌细胞系.将其用丝裂霉素(MMC)处理后,行混合淋巴细胞肿瘤细胞培养(mixed lymphocyte tumor cell culture,MLTC)以诱导HLA-A2 的健康人外周血淋巴细胞(peripheral blood lymphocyte, PBL)产生肿瘤特异性CTL,并通过特异性杀伤实验、单克隆抗体阻断实验、淋巴细胞增殖实验进行检验.结果 HLA-A·0201基因在A549细胞中的瞬时与稳定表达率分别为0.32、0.91.成功获得稳定表达HLA-A2的A549细胞株.MLTC诱导最终产生4组T淋巴细胞,且符合肿瘤特异性CTL的形态学特征与大量增殖的特点,但CTL杀伤实验和单克隆抗体阻断实验结果示肿瘤细胞未被有效杀伤,可能与肿瘤特异性CTL的数量较少及活性较低有关.结论 本研究通过构建表达HLA-A2的肺癌细胞系,对建立肿瘤特异性CTL模型作了初步探讨,为进一步完善肺癌特异性CTL细胞模型并为研究肿瘤免疫逃避、免疫耐受及反杀伤淋巴细胞等生物学行为打下基础.     

负载乳腺癌细胞冻融抗原的DC对CTL体外特异性杀伤活性的影响

目的：研究乳腺癌患者腋下淋巴结单个核细胞来源的DC,经自体肿瘤细胞冻融抗原刺激后,对CTL体外特异性杀伤活性的影响。方法：以细胞因子分别诱导、培养乳腺癌腋下淋巴结单个核细胞中的DC及TDLNC,用自体癌细胞冻融抗原刺激DC,并和TDLNC共培养,诱导成为肿瘤抗原特异性CTL;检测特异性CTL对自体乳腺癌细胞及MCF-7细胞的体外杀伤活性。结果：DC-Ag-TDLNC、DC-TDLNC和TDLNC对自体乳腺癌细胞的杀伤率分别为67.64%、31.25%和26.36%,P〈0.001;三种TDLNC细胞对MCF-7细胞的杀伤率无明显差异（P〉0.05）。结论：乳腺癌患者腋下引流淋巴结中的单个核细胞在细胞因子的诱导、活化及自体肿瘤抗原刺激下,可分化为成熟DC,DC可促进TDLNC增殖、分化为特异性CTL,后者对自体肿瘤细胞有较高的杀伤活性。     

MUC1基因疫苗对膀胱肿瘤抑制的免疫学实验研究

目的：研究人粘蛋白MUC1基因DNA疫苗诱导小鼠细胞毒性T细胞（CTL）及体液免疫应答的作用，为膀胱癌的疫苗治疗提供实验资料。方法：接种pcDNA3．1（＋）-MUC1质粒，通过ELISA法检测小鼠血清MUC1抗体生成和脾细胞产生IL-2和1FN-7的量，通过LDH释放法测定CTL对BIU-87细胞的杀伤活性。结果：接种pcDNA3．1（＋）-MUC1疫苗后，小鼠血清中抗MUC1抗体水平明显升高，与对照组相比有显著性差异（P〈0．01）。脾细胞产生的IL-2和IFN-7水平较对照组高（P〈0．01）。在效靶比为100：1、50：1、25：1、12．5：1时，MUC1基因疫苗免疫组小鼠CTL对BIU-87杀伤率（分别为58．4％、47．2％、35．7％、27．4％），较空载体pcDNA3．1（＋）组小鼠和灭菌生理盐水组小鼠（分别为11％、19．2％、9．5％、14％和16．5％、11．9％、8．6％、10．3％）均明显升高，且有显著性差异（P〈0．01）。结论：MUC1基因DNA疫苗能够诱导小鼠产生抗MUC1特异性抗体，诱导产生杀伤表达MUC1细胞的CTL，为MUC1基因疫苗用于膀胱肿瘤生物治疗提供了一定的实验依据。     

