【作者】 靳斌；

【导师】 吴开春； 王新；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 胃癌是最常见的恶性肿瘤之一,其死亡率居肿瘤的第二位。对胃癌的早期诊断和治疗是胃癌研究的重大课题。胃癌早期无明显症状,发现时已到晚期,预后极差。以往的胃癌标志物如CEA、CA50等对胃癌的敏感性和特异性较低,早期诊断受到限制。因此,寻找新的组织学、细胞学、血清学的胃癌新抗原,对胃癌诊治的意义重大。本实验室樊代明教授在上世纪八十年代研制出一系列胃癌单克隆抗体,命名为MG系列。其中,MG7-Ag对胃癌的敏感性特异性都很高。后续本实验室及国内医学专家对此进行了一系列相关深入的研究,包括胃癌单克隆抗体MG7对胃癌的诊断、评价预后及动态监测作用、MG7靶向治疗作用、特异性纳米疫苗的制备等等,在国内外发表了几十篇很有影响力的文章。但是,MG7-Ag尚未可知,MG7单抗在国际上的推广及进一步研究受到限制。香港科技大学学者鉴定MG7-Ag为hnRNPA2?B1,但是其性质与我们的以往研究的MG7-Ag不符。本研究使用免疫沉淀、双向电泳、质谱分析等技术鉴定MG7-Ag,对于发现新的癌抗原或癌蛋白,对于胃癌新的分子机制的研究有着不可估量的重要意义。此外,本研究拟建立一个简便的胃癌血清学早期诊断方法,用于胃癌高危人群的普查。MG7单抗对胃癌细胞凋亡作用的研究,为今后的临床药物治疗提供新的思路。总之,本项研究有深刻的理论意义和临床应用前景。【目的】1、免疫组织化学检测MG7-Ag在胃粘膜组织中的表达与分布特点及与临床特征的联系;2、研究MG7-Ag与hnRNPA2/B1的表达区别;3、MG7-Ag的鉴定;4、建立检测MG7-Ag的简便的胃癌血清学早期诊断方法;5、MG7单抗对胃癌细胞凋亡及细胞周期的影响。【方法】1、通过细胞培养技术复苏MG7杂交瘤细胞,制备MG7单抗;2、应用免疫组化技术检测MG7-Ag在胃粘膜组织中的表达与分布特点及与临床特征的联系;3、应用免疫组化技术观察MG7-Ag与hnRNPA2/B1在胃癌组织中的表达区别;4、应用免疫细胞化学技术和扫描共聚焦显微镜观察MG7-Ag与hnRNPA2/B1在胃癌细胞中的表达区别;5、通过Western blot技术检测MG7-Ag与hnRNPA2/B1胃癌细胞中的表达区别;6、应用免疫沉淀,Western blot,双向电泳,质谱技术鉴定MG7-Ag;7、通过酶联免疫吸附方法建立检测MG7-Ag的胃癌血清学早期诊断方法;8、通过MG7-Ag的胃癌血清学早期诊断方法检测胃癌,胃癌前病变和其它各种肿瘤病人;9、流式细胞仪检测MG7抗体对胃癌细胞凋亡和细胞周期的影响。【结果】1、MG7-Ag在胃粘膜组织中的表达与分布及与临床特征的联系检测了MG7-Ag在胃粘膜组织(胃癌116例;浅表性胃炎,肠化,增生、萎缩性胃炎等84例)石蜡组织标本中的表达与分布情况,发现MG7-Ag阳性表达率是随着浅表性胃炎,肠化,增生,胃癌,逐渐增高的。MG7-Ag阳性分布:1.腺上皮细胞的胞浆上极和胞膜;2.胃腺腔内。胃癌旁切片染色提示:胃黏膜腺体底部上皮细胞MG7-Ag几乎全部阳性,向顶端方向阳性细胞渐减少,而顶端腔面上皮细胞MG7-Ag又多为阳性。在胃癌患者中,MG7-Ag表达与年龄、性别无关,与分化和临床分期有关。2、MG7-Ag与hnRNPA2/B1在胃癌细胞中表达存在明显区别我们应用免疫组化技术检测同一胃癌石蜡切块中MG7-Ag与hnRNPA2/B1在胃癌细胞中的表达,发现MG7-Ag主要分布在细胞膜,细胞核中未见阳性表达,而hnRNPA2/B1主要分布在细胞核中。应用免疫细胞化学技术和扫描共聚焦观察MG7-Ag与hnRNPA2/B1在胃癌细胞SGC7901,MKN45中的表达区别,发现MG7-Ag主要分布在细胞膜上,细胞核中未见阳性表达,而hnRNPA2/B1主要分布在细胞核中。通过Western blot技术检测MG7-Ag与hnRNPA2/B1在胃癌细胞MKN45中的区别,MG7-Ag表现为2条带,分子量分别约100KD、120KD;hnRNPA2/B1表现为2条带,分子量分别约34KD、37KD。3、MG7-Ag的鉴定我们通过免疫沉淀方法,将MG7抗体分别与胃癌细胞系SGC7901,MKN45,胃癌组织交联,行单向或双向电泳。与Western blot相匹配的蛋白点从银染或考马斯亮蓝染色的胶中切下,转到离心管中做质谱检测。免疫沉淀的单向电泳,银染的表观分子量为100KD的胃癌组织条带和胃癌细胞系MKN45条带,LC-MS/MS分析鉴定最可能为anti-colorectal carcinoma heavy chain;考马斯亮蓝染色的表观分子量为100KD的胃癌细胞系MKN45条带和SGC7901条带,ESI-MS/MS分析鉴定最可能为anti-human CD19 monoclonal antibody 4G7 immunoglobulin gamma1 heavy chain。免疫沉淀的双向电泳,考马斯亮蓝染色的表观分子量为100KD的胃癌组织条带,MALDI-TOF/TOF分析鉴定为IGHG3 protein。通过Western blot技术,抗人IgG抗体可以与MG7-Ag结合,说明MG7-Ag可能为免疫球蛋白重链样分子。4、研发检测MG7-Ag的胃癌血清学早期诊断方法及应用应用间接酶联免疫吸附方法,生物素-亲和素放大系统,选择合适的包被条件和抗体浓度,检测胃癌手术前后,胃癌前病变和其他肿瘤患者的血清中MG7-Ag。PBS(0.01M)为阴性对照。大于阴性对照的A值的2.1倍为阳性。116例胃癌术前患者的阳性率为83.6%,明显高于胃癌术后(总63例,47.6%),胃癌前病变(总78例,12.8%)和其它肿瘤组。统计学分析差别有显著意义(P<0.01)。MG7-Ag在其他肿瘤患者血清中也有不同程度的阳性表达,如肺癌(45.2%),直肠癌(45.5%),结肠癌(17.6%),乳腺癌(14.2%),而子宫癌,淋巴瘤,急性白血病患者中未检测出。胃癌术前患者血清检测MG7-Ag的的阳性表达率随着TNM分期进展逐渐升高:Ⅰ期(总6例,33.3%),Ⅱ期(总46例,78.3%),Ⅲ期(总62例,91.9%),Ⅳ期(总2例,100%),统计学显示各组间差别有显著意义(P<0.01);N0(总30例,60.0%),≥N1(总86例,91.9%),统计学显示差别有显著意义(P<0.01)。以上结果提示,MG7-Ag是胃癌较特异性的标志,MG7-Ag的表达率随胃癌的进展而升高。间接酶联免疫吸附实验检测患者血清MG7-Ag可以作为胃癌的早期诊断的简便方法。5、MG7单抗对胃癌细胞凋亡及细胞周期的影响。应用流式细胞仪检测MG7单抗对胃癌细胞系KATOⅢ的凋亡影响,发现MG7单抗明显促进胃癌细胞的早期凋亡(P<0.05)。细胞周期检测,MG7单抗主要使胃癌细胞阻滞在G1期。【结论】本研究提示:MG7-Ag的表达水平与胃癌的分期和恶性程度相关,可能在胃癌的发生和进展中起重要的作用;MG7-Ag与hnRNPA2/B1在胃癌细胞表达中存在明显区别,MG7-Ag是hnRNPA2/B1的可能性不大;MG7-Ag可能为免疫球蛋白重链样分子;建立了MG7-Ag的胃癌血清学早期诊断方法,预计有较广阔的应用前景。MG7单抗对胃癌细胞有促进凋亡的作用,对细胞周期的影响主要在G1期。更多还原

