RegⅣ抗体制备及组织表达谱与应用研究
摘要
结直肠癌是严重危害人类健康的常见恶性肿瘤之一。在我国由于结直肠癌的发病率不断攀升,对其发生机制的研究已经引起人们的极大重视。肿瘤的发生发展是基因改变不断发生、不断累积的结果。研究显示,腺瘤是结直肠癌发生的早期阶段,多数结直肠癌是从腺瘤癌变而来的。切除腺瘤可以有效地降低结直肠癌的发病率。然而,约30%～46%的腺瘤患者在接受治疗后2.5～7年内会复发。目前尚缺乏有效的指标来预测腺瘤的进一步演进或复发。因此,腺瘤的发生机制及其进展为癌的机制的研究对于结直肠癌的预防及治疗具有重大意义。尽管结直肠癌发生的分子机理相对于其它实体肿瘤的研究较为深入,但细胞的恶性转变是一个极其复杂的、多步骤过程,许多细节至今还不清楚。除了少数已知基因外,许多未知基因也参与了细胞恶性转化的过程,其在转变过程中的作用还有待进一步鉴定。因此,结直肠腺瘤、结直肠癌发生相关差异表达基因的识别、分离和鉴定,结直肠癌发生不同阶段转变过程中的关键基因的确定,将为结直肠腺瘤及结直肠癌发生的机理的深入了解奠定基础,同时,为早期诊断和治疗提供依据。
1999年,本实验室应用抑制性差减杂交法(SSH)(利用一个同时患有结肠腺瘤和腺癌的病例)构建了三个cDNA差减文库,分别为:腺瘤相对于正常粘膜的差异片段文库(A-N)、腺癌相对于腺瘤的差异片段文库(T-A)、腺癌相对于正常粘膜的差异片段文库(T-N)。在分析A-N文库时,我们发现RegⅣ是在腺瘤中相对高表达的基因。
Reg基因家族,即再生基因(Regenerating Gene)家族,属于C型植物血凝素超家族。该基因家族成员,均为小分子量的分泌性蛋白质,在结构上具有相似的钙离子依赖性血凝素结构域。它们具有急性期反应蛋白,凝集素,及抗凋亡或生长因子样的作用,参与炎症,损伤/修复过程,并与糖尿病及恶性肿瘤的发生发展有关。迄今为止,已经发现的与人类相关的Reg基因家族成员一共有4个,即RegⅠα,RegⅠβ,RegⅢ及RegⅣ。RegⅣ又称为再生蛋白样蛋白(regeneratingprotein-like protein,RELP),或胃肠分泌蛋白(gastrointesinal protein,GISP),是2001年从炎症性肠病中筛选出来的新基因,属于最新一类Reg基因家族成员。不久之后,我们便从A-N文库中,筛选到RegⅣ在腺瘤中高表达。与其他人类Reg家族成员不同的是,RegⅣ定位于第一号染色体上,而其余成员均定位于第二号染色体。但RegⅣ与Reg基因家族的其他成员在结构及功能上具有相似性。近年来的研究显示,RegⅣ与胃、结直肠及胰腺肿瘤相关,并可在前列腺癌中高表达。Violette等发现相对于药物敏感性细胞系,RegⅣ在耐药性的结直肠细胞系中表达水平高。有关RegⅣ功能机制的研究只有一篇有关它参与EGFR/AKT/AP-1信号通路的报道。RegⅣ作为一个从结直肠腺瘤的cDNA文库中筛选出来的高表达基因,该基因的编码产物在结直肠组织中的定位及表达情况是未知的;作为一个在癌前病变中高表达的基因,RegⅣ是否可作为结直肠癌早期诊断工具是未知的;作为一个较新的基因,RegⅣ的表达谱从未有人做详尽的报道。
为更多地了解RegⅣ,并为深入研究RegⅣ在恶性肿瘤发生发展中的作用提供数据支持,本课题拟制备RegⅣ蛋白的多克隆抗体及单克隆抗体;研究RegⅣ基因在结直肠癌中的表达情况;利用自制抗体,通过免疫组化的方法,分析RegⅣ在全身各器官正常及病变组织中的表达谱,以期为其功能的阐释及应用前景的描述提供新的视角。
我们利用原核系统诱导表达RegⅣ全长重组蛋白,将其纯化后与RegⅣ肽段蛋白同时免疫新西兰大白兔,制备多克隆抗体;用RegⅣ全长重组蛋白免疫BALB/c小鼠,与小鼠骨髓瘤SP2/0细胞体外融合,通过筛选、克隆化稀释及鉴定,获得稳定分泌RegⅣ单克隆抗体的杂交瘤细胞株。将自制的兔抗RegⅣ肽段多抗、自制兔抗RegⅣ重组蛋白多抗及商业化羊抗RegⅣ重组蛋白多抗的检验效能进行比较。利用自制兔抗RegⅣ重组蛋白多抗进行免疫组织化学分析RegⅣ在结直肠正常粘膜,腺瘤及癌配对组织中的表达定位情况,并用实时荧光定量PCR及Western-blot方法进行验证。我们通过对65例结直肠正常粘膜及匹配癌组织的免疫组化分析,研究RegⅣ的表达情况及其表达水平与结直肠癌临床病理参数之间的关系。随后,利用自制的单克隆抗体检测全身24种正常器官组织及40种良恶性病变组织中RegⅣ的表达分布。另外,检测18种神经内分泌肿瘤中RegⅣ的表达情况。
研究结果显示我们制备的多克隆抗体及单克隆抗体,效价好,特异性强,可用于ELISA、Western-blot、及免疫组化检测。相比于肽段蛋白作为抗原制备的多抗,全长重组蛋白的多抗作为检测工具更为可靠。免疫组化,Real-time PCR及Western-blot结果显示RegⅣ在结直肠腺瘤中的表达显著高于配对的正常粘膜与癌组织(p＜0.05),而在正常粘膜与癌组织中的表达未达到统计学差异(p＞0.05)。RegⅣ在结直肠腺瘤中的阳性染色率为87.5%,在癌旁粘膜中为100%,而在癌组织中为30.8%。RegⅣ的表达与结直肠癌组织学类型(粘液腺癌/印戒细胞癌,8/11,72.7%,p=0.004)及分化程度(低分化,7/11,63.6%,p=0.007)有关。在全身正常组织中,RegⅣ除较特异地表达于胃肠道外,还可在肾上腺与乳腺中被检测到。在所有被检恶性疾病组织中,RegⅣ倾向于在组织学类型为腺癌的恶性肿瘤中表达,其中在胰腺、胃、结肠、前列腺及肝脏转移性腺癌中呈中度或强阳性表达,但是在肺腺癌中RegⅣ表达为阴性。在18种类型的神经内分泌肿瘤中,RegⅣ主要表达于胃肠道神经内分泌肿瘤包括胰腺、胃、结直肠及阑尾,很少见于胃肠道外的神经内分泌肿瘤,除嗜铬细胞瘤(大多为中强阳性染色)及甲状腺髓样癌(一般为弱阳性)。CgA与RegⅣ染色比较发现,CgA表达阴性的胃肠道神经内分泌肿瘤中,RegⅣ可呈阳性表达。