胰腺癌高表达抗原MUC4 CTL表位肽的筛选与改造

目的:观察胰腺癌高表达抗原黏蛋白4(MUC4)的改造表位是否有HLA-A2限制性抗肿瘤能力。方法:首先运用RT-PCR和Western blot方法检测MUC4在胰腺癌细胞系CAPAN-2和ASPC-1的表达情况。通过Net CTL 1.2、BIMAS、SYFPEITHI和IEDB软件预测打分来选取MUC4的HLA-A2限制性表位;替换MUC4抗原锚定位点氨基酸获得改造肽;候选表位肽的合成方法为标准的Fmoc化学合成法,结合力实验用于检测候选表位与T2A2细胞表面HLA-A2分子的结合能力,ELISPOT实验检测候选表位肽诱导细胞毒性T淋巴细胞(CTL)分泌IFN-γ的能力,体外细胞毒实验活性检测候选肽诱导CTL的能力。结果:MUC4在胰腺癌细胞系CAPAN-2和ASPC-1均有表达。P1944-1Y、P1944-2L、P1944-1Y2L、P2004和P2004-1Y9V具有较好的结合力,且P2004-1Y9V、P1944-1Y2L等改造肽与HLA-A2的结合力高于原肽。ELISPOT实验结果显示表位肽P1944、P1944-1Y2L、P2004和P2004-1Y9V诱导的CTL具有分泌IFN-γ的能力。P1944-1Y2L和P2004-1Y9V诱导特异性T细胞免疫分泌的IFN-γ略高于原肽。细胞毒实验结果显示表位P1944、P1944-1Y2L、P2004和P2004-1Y9V对CAPAN-2细胞均有一定的杀伤作用。P1944-1Y2L和P2004-1Y9V特异性CTLs对CAPAN-2细胞杀伤率高于原肽特异性CTLs。结论:MUC4抗原改造表位P1944-1Y2L、P2004-1Y9V与天然表位P1944、P2004相比有更高的HLA-A2分子亲和力,保留了原有的免疫原性,并且改造肽抗肿瘤免疫效应强于天然表位。P1944-1Y2L和P2004-1Y9V是优秀的MUC4抗原的HLAA2限制性CTL候选表位,可以成为新的抗肿瘤多肽免疫治疗疫苗的候选表位。     

MUC1/Y胞外段在大肠杆菌中的表达和鉴定

目的：获得MUC1／Y胞外段重组蛋白，研究其生物学功能，为肿瘤治疗提供实验依据。方法：利用RT—PCR从MCF7细胞中获得MUC1／Y胞外段编码基因，将其克隆到原核表达载体pET-32a中，并在BL21（DE3）大肠杆菌中进行表达；以亲和层析法对MUC1／Y重组蛋白进行纯化；利用纯化的MUC1／Y蛋白免疫家兔制备MUC1／Y多克隆抗体，然后对乳腺癌组织进行组化染色。结果：在大肠杆菌BL21（DE3）中成功表达了分子量为30000的Trx—MUC1／Y融合蛋白，经镍亲和层析一步纯化所获得的蛋白质纯度〉90％，用Trx-MUC1／Y融合蛋白免疫家兔获得的抗血清对乳腺癌组织的初步组化检测证明MUC1／Y融合蛋白具有很好的生物学活性。结论：成功表达并纯化了具有生物学活性的MUC1／Y胞外段重组蛋白。     

MUC1 and MUC2 in pancreatic neoplasia

MUCs are glycoproteins with various roles in homeostasis and carcinogenesis. Among other actions, MUC1 may inhibit cell-cell and cell-stroma interactions and function as a signal transducer, participating in cancer progression. In contrast, MUC2 is normally found only in goblet cells, where it contributes to the protective barrier function of these cells. Recently, a tumour suppressor role has been demonstrated for MUC2, and both MUC1 and MUC2 appear to have important roles in pancreatic neoplasia. MUC1 appears to be a marker of aggressive phenotype and may facilitate the vascular spread of carcinoma cells. In contrast, MUC2 is rarely detectable in aggressive pancreatic tumours, but is commonly expressed in intraductal papillary mucinous neoplasms (IPMNs), which are rare, indolent tumours, in intestinal IPMNs, and in indolent colloid carcinomas. MUC2 appears to be not only a marker of this indolent pathway, but also partly responsible for its less aggressive nature. Thus, in pancreatic neoplasia, MUC1 and MUC2 have potential diagnostic and prognostic value as markers of aggressive and indolent phenotypes, respectively, and have potential as therapeutic targets.     