【Abstract】 Gastric cancer is one of the most prevalent malignancies worldwide, and is the second cause of tumor-related death. In gastric cancer researches,early diagnosis and treatment is of the most importance. Few overt symptoms are shown at the early stage of gastric cancer, while most of the diagnosis are made at the advanced stage with poor prognosis. The low sensitivity and specificity of many available cancer markers, including carcinoembryonic antigen (CEA), CA 50,etc. have hampered their use in detecting gastric carcinoma. Therefore, it is vital to seek new gastric cancer antigen at the histological, cytological and serological levels for diagnosis and treatment of gastric cancer.A series of gastrointestinal cancer specific mAb named MG series were discovered in our lab by Prof. Fan Danming in 1980s. Among these specific antibodies, MG7 showed the highest specificity and sensitivity. Further studies of MG7, including the effects of MG7 in diagnosis, evaluation of prognosis, dynamic follow-up study and targeting therapy of gastric cancer, as well as preparation of gastric cancer specific nanovaccine, et al., have been carried out by medical experts in and out of our lab, with dozens of papers subscribed in both domestic and foreign journals.However, up to now, little is known about MG7-Ag, which has limited the international generalization of MG7-Ab and hindered further researches. The scientists in the Hong Kong Polytechnic University have identified MG7-Ag as hnRNPA2/B1, the characteritics of which are different from that of MG7-Ag in our prevous resrarch.In the present study we used immunoprecipitation, 2-D PAGE, mass spectrometry and other methodes to identify MG7-Ag. The discovery of new gastric cancer antigens or proteins may greatly facilitate the studies of molecular mechanisms of gastric cancer.Besides, attempts were also made in our study to establish a convenient serologic method for gastric cancer screening in the high risk population. The studies of MG7 mAb effect on gastric cancer cell apoptosis may help to find new methods in clinical pharmacal therapies.In conclusion, our study has deepgoing theoretical values and good clinical prospects.【Objectives】(1) To investigate the expression or distribution of MG7-Ag in different gastric mucosal tissues and the relationship between expression intensity of MG7-Ag with the clinicopathologic characteristics of gastric cancer; (2) To examine the expression difference between MG7-Ag and hnRNPA2/B1 protein; (3) To identify MG7-Ag; (4) To establish a convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag;(5) To investigate the effects of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line.【Methods】(1) MG7 hybridoma cells were resuscitated and cultured for MG7-Ab preparation. (2)The expression or distribution characteristics of MG7-Ag in different gastric mucosal tissues were determined by immunohistochemistry assay, and the relationship between MG7-Ag expression intensity with the clinicopathologic characteristics were eveluated. (3)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer tissue was investigated by immunohistochemistry assay. (4)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells was detected by immunocytochemistry assay and laser scanning confocal microscope. (5)The expression difference between MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells was detected by Western blot. (6) MG7-Ag identification was carried out by Immunoprecipitation, Western blot,2-D PAGE and mass spectrometry.(7) Enzyme-linked immunoadsorption assay was used to establish a convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag. (8) The convenient serologic method for early diagnosis of gastric cancer by detecting MG7-Ag was applied in detection of gastric cancer, precancerous lesion and other tumors. (9) The effects of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line were evaluated by flow cytometry.