摘要
【背景】
MGb2是本研究所建立的一种胃肠道肿瘤特异性单克隆抗体。利用该抗体检测胃癌敏感性高、特异性强,对胃印戒细胞癌尤为明显。但是,该抗体建立20年来,其识别抗原MGb2-Ag一直没有鉴定成功,故对其的认识非常有限。
【目的】
进一步观察MGb2-Ag在人体多种组织中的表达分布特征,明确其在胃癌发生发展中的临床病理诊断价值,鉴定MGb2-Ag,并研究其在肿瘤细胞中可能的作用。
【方法】
(1)用免疫组化方法检测人体多种组织中MGb2-Ag的表达分布特征。
(2)以141例胃癌(n=81)和非胃癌患者(n=60)的胃组织石蜡切片标本作为研究对象,用免疫组化方法评价MGb2-Ag在胃癌发生发展中的临床病理诊断价值。免疫组化染色结果判断结合染色细胞数量和染色程度进行综合评分分析。-2-
(3)用Western blotting检测7种胃癌细胞系和1种胃黏膜上皮细胞永生化细胞系GES-1中MGb2-Ag的表达,并初步确定目的蛋白的分子量。
(4)以胃癌细胞系和新鲜胃癌组织为材料,用免疫沉淀技术(IP)富集MGb2-Ag,SDS-PAGE胶分离目的抗原后切胶进行MALDI-TOF质谱分析和生物信息学分析,获得候选分子为TRAK1(Trafficking protein, kinesin-binding 1)。
(5)以下列方法进一步鉴定MALDI-TOF质谱分析结果:用抗TRAK1抗体和MGb2抗体检测原核表达的TRAK1蛋白,用两种抗体分别检测组织连续切片中TRAK1和MGb2-Ag蛋白的组织表达特征和亚细胞定位特征。
(6)借助RNAi(RNA interference)技术,设计Trak1 siRNAs,构建其表达质粒,转染胃癌细胞系MKN-45,筛选有效靶序列,观察转染Trak1 siRNAs对肿瘤细胞的细胞周期和细胞凋亡的影响。
【结果】
(1)MGb2-Ag在人体多种组织中的表达特征:组织和器官不同,MGb2-Ag表达水平不同;在人癌组织,特别是胃癌中高表达;在处于癌前状态的组织中(胃、结肠和胆囊等)弱表达;正常组织中几乎不表达;在正常胰腺的胰岛和脑灰质神经元中,能够观察到特异性的表达。
(2)胃癌组织分化越差(P = 0.015),肿瘤细胞侵袭深度越深(P < 0.001),肿瘤细胞淋巴结转移的发生越多(P = 0.011),MGb2-Ag表达水平越高。随机挑选胃癌患者癌旁组织标本(n = 16)检测MGb2-Ag,发现其在癌组织中的表达高于相应癌旁组织(P = 0.001)。同时,本研究观察到慢性浅表性胃炎组织中MGb2-Ag的表达低于其在癌前状态组织(本研究包括萎缩性胃炎、肠上皮化生、胃息肉)中的表达(P = 0.005)。MGb2-Ag在胃癌中阳性率为81.48%(66/81),其中,在胃印戒细胞癌和粘液细胞癌的中阳性率为100% (23/23);MGb2-Ag在癌前状态组织中阳性率为13.16% (5/38) ,在慢性浅表性胃炎组织中阳性率为0%(0/22);胃癌组织中MGb2-Ag的阳性率高于所有非癌组织(P < 0.001)。
(3)MGb2-Ag在7种胃癌细胞系中呈不同程度的表达,但在人永生化胃黏膜细胞系GES-1中的表达很弱。该抗原用Western blotting可检测到2条蛋白质条带,分子量分别为~115 kDa和~100 kDa,所有细胞样品皆有的条带是~100 kDa。明显呈两条条带的细胞是MKN-45、SGC-7901和AGS细胞。
(4)切取从MKN-45细胞、KATOIII细胞和胃癌组织中富集到的目的抗原MGb2-Ag条带(~100 kDa),MALDI-TOF质谱分析结果均显示候选分子均为TRAK1。原核表达的TRAK1蛋白能够被MGb2单抗和羊TRAK1多抗所识别。用MGb2单抗和羊TRAK1多抗检测胃癌、肠上皮化生、慢性浅表性胃炎等组织的连续切片,发现染色定位、细胞类型等特征高度一致。
(5)成功构建Trak1 siRNA表达载体,筛选出2对针对Trak1的siRNA,瞬时转染MKN-45细胞,与对照siRNA比较,两者均显著减少细胞凋亡,促进细胞周期从G1期向S期进展。
【结论】
本研究进一步观察了MGb2-Ag在人体多种组织的分布特征;发现MGb2-Ag是胃癌发生发展过程中一种有意义的病理诊断标志物,MGb2-Ag表达增加可能是胃癌发生的早期事件;成功鉴定MGb2-Ag为一种新分子TRAK1,首次发现TRAK1是一种新的胃癌诊断标志物;本研究还借助RNAi介导的TRAK1低表达效应,首次研究了TRAK1在肿瘤细胞中的功能,发现转染Trak1 siRNAs抑制胃癌细胞系MKN-45细胞凋亡,促进其细胞周期从G1期向S期进展。这提示MGb2-Ag/TRAK1在肿瘤细胞中高表达,可能在肿瘤生长与侵袭中发挥负调控作用。
【Background】
MGb2 is a gastrointestinal cancer specific monoclonal antibody developed in our laboratory in 1980s. It can be used to detect gastric cancer with high sensitivity and specificity, especially for gastric signet-ring cell carcinoma. However, its target antigen MGb2-Ag has not been identified.
【Objectives】
The present study aimed: (a) to assess the distribution of gastrointestinal cancer associated antigen MGb2-Ag in human tissues, (b) to determine the clinical pathological significance of MGb2-Ag in gastric cancer, (c) to identify the physiochemicl nature of MGb2-Ag and to explore biological function of MGb2-Ag in gastric carcinogenesis.