MUC1, MUC2 and MUC5AC expressions in cardiac myxoma

Background Cardiac myxoma, the most common primary tumor of the heart, has a variable clinical presentation and immunohistochemical profile. An abundant mucopolysaccharidic matrix exists, including mucin, within cardiac myxoma. This investigation first reports the expressions of mucin genes in cardiac myxoma.Methods A retrospective study was conducted between December 1976 and February 2003, comprising 101 consecutive patients with cardiac myxoma who were treated with surgical excision. Detailed clinical parameters also were reviewed. Mucin genes, namely MUC1, MUC2 and MUC5AC, were studied immunohistochemically in 47 randomly selected patients.Results The study group contained 57 (57%) women and 44 (43%) men, with a mean age of 38±21 years. Their presentations included: asymptomatic (41%), dyspnea (35%), stroke (23%), chest pain (7%), fever (6%), syncope (5%) and tricuspid regurgitation (70%). The sample included 90 myxoma located in the left atrium, 3 (3%) recurrent myxoma and 8 (8%) myxoma not located in the left atrium. The myxoma did not differ with location or clinical event in terms of pathological scores, such as vascular proliferation, inflammation, cellularity, hyaline, calcification and thrombosis. Cardiac myxoma is characterized by excessive mucus secretion. Expression of membrane-associated MUC1 was considerably higher than that of the secreted mucins, MUC2 and MUC5AC (P<0.05). Furthermore, expression of MUC5AC is related to lesser embolism (P<0.05).Conclusions This work first examined the immunohistochemical expression of mucin (MUC1, MUC2 and MUC5AC) in cardiac myxoma. This investigation then showed that the expression of representative membrane-associated mucin, MUC1, and/or secretory mucins, MUC2 and MUC5AC, in cardiac myxoma was associated with important tumor clinicopathological characteristics. Moreover, MUC5AC appears related to lesser embolism. This approach can help distinguish the potential roles of secretory mucins versus membrane-associated mucins in the development of cardiac myxoma.     

MUC1, MUC2, MUC4, MUC5AC and MUC6 Expression in the Progression of Prostate Cancer

Molecular changes are vital for the development of prognostic markers and therapeutic modalities of prostate cancer (CaP). There is growing interest in mucins as treatment targets in human malignancies, including CaP. The role of their expression in the progression of CaP is however unclear. We examined the expressions MUC1, MUC2, MUC4, MUC5AC and MUC6 in CaP tissues using tissue microarrays (TMAs) to look for tumor-associated antigens (TAAs) for targeted therapy. In this study, 120 paraffin-embedded specimens were selected from patients who underwent radical retro-pubic prostatectomy (RRP) or trans-urethral-resection of the prostate (TURP) for primary, untreated CaP and 10 matched lymph node metastases. A series of MUC monoclonal antibodies (mAbs) was used on TMAs by standard immunohistochemistry. Our results indicate that the over-expression of MUC1 was detected in 58% of primary CaP tissues and 90% of lymph node metastases but not in normal prostate or benign tissues, while the expression of MUC2, MUC4, MUC5AC and MUC6 was found to be negative in both normal and cancer tissues. Of the MUC1 positive tumors 86% were Gleason grade 7 or higher. Over-expression of MUC1 was found in late stage CaP while MUC2, 4, 5AC and 6 were negative in CaP. MUC1 is a TAA that is highly related to tumor progression in CaP patients. This antigen is ideal for targeted therapy to control micrometastases and hormone refractory disease but additional studies are necessary to assess its usefulness in patient biopsies and CaP bone metastases before clinical trial.     

Carbohydrate/peptide mimics: effect on MUC1 cancer immunotherapy

 Recent clinical studies with mannan mucin immunotherapeutic agents indicate that patients produce predominantly antibody responses while mice produce a high cytotoxic T lymphocyte response. In studying the reason for the ’immune deviation’ occurring in mice to humans from cellular to antibody responses, it has been found that natural anti-Galα(1,3)Gal antibodies, present in all humans, react with the mucin component of the agent, providing an example of a carbohydrate-peptide mimic. The immune deviation can be overcome by in vitro sensitization of antigen-presenting cells in the absence of anti-Gal antibodies – at least in mice. The review examines the background of these observations and discusses other peptide carbohydrate mimics and immune deviation Received: 11 January 1999 / Accepted: 22 March 1999