CN110045109A - 一种多肽在临床早期结直肠癌及癌前病变诊断中的应用 - Google Patents

一种多肽在临床早期结直肠癌及癌前病变诊断中的应用 Download PDF

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CN110045109A
CN110045109A CN201810030595.3A CN201810030595A CN110045109A CN 110045109 A CN110045109 A CN 110045109A CN 201810030595 A CN201810030595 A CN 201810030595A CN 110045109 A CN110045109 A CN 110045109A
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colorectal cancer
tcp
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王琰
曹之宪
陈晓光
杨潇骁
李燕
温宝莹
张森
冯茹
黄敏
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Institute of Materia Medica of CAMS
Chinese University of Hong Kong CUHK
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract

本发明公开了一种多肽在临床早期结直肠癌及癌前病变诊断中的应用，所述的多肽是如通式Ⅰ所示的9肽TCP‑1(CTPSPFSHC)。

Description

一种多肽在临床早期结直肠癌及癌前病变诊断中的应用
技术领域
本发明涉及与支持消化道肿瘤的血管系统选择性结合的9肽在临床早期结直肠癌及癌前病变诊断中的应用。
背景技术
结直肠癌(Colorectal cancer,CRC)严重威胁人类健康，长期处于世界常见恶性肿瘤的前三位。在早期即诊断出结直肠癌，病人的五年生存率会在85-90％之间，而如果在第四期发现，患者的五年生存率骤降到不足5％，这些充分证明了早期诊断的重要性。
在中国发明专利(发明名称：“用于肿瘤血管系统的多肽”，申请号：201080031426.3；授权号CN 102471371 B；公告日2014.04.23)中公开了一种用于肿瘤血管系统的多肽，公开了这种多肽被用作用于癌症的药物载体和成像剂。没有公开其对于临床早期结直肠癌及癌前病变组织诊断中的应用。
发明内容
本发明要解决的技术问题是提供一种多肽在临床早期结直肠癌及癌前病变诊断中的应用。
为解决本发明的技术问题，本发明提供如下技术方案：
本发明技术方案提供了一种多肽在临床早期结直肠癌及癌前病变诊断中的应用，其特征在于，所述的临床早期结直肠癌及癌前病变包括结直肠癌临床Ⅰ或Ⅱ期、高级别上皮内瘤变的早期病变组织。所述的临床Ⅰ和Ⅱ期是基于TNM分期系统的结直肠肿瘤分期。所述的高级别上皮内瘤变的早期病变组织包括重度异型增生和确诊癌变的腺瘤或息肉
有益技术效果
本发明公开了多肽TCP-1对于不同临床分期结直肠肿瘤的诊断率，可见TCP-1对于临床早期，即临床Ⅰ和Ⅱ期结直肠肿瘤具有很好的靶向性和检测灵敏度。
本发明公开了多肽TCP-1对于不同结直肠早期病变组织的诊断能力，可见TCP-1对于癌变风险大的高级别上皮内瘤变组织具有较好的结合阳性率，对癌变风险小的炎性息肉无特异结合性，具有较好的特异靶向性和检测灵敏度。
附图说明
图1.TCP-1以及对照肽的人结直肠组织免疫荧光染色结果
图2.临床分期确切的结直肠肿瘤样本免疫荧光染色结果统计(A)TCP-1对不同临床分期结直肠肿瘤组织结合阳性率；(B)TCP-1对临床Ⅰ和Ⅱ期、Ⅲ和Ⅳ期结直肠肿瘤组织结合阳性率；(C)TCP-1对T分期结直肠肿瘤组织结合阳性率；(D)TCP-1对N分期结直肠肿瘤组织结合阳性率；(E)TCP-1对M分期结直肠肿瘤组织结合阳性率
图3.结直肠早期病变样本免疫荧光染色结果统计(A)TCP-1对早期病变结直肠组织的结合阳性率；(B)TCP-1对炎性息肉无特异结合
具体实施方式
药理实验
在我们的研究中，多肽TCP-1对51个临床结直肠肿瘤标本的检测结果表明，TCP-1靶向结合的总阳性率为60.78％，其中对临床Ⅰ和Ⅱ期结直肠癌标本靶向阳性率达82.35％(17个标本)，提示TCP-1可用于结直肠肿瘤的早期诊断。此外，对2例分别确诊为原位癌或已癌变的腺瘤或息肉样本的检测，表明TCP-1靶向结果呈阳性，提示TCP-1可能进一步用于早期结直肠癌前病变的诊断。
实验例1.TCP-1体外靶向人结直肠肿瘤组织
收集人结直肠组织，包括肿瘤及癌旁组织、正常黏膜组织，经OCT包埋后制备冰冻切片(5μm)，95％乙醇固定10min后PBS洗3遍，5min/次，人组织冰冻切片10％山羊血清封闭1h后，加入100μL浓度为10μg/mL Biotin-TCP-1或Biotin-Control peptide(10％山羊血清配置)，同时加入100μL mouse anti-human CD31 monoclonal antibody(10％山羊血清1:50稀释)，4℃孵育过夜。PBS洗3次，5min/次,加入100μL Alexa Fluor 488 streptavidinantibody(10％山羊血清1:100稀释)及100μL Alexa Fluor 568 goat anti-mouse IgG(H+L)(10％山羊血清1:100稀释)，37℃孵育1h。PBS洗3次，5min/次，用含有DAPI及防荧光猝灭剂的封片剂封片，荧光倒置显微镜观察。