【Methods】
Distribution of MGb2-Ag was detected by immunohistochemistry. MGb2-Ag was enriched using immunoprecipitation followed by confirmation with Western blotting and silver staining. The target protein band was detected by MALDI-TOF MS followed by bioinformatics analysis. Prokaryotic expression system and immunohistochemistry provided the further supporting evidences. siRNAs against Trak1 were subcloned into plasmid vector. Cell cycle analysis and apoptosis detection were used to explore the role of siRNAs in cancer cell line using transient transfection.
【Results】
This study provided further evidences on distribution characteristics of MGb2-Ag in human cancers and non-cancerous tissues. Immunostaining analysis demonstrated that the presence of over-expression of MGb2-Ag was correlated with an increased aggressiveness of the tumor, including poorer differentiation, deeper penetration and metastasis of gastric cancer cells. The positive rate of MGb2-Ag was 81.48% (66/81) in gastric cancer, 100% (23/23) in gastric signet-ring cell carcinoma and mucinous adenocarcinoma, 13.16 (5/38) in gastric precancerous conditions, and 0% (0/22) in chronic superficial gastritis. Approximately 106 kDa protein immunoreactive with MGb2 antibody in gastric cancer cell lines KATOIII, MKN-45 or gastric cancer tissues, was identified as Trafficking protein, kinesin-binding 1(TRAK1), which has been reported as a new molecular gained limited recognization. Both anti-TRAK1 antibody and MGb2 antibody could recognize prokaryotic expressed TRAK1. The immunostaining characteristics of TRAK1 in paraffin-embedded tissues of gastric cancer, intestinal metaplasia or chronic superfacial gastritis were highly identical with MGb2-Ag in the continuous sections of these samples. Transfection of two siRNAs targeting Trak1 into gastric cancer cell line MKN-45 decreased apoptosis and promoted cell cycle G1 to S stage transition.
【Conclusions】
The present study assessed the distribution of MGb2-Ag in human cancer and non-cancerous tissues and analyzed the clinical pathological significance of MGb2-Ag in gastric cancer. The results showed that the high expression of MGb2-Ag was associated with the poorer differentiation, deeper penetration and presence of lymphonde metastasis of gastric cancer cells. MGb2-Ag was identified as TRAK1, and MGb2-Ag/TRAK1 might be a promising gastrointestinal cancer biomarker and act as an anti-tumor target.
引文
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