通过以上研究,我们得出以下结论:
我们成功制备了RegⅣ的多克隆抗体及单克隆抗体,从而为RegⅣ表达谱及应用前景的研究奠定了良好的基础;
RegⅣ在结直肠腺瘤中高表达,可能参与了结直肠癌早期阶段的分子改变,而RegⅣ在结直肠癌中的表达与低分化及粘液腺癌有关,说明其可能在结直肠癌的发生发展过程中有多重作用;
在正常组织中,RegⅣ的表达部位主要局限于消化系统;恶性病变中,RegⅣ富集表达于腺癌中,可见,RegⅣ的表达具有一定的组织特异性,它可能在腺上皮肿瘤尤其是消化系统腺上皮肿瘤的发生发展中发挥作用。另外,RegⅣ在结直肠腺癌及其他多种组织来源的腺癌中呈阳性表达而在肺腺癌中完全阴性表达,使其有可能成为一个鉴别转移性腺癌原发灶为肺或肺外器官的辅助指标;
在神经内分泌肿瘤中,除嗜铬细胞瘤外,RegⅣ特异性地表达于胃肠道神经内分泌肿瘤,并可在CgA阴性表达的胃肠道神经内分泌肿瘤中阳性表达,表明RegⅣ可能具有辅助诊断胃肠道神经内分泌肿瘤的应用前景,为该类型肿瘤的明确诊断增加筹码。
Colorectal Cancer(CRC) is one of the commonest malignancies which threaten the health of human.Understanding of the mechanism of colorectal carcinogenesis has been gaining momentum for some years on account of its high incidence.Tumors carinogenesis is a process of multigene and multistep change.The adenoma-carcinoma sequence is now widely accepted as the major pathway for the colorectal cancer development in the general population.Colorectal adenoma is recognized as the precancerous lesion for most cases of colorectal cancer.Removal of these adenomas resulted in a markedly lower colorectal cancer occurrence than that without polypectomy.A 30%-46%recurrence rate of adenomas has been reported in researches with a follow-up period of 2.5-7 years.There is no effective criterion in evaluating whether adenoma will develop cancer or recur.A better understanding of the underlying mechanism of adenoma development is crucial for prevention and early diagnosis of colorectal cancer.Although many genes have been reported to be in close association with the progression of colorectal carcinogenesis,they are far from enough to explain the malignant transformation.A lot of other genes,known or unknown,remain to be discovered.Identification and characterization of genes expressed exclusively or preferentially in human tumor tissue will,hopefully,shed light on the mechanism of colorectal carcinogenesis and provide useful biological markers for early diagnosis and therapy.
