血清MUC1粘蛋白IRMA及其初步临床应用

建立有效的ＭＵＣ１粘蛋白免疫放射分析法（ＩＲＭＡ），并初步用于乳腺肿瘤的诊断。利用纯化的ＭＵＣ１及其多抗，建立ＭＵＣ１双抗夹心ＩＲＭＡ法，对５５例健康女性、１１例乳腺良性肿瘤、２７例乳腺癌及２８例乳腺癌术后患者进行血清ＭＵＣ１的测定。结果：血清ＭＵＣ１ＩＲＭＡ的灵敏度为０７４ｋＵ／Ｌ；健康女性、乳腺良性肿瘤、乳腺癌及乳腺癌术后患者血清ＭＵＣ１的平均含量分别为７２３±３６７、１７８０±１０５６、３００４±２３８４和３１８４±２４７９ｋＵ／Ｌ；正常值上限为１３５４ｋＵ／Ｌ，假阳性率为１８２％；乳腺肿瘤患者血清ＭＵＣ１水平较健康女性明显提高；乳腺癌患者血清ＭＵＣ１水平高于乳腺良性肿瘤患者，但两者的阳性率（７７８７％和７２７０％）差异无显著性（χ２＝０１５４，Ｐ＞００５）；乳腺癌与乳腺癌术后患者血清ＭＵＣ１水平差异无显著性（ｔ＝－０２２，Ｐ＞００５）。ＭＵＣ１血清水平与肿瘤恶性程度成正相关，对于乳腺癌的诊断具有重要参考价值。     

地塞米松和组胺对人鼻息肉组织黏蛋白MUC5AC mRNA和蛋白表达的影响

目的:研究地塞米松和组胺对培养的人鼻息肉组织黏蛋白MUC5AC mRNA和蛋白表达的影响。方法:实验分为对照组、组胺刺激组和地塞米松干预组,采用RT-PCR和免疫组织化学技术分别检测鼻息肉组织中MUC5AC mRNA和蛋白的表达。结果:组胺刺激组鼻息肉组织MUC5AC mRNA和蛋白表达分别为0.6389±0.0526和0.1934±0.0137,均较对照组(分别为0.3495±0.0357和0.1172±0.0173)明显增加(均P<0.01),地塞米松抑制后两者均明显降低(分别为0.4988±0.0603和0.1444±0.0075)(均P<0.05)。结论:炎性递质组胺可上调黏蛋白表达;地塞米松可下调黏蛋白表达,这可能是它抑制鼻息肉黏液过度分泌的途径之一。     

呼吸道纤毛上皮细胞的组织块法培养

目的建立组织块法呼吸道纤毛上皮细胞原代培养模型,观察细胞生长分化情况,为深入研究黏液纤毛传输系统提供方法学基础。方法应用组织块法在鼠尾胶原上培养兔气管纤毛上皮细胞,对培养7天的标本行苏木素-伊红染色、黏蛋白5AC(mucin-5AC,MUC5AC)单克隆抗体免疫组织化学染色、扫描电镜及透射电镜观察、纤毛摆动频率测量。结果①细胞培养7天后可以见到大量的纤毛细胞和杯状细胞,纤毛形态正常,摆动活跃;②苏木素-伊红染色和黏蛋白MUC5AC单克隆抗体免疫组织化学染色可见分化良好的纤毛细胞和杯状细胞;③扫描电镜及透射电镜观察见纤毛细胞分化良好,纤毛形态正常;④(30±1)℃下测得纤毛摆动频率为(13.2±0.9)次/s。结论应用组织块法进行兔气管纤毛上皮细胞培养,可以得到分化良好的纤毛细胞和杯状细胞,纤毛摆动活跃,可应用此模型对黏液纤毛传输系统进行深入研究。     

钙激活的氯通道gob-5和粘蛋白基因Muc5ac在小鼠哮喘模型肺部的表达

目的：研究“钙激活的氯通道”成员之一gob-5和粘蛋白基因Muc5ac在小鼠哮喘模型肺部的表达。 方法： 22只清洁级BALB/c小鼠随机分成哮喘组和对照组，用逆转录聚合酶链反应法（RT-PCR）和免疫组化法分别测定肺组织gob-5 mRNA和粘蛋白基因Muc5ac蛋白的表达。 结果： 小鼠哮喘组肺组织gob-5 mRNA表达阳性（0.2297±0.0186，A），而对照组未出现gob-5 mRNA的表达(P<0.01)；哮喘组Muc5ac蛋白表达（0.6637±0.0127，A）明显高于对照组（0.2060±0.0179，A）（P<0.01）；哮喘组肺组织gob-5 mRNA表达与Muc5ac蛋白表达呈直线正相关（r=0.864, P<0.05）。 结论： Gob-5 mRNA表达的上调可能在哮喘小鼠气道粘液过度分泌中起着关键作用；抑制gob-5的表达将为哮喘的治疗提供新的策略。     