【Results】1. The expression or distribution characteristics of MG7-Ag in different gastric mucosal tissues and the relationship between MG7-Ag expression intensity with the clinicopathologic characteristics of gastric cancer. We detected the expression and distribution of MG7-Ag by immunohistochemistry analysis in different gastric mucosal tissues(gastric cancer specimens,116; superficial gastritis,intestinal metaplasia, atypical hyperplasia and atrophic gastritis specimens, 84) and we found that the positive expression rates of MG7-Ag gradually increased in superficial gastritis, intestinal metaplasia,atypical hyperplasia and gastric cancer (P<0. 01). It indicated that the expression of MG7-Ag in gastric cancer mainly located in the upper part of kytoplasm or cell membrane in glandular epithelium cells and gastric gland intracavity. The MG7-Ag staining was positive in most of the basal epithelial cells of gastric mucosa gland, and the positive rate decreased gradually towards the top, but the MG7-Ag expression on the top cavosurface epithelial cells was mostly positive. The MG7-Ag expression was closely related with tissue differentiation and clinical phases, but not with age or sex.2. Significant difference exists between the expression of MG7-Ag and hnRNPA2/B1 protein in gastric cancer cellsImmunohistochemistry was carried out to determine the expression of MG7-Ag and hnRNPA2/B1 on the same paraffin-embedded gastric cancer section, and the results showed that MG7-Ag expressed mainly on the cell membrane, and little in the nucleus.In contrast, the expression of hnRNPA2/B1 mainly distributed in the cell nucleus. Immunocytochemistry and laser scanning confocal microscope were carried out to detect the expression of MG7-Ag and hnRNPA2/B1 in gastric cancer cell lines SGC7901 with MKN45.The results showed that the former mainly distributed on the cell membrane and little in the nucleus, while the latter mainly distributed in the cell nucleus. Western blot carried out on gastric cancer cell line MKN45 showed that two strips with Mr of 100 kDa and 120 kDa were detected by MG7 antibody, compared with another two strips with Mr of 34 kDa and 37 kDa detected by hnRNPA2/B1 antibody.3. The identification of MG7-AgMG7-Ab was crosslinked to gastric cancer cell line SGC7901, MKN45, and gastric cancer tissue respectively by immunoprecipitation technique, and 1-D or 2-D polyacrylamide gel electrophoresis was proceeded. Protein spots, which were recognized and matched to those detected on the Western blot, were excised from a sliver stained gel or a coomassie blue stained gel and transferred to microcentrifuge tubes for mass spectrometry analysis.The two strips with Mr of 100 kDa were excised from a sliver stained gel by 1-D polyacrylamide gel electrophoresis, which were immunoprecipitate of gastric cancer tissue and MG7-Ab, immunoprecipitate of gastric cancer cell line MKN45 and MG7-Ab, respectively. Both of the strips were identified as anti-colorectal carcinoma heavy chain by LC-MS/MS analysis. The two strips with Mr of 100 kDa were excised from a coomassie blue stained gel by 1-D polyacrylamide gel electrophoresis, which were immunoprecipitate of gastric cancer cell line MKN45 and MG7-Ab, immunoprecipitate of gastric cancer cell line