In 1999,we constructed three subtracted cDNA libraries by SSH.They were colonic adenocarcinoma-normal mucosa cDNA subtracted library(subtracted T-N library),colonic adenoma-normal mucosa cDNA subtracted library(subtracted A-N library),and colonic adenocarcinoma-adenoma cDNA subtracted library(subtracted T-A library).RegⅣgene was found in A-N library,the expression level of which was higher in adenoma than in normal mucosa.
Reg gene family,namely regenerating gene family,belongs to C-type lectin superfamily.The coding product of Reg gene family is a group of small secertory proteins which share similar C-type lectin domain structurally.Studies on Reg gene family revealed that they may serve as tissue mitogen,which are active in injury response and inflammation,and play a role in diabetes and tumors.Up to now,there are four human Reg gene family members,namely,RegⅠα,RegⅠβ,RegⅢand RegⅣ. Regenerating geneⅣ,the most recently discovered member of the family,was also named as regenerating protein-like protein(RELP) and gastrointesinal protein(GISP). Different from other Reg gene family members,which locate in chromosome 2,RegⅣlocates in chromosome 1.However,RegⅣshares common features with other members structurally and functionally.Current data show that RegⅣwas detected as a differentially expressed gene in drug-resistant colorectal cancer cell lines compared with the sensitive ones.Similar with other members of Reg gene family,RegⅣis overexpressed in inflammatory bowel disease and tumors of stomach,colorectum, prostate and pancreas.Till now,little has been revealed about underlying mechanism that how RegⅣfunctions,except that it might be involved in EGFR signal pathway.To have a better knowledge,we explored the expression profile of RegⅣby IHC analysis in normal and benign/malignant human tissues,which covered a much broader range than that in existing reports.After that,we demonstrated RegⅣexpression in colorectal tumors and analyzed its association with clinical features.Since RegⅣmay be associated with neuroendocrine differentiation,we studied its distribution in various neuroendocrine tumors.
The recombinant RegⅣprotein was expressed in prokaryotic system,after that it was purified by affinity chromatograph beads.The purified recombinant protein and chemically synthesized RegⅣpolypeptide were both used to immunize New Zealand Rabbits to produce antiserum.Meanwhile,BALB/c mice were immunized by RegⅣrecombinant protein,the spleen cells of which were fused with mouse myeloma cell SP2/0 to produce RegⅣmonoclonal antibody.Self-prepared rabbit anti-popypeptide RegⅣantibody,self-prepared rabbit anti-recombinant human RegⅣantibody and commercial goat anti-recombinant human RegⅣantibody were compared on their detection efficacy of RegⅣpositive cases.Self-prepared rabbit anti-recombinant human RegⅣantibody was chosen to analyse RegⅣexpression in colorectal normal mucosa,adenoma and colorectal carcinoma tissue by immunohistochemistry. Quantitative PCR and Western-blot were conducted to verify RegⅣexpression level. An additional set of 65 CRC cases was used to evaluate RegⅣexpression and its association with clinicopathological parameters.Then,24 types of human normal tissues and 40 types benign and malignant tissues were involved in the study to detect RegⅣexpression profile with the application of RegⅣmonoclonal antibody.Besides that,immunohistochemistry was also done on 18 types of neuroendocrine tumors to analyze RegⅣdistribution in neuroendocrine tumors.