免疫荧光染色结果显示，TCP-1在人肿瘤组织中与CD31共定位，但在正常结直肠组织不显示荧光。Biotin-对照肽在人肿瘤组织与正常组织中均未检测到荧光。(结果如图1所示)
为考察TCP-1对临床不同分期的人结直肠癌组织的靶向情况，根据不同组织样本特点，分别对染色结果进行整理统计。
目前，已收集人结直肠组织样本累计共129个，其中包括83个肿瘤样本、4个癌旁组织和8个正常黏膜。在83个人结直肠癌肿瘤组织中，TCP-1靶向总阳性率为62.65％，TCP-1对正常组织无靶向性。(样本统计信息见表1)
表1.结直肠肿瘤样本免疫荧光染色结果统计
TNM分期系统是目前国际上最为通用的恶性肿瘤分期系统。TNM分期系统中：T(肿瘤一词英文Tumor的首字母)指肿瘤原发灶的情况，随着肿瘤体积的增加和邻近组织受累范围的增加，依次用T1～T4来表示。N(淋巴结一词英文Node的首字母)指区域淋巴结(regional lymph node)受累情况。淋巴结未受累时，用N0表示。随着淋巴结受累程度和范围的增加，依次用N1～N2表示。M(转移一词英文metastasis的首字母)指远处转移，没有远处转移者用M0表示，有远处转移者用M1表示。在此基础上，用TNM三个指标的组合(grouping)划出特定的分期(stage)。根据以上不同组合分为Ⅳ期：
0期：TisN0M0。
Ⅰ期：T1N0M0或T2N0M0。
Ⅱ期：T3N0M0或T4N0M0。
Ⅲ期：任何TN1M0或任何TN2M0。
Ⅳ期：任何T任何NM1。
对51个临床分期确切的结直肠肿瘤样本，TCP-1靶向临床Ⅱ期阳性率高达92.31％，综合临床Ⅰ和Ⅱ期结直肠癌标本，TCP-1靶向阳性率达82.35％(17个标本)；分别地，针对T分期，即原发瘤侵及程度，TCP-1靶向T3分期阳性率为62.50％，靶向T4分期阳性率为71.43％；针对N分期，即区域淋巴结转移，TCP-1靶向N0分期阳性率为82.35％；针对M分期，即远处转移，TCP-1靶向M0分期阳性率为61.70％。(样本统计信息和结果见表2、图2)
表2.临床分期确切的结直肠肿瘤样本免疫荧光染色结果统计
实验例2.TCP-1体外靶向人结直肠早期病变组织
收集人结直肠早期病变组织，包括腺瘤、息肉组织，经OCT包埋后制备冰冻切片(5μm)，95％乙醇固定10min后PBS洗3遍，5min/次，人组织冰冻切片10％山羊血清封闭1h后，加入100μL浓度为10μg/mL Biotin-TCP-1或Biotin-Control peptide(10％山羊血清配置)，同时加入100μL mouse anti-human CD31 monoclonal antibody(10％山羊血清1:50稀释)，4℃孵育过夜。PBS洗3次，5min/次,加入100μL Alexa Fluor 488 streptavidinantibody(10％山羊血清1:100稀释)及100μL Alexa Fluor 568 goat anti-mouse IgG(H+L)(10％山羊血清1:100稀释)，37℃孵育1h。PBS洗3次，5min/次，用含有DAPI及防荧光猝灭剂的封片剂封片，荧光倒置显微镜观察。
肠黏膜的异型增生为大肠癌的癌前病变，国内外普遍分为轻、中、重三级。“异型增生”是一种明确的肿瘤性生长,代表肿瘤生长的起始阶段,属于癌的浸润前期。结直肠息肉是指所有向肠腔内隆起的病变。按组织学类型不同可分为腺瘤性息肉、错构瘤性息肉、增生性息肉、炎性息肉等。息肉在形态上可分为有蒂、无蒂、广基、扁平状等。研究证明影响腺瘤性息肉与结直肠癌发病的危险因素基本一致。增生性息肉或炎性息肉则与感染和损伤相关。
根据病理学知识，轻、中度异型增生组织可归为低级别上皮内瘤变样本，重度异型增生和已确诊癌变的组织可归为高级别上皮内瘤变样本。对于目前收集到的24个结直肠早期病变样本，包括21个腺瘤和3个息肉样本，根据免疫荧光染色结果，TCP-1对癌变风险大的高级别上皮内瘤变的组织样本结合阳性率为66.67％。对目前收集到的2个炎性息肉样本，TCP-1对癌变风险小的炎性息肉未见特异靶向性。(统计结果如表3、图3所示)
表3.结直肠早期病变样本免疫荧光染色结果统计

Claims (4)

1.如通式Ⅰ所示9肽TCP-1在临床早期结直肠癌及癌前病变诊断中的应用
2.根据权利要求1的应用，其特征在于，所述的临床早期结直肠癌及癌前病变包括结直肠癌临床Ⅰ或Ⅱ期、高级别上皮内瘤变的早期病变组织。
3.根据权利要求2的应用，其特征在于，所述的临床Ⅰ和Ⅱ期是基于TNM分期系统的结直肠肿瘤分期。
4.根据权利要求2的应用，其特征在于，所述的高级别上皮内瘤变的早期病变组织包括重度异型增生和确诊癌变的腺瘤或息肉。
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Application publication date: 20190723