SGC7901 and MG7-Ab, respectively. The both strips were identified as anti-human CD19 monoclonal antibody 4G7 immunoglobulin gamma1 heavy chain by ESI-MS/MS analysis.The strip with Mr of 100 kDa was excised from a coomassie blue stained gel by 2-D polyacrylamide gel electrophoresis, which was immunoprecipitate of gastric cancer tissue and MG7-Ab.The strip was identified as IGHG3 protein by MALDI-TOF/TOF analysis. MG7-Ag was detected using IgG antibody (anti-human) by Western blot, which indicated that MG7-Ag is Ig heavy chain- like molecular.4. The development application of the serologic method in early diagnosis of gastric cancer by detecting MG7-AgUsing proper coating condition and antibody concentration, indirect BA-ELISA (biotin-avidin ELISA) was carried out before and after the operation in patients with gastric cancer and patients with precancerous lesions or other tumor groups to detect MG7-Ag in sera. PBS (0.01M) was taken as the negative control. A sample was considered“positive”if its absorbance was 2.1-fold of or higher than the negative value. The positive rate of gastric cancer-related MG7-Ag determined by ELISA was 83.6% in preoperative gastric cancer patients (97 of 116),which is higher than that of postoperative patients(63, 47.6%), precancerous lesions patients(78,12.8%), and other tumor groups, respectively (P <0.01). MG7-Ag can also be detected in sera of patients with other tumors (45.2% for lung cancer, 45.5% for rectal cancer, 17.6% for colonic cancer, and 14.2% for breast cancer), but no MG7-Ag was detected in sera of patients with endometrial cancer, lymphoma, or acute leukemia. The expression level of circulating MG7-Ag was significantly associated with TNM stage (P < 0.01) and lymph node metastasis (N0, 30, 73.3%;≥N1, 86,95.3%,P < 0.01). The positive rate of circulating MG7-Ag in stageⅠ(6, 50%),Ⅱ(46, 84.8%),Ⅲ(62, 96.8%) andⅣ(2, 100%)increased gradually. The above results indicated that MG7-Ag was a relatively specific marker in gastric cancer, and the expression of MG7-Ag was positive gradually along with the advancement of gastric cancer. The indirect BA-ELISA (biotin-avidin ELISA) method carried out to detect MG7-Ag in sera of patients may be taken as a convenient method for early diagnosis of gastric cancer.5. The effect of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell lineFlow cytometry was applied to detect the effect of MG7 monoclonal antibody on apoptosis and cell cycle in human gastric cancer cell line KATOⅢ.The results showed that MG7 monoclonal antibody would enhance the early apoptosis in human gastric cancer cell(P<0.05) and induce cell cycle arrest at G1 phrase remarkably.【Conclusions】In conclusion, the expression of MG7-Ag was statistically correlated with the differentiation level (P <0.01) and pathological stage (P <0.01) of gastric cancer. It indicated that MG7-Ag may play an important role in gastric cancer carcinogenesis. There was significant difference between the expression of MG7-Ag and hnRNPA2/B1 protein in gastric cancer cells. It indicated that MG7-Ag and hnRNPA2/B1 were not the same. MG7-Ag may be Ig heavy chain- like molecular. A serologic method for early diagnosis of gastric cancer was established by enzyme-linked immunoadsordent assay, with promising values in clinical use. MG7 monoclonal antibody would enhance the early apoptosis in human gastric cancer cells (P<0.05) and may induce cell cycle arrest at G1 phrase remarkably.更多还原