肠三叶因子和黏蛋白对烧伤血清所致肠上皮细胞免疫功能变化的影响

目的 观察肠三叶因子(ITF)和黏蛋白对烧伤血清所致肠上皮细胞免疫功能变化的影响.方法 (1)体外培养大鼠小肠上皮细胞株IEC-6,根据培养液添加物质不同,按照随机数字表法将细胞分为5组:①正常对照组,培养液中含10％(指体积分数,下同)小牛血清;②烧伤对照组,培养液含10％烧伤血清;③ITF+烧伤血清组,培养液含10％烧伤血清及终浓度25μg/mL ITF;④黏蛋白+烧伤血清组,培养液含10％烧伤血清及终浓度250 μg/mL黏蛋白;⑤ITF+黏蛋白+烧伤血清组,培养液含10％烧伤血清及终浓度25 μg/mL ITF、250 μg/mL黏蛋白.向各组细胞加入上述培养液的同时,加入大肠杆菌菌液(1×108 CFU/mL,200 μL).继续培养15 min、30 min、1h、2h、3h后,行瑞氏-吉姆萨染色,于显微镜下观察并统计黏附在细胞上的细菌数;采用锥虫蓝染色法观察并统计细胞存活率.每组每时相点样本数均为20.(2)将IEC-6细胞按照随机数字表法分为4组:烧伤对照组、ITF+烧伤血清组、黏蛋白+烧伤血清组、ITF+黏蛋白+烧伤血清组,分别同前加入相应的培养液(不加菌液)培养3、6、12、24、48 h.采用放射免疫分析法测定各时相点培养上清液中TNF-α、IL-6和IL-8 含量,每组每时相点样本数均为6.对实验数据行t检验.结果 (1)烧伤对照组细胞各时相点细菌黏附数量较正常对照组明显增多(t值为2.947 ～8.149,P值均小于0.01).与烧伤对照组比较,其余3个烧伤血清组在加菌后多数时相点细菌黏附的数量明显偏少(t值为-4.733～-2.180,P＜0 05或P ＜0.01).烧伤对照组细胞各时相点存活率与正常对照组比较均明显降低(t值为-4.126～-2 363,P值均小于0 05).ITF+烧伤血清组、黏蛋白+烧伤血清组细胞存活率在部分时相点明显高于烧伤对照组(t值为2.120～3.423,P＜0.05或P＜0.01).ITF+黏蛋白+烧伤血清组细胞存活率加菌后15 min为(96.7±2 4)％,明显高于ITF+烧伤血清组[(94 5±3 1)％,t=2.507,P＜0.05];在3h时为(84.0±6 7)％,明显高于黏蛋白+烧伤血清组[(77 1±8 2)％,t=2.934,P＜0.01].(2)ITF+黏蛋白+烧伤血清组培养6、12、24、48 h,TNF-α含量均低于其余3组(t值为-6.914 ～ -2.889,P＜0.05或P＜0.0i).ITF+黏蛋白+烧伤血清组IL-6含量在部分时相点明显低于其余3组(t值为-7.657～-2.580,P＜0.05或P＜0.01).ITF+黏蛋白+烧伤血清组在培养6、12、24、48 h IL-8含量明显低于烧伤对照组和黏蛋白+烧伤血清组(t值为-8.802 ～ -3.640,P值均小于0.01);在培养12、24 h明显低于ITF+烧伤血清组(t值分别为-2.786、-2.740,P值均小于0.05).结论 ITF能维护肠上皮细胞功能,抵御细菌黏附,降低细胞死亡率,同时能维护细胞免疫稳态,减少炎症介质释放;ITF与黏蛋白联用效果更明显.     

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium

ObjectivesThe mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium.DesignMUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells.ResultsThe cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2 h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p < 0.05) or by immunocytochemistry (+44%, p < 0.001).ConclusionThe membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.     

Role of human nucleoside transporters in pancreatic cancer and chemoresistance

The prognosis of pancreatic cancer is poor with the overall 5-year survival rate of less than 5% changing minimally over the past decades and future projections predicting it developing into the second leading cause of cancer related mortality within the next decade. Investigations into the mechanisms of pancreatic cancer development, progression and acquired chemoresistance have been constant for the past few decades, thus resulting in the identification of human nucleoside transporters and factors affecting cytotoxic uptake via said transporters. This review summaries the aberrant expression and role of human nucleoside transports in pancreatic cancer, more specifically human equilibrative nucleoside transporter 1/2 (hENT1, hENT2), and human concentrative nucleoside transporter 1/3 (hCNT1, hCNT3), while briefly discussing the connection and importance between these nucleoside transporters and mucins that have also been identified as being aberrantly expressed in pancreatic cancer. The review also discusses the incidence, current diagnostic techniques as well as the current therapeutic treatments for pancreatic cancer. Furthermore, we address the importance of chemoresistance in nucleoside analogue drugs, in particular, gemcitabine and we discuss prospective therapeutic treatments and strategies for overcoming acquired chemoresistance in pancreatic cancer by the enhancement of human nucleoside transporters as well as the potential targeting of mucins using a combination of mucolytic compounds with cytotoxic agents.     