Our data showed that the self-prepared RegⅣpolyclonal and monoclonal antibody were qualified by their specificity and sensitivity for ELISA,Western-blot and immunohistochemistry assays.High expression level of RegⅣin colorectal adenoma was demonstrated by Q-PCR,Western-blot and IHC analysis(p<0.05),while RegⅣexpression in normal mucosa and carcinoma tissue were of no significant difference(P>0.05).RegⅣpositive staining was found in evident association with poor differentiated tumors(7/11,63.6%,p=0.007) and tumors with histological type of mucinous adenocarcinoma/signet-cell carcinoma(8/11,72.7%,p=0.004).In human normal tissues,besides its relative specificity to gastrointestinal tract,RegⅣcould also be detected in normal adrenal gland and mammary gland.Among all the malignancy of various histology types under evaluation,RegⅣshowed predominance in adenocarcinomas,which was intensively positive in adenocarcinoma of pancreas, stomach,colon,and prostate,but negative in that of lung.Of the 18 types neuroendocrine disorders,RegⅣexpression was positive in gastrointestinal neuroendocrine tumors including stomach,colon,rectum and pancreas but not in non-digestive system except for medullary thyroid cancer(usually weak) and pheochromocytoma(usually moderate/strong).Comparasion between Chromogranin A(CgA) and RegⅣstaining in neuroendocrine tumors revealed that RegⅣcould detect neuroendocrine tumors that CgA could not.
Conclusion:
We successfully prepared RegⅣpolyclonal and monoclonal antibody,which are basic tools for RegⅣ's expression profile and for the assessment of its application potential;
RegⅣis overexpressed in colorectal adenoma.Therefore,RegⅣmay play an important role in early stage of colorectal carcinogensis.However,interestingly,RegⅣexpression in CRCs was in association with poor differentiation,suggesting that RegⅣmay have multiple roles in colorectal carcinogenesis.
In normal tissues,RegⅣexpression is mainly restricted to digestive system; among different types of malignant lesion,RegⅣis preferentially expressed in adenocarcinoma,which suggests that RegⅣexpression was of organ and histology specificity,and it may be involved in tumorogenesis of glandular epithelial carcinoma especially in that of digestive system epithelial carcinoma.
The adenocarcinoma of stomach,colorectum,pancreas,and prostate are RegⅣstrong positive,but lung adenocarcinoma is definitely negative,which indicates RegⅣ is a possibly good marker in determining the origin of metastatic adenocarcinoma from lung or outside lung organs.
RegⅣmay widen the arsenal for diagnosis of neuroendocrine tumors with a restricted expression pattern in digestive tract neuroendocrine tumors and pheochromocytoma.
1999年,本实验室应用抑制性差减杂交法(SSH)(利用一个同时患有结肠腺瘤和腺癌的病例)构建了三个cDNA差减文库,分别为:腺瘤相对于正常粘膜的差异片段文库(A-N)、腺癌相对于腺瘤的差异片段文库(T-A)、腺癌相对于正常粘膜的差异片段文库(T-N)。在分析A-N文库时,我们发现RegⅣ是在腺瘤中相对高表达的基因。
Reg基因家族,即再生基因(Regenerating Gene)家族,属于C型植物血凝素超家族。该基因家族成员,均为小分子量的分泌性蛋白质,在结构上具有相似的钙离子依赖性血凝素结构域。它们具有急性期反应蛋白,凝集素,及抗凋亡或生长因子样的作用,参与炎症,损伤/修复过程,并与糖尿病及恶性肿瘤的发生发展有关。迄今为止,已经发现的与人类相关的Reg基因家族成员一共有4个,即RegⅠα,RegⅠβ,RegⅢ及RegⅣ。RegⅣ又称为再生蛋白样蛋白(regeneratingprotein-like protein,RELP),或胃肠分泌蛋白(gastrointesinal protein,GISP),是2001年从炎症性肠病中筛选出来的新基因,属于最新一类Reg基因家族成员。不久之后,我们便从A-N文库中,筛选到RegⅣ在腺瘤中高表达。与其他人类Reg家族成员不同的是,RegⅣ定位于第一号染色体上,而其余成员均定位于第二号染色体。但RegⅣ与Reg基因家族的其他成员在结构及功能上具有相似性。近年来的研究显示,RegⅣ与胃、结直肠及胰腺肿瘤相关,并可在前列腺癌中高表达。Violette等发现相对于药物敏感性细胞系,RegⅣ在耐药