Cadherin-catenin adhesion system and mucin expression: a comparison between young and older patients with gastric carcinoma

BACKGROUND: Young patients are thought to develop gastric carcinomas with a molecular genetic profile that is distinct from that of gastric carcinomas occurring at a later age. The aim of this study was to compare the clinicopathological features and expression patterns of the markers E-cadherin and beta-catenin, and mucins (MUC1, MUC2, MUC5AC, and MUC6) in young and older patients. METHODS: The clinicopathological features and overall survival data of 62 young patients (age 40 years). A tissue microarray method and immunohistochemistry were used in order to analyze marker expression in paraffin-embedded tissue blocks obtained from both groups. RESULTS: The young group presented a higher percentage of diffuse-type tumors in comparison to the older group (P<0.01). The rates of positivity for E-cadherin and beta-catenin membranous expression patterns and mucin (MUC2, MUC5AC and MUC6) positivity were higher in the young group (P<0.01). Although young patients showed a lower frequency of alterations in marker expression and had significantly better survival rates than the older patients, neither age nor the marker expression pattern were found to be independent prognostic factors of survival. Only stage, tumor size, and tumor location persisted as prognostic factors for patients with gastric cancer. CONCLUSION: Biological markers of cellular adhesion and gastric differentiation were differently expressed in young and older patients. Our findings support the hypothesis that young patients develop carcinomas with a different genetic pathway compared to the pathway of tumors occurring at a later age, and we suggest further investigations to assess the prognostic relevance of the markers to specific subgroups.     

Expression of Mucins in Mucoid Otitis Media

A hallmark of mucoid otitis media (MOM, i.e., chronic otitis media with mucoid effusion) is mucus accumulation in the middle ear cavity, a condition that impairs transduction of sounds in the ear and causes hearing loss. The mucin identities of mucus and the underlying mechanism for the production of mucins in MOM are poorly understood. In this study, we demonstrated that the MUC5B and MUC4 were major mucins in MOM that formed distinct treelike polymers (mucus strands). The MUC5B and MUC4 mRNAs in the middle ear mucosa with MOM were up regulated 5-fold and 6-fold, compared with the controls. This upregulation was accompanied by the extensive proliferation of the MUC5B- and MUC4-producing cells in the middle ear epithelium. Further study indicated that the mucin hyperproduction was significantly linked to CD4⁺ and CD8⁺ T cells and/or CD68⁺ monocyte macrophages. It suggests that MUC5B and MUC4 expression may be regulated by the products of these cells.     

Simple mucin-type carbohydrate antigens (Tn, sialosyl-Tn, T and sialosyl-T) and gp 230 mucin-like glycoprotein are candidate markers for neoplastic transformation of the human cervix

Mucins and simple mucin-type carbohydrates are cancer-associated antigens in several human tumors. Expression of Tn, sialosyl-Tn, Thomsen-Friedenreich (T), sialosyl-T and of a recently identified mucin-like glycoprotein (gp230) has not yet been thoroughly investigated in human cervix carcinogenesis. In the present study sections from normal cervix (n=10), CIN III lesions (n=10), and invasive carcinomas (n=47) were evaluated immunohistochemically using monoclonal antibodies. In normal cervix there was: cytoplasmatic expression of Tn in 1 case (10%); membranous expression of STn in 8 cases (80%); no expression of T and cytoplasmatic expression of ST in 1 case (10%); gp 230 was expressed in all cases with a membranous pattern. In CIN III lesions there was cytoplasmatic and membranous expression of Tn in 3 cases (30%) and of STn in 9 cases (90%); T and ST were not expressed; gp 230 was expressed in 5 cases (50%) both in the cytoplasm and at the cell membrane. In invasive carcinomas we observed Tn expression in 30 cases (63.8%) and STn in 31 cases (66%); T antigen was not expressed; expression of both ST and gp 230 in 24 cases (51.1%); all antigens showed membranous and cytoplasmatic staining. Our results show that Tn and ST are good markers of invasive carcinomas of the human cervix. We have also shown that loss of expression of the mucin-like glycoprotein gp 230 is associated with malignant transformation at a preinvasive stage. Received: 16 December 1999 / Accepted: 9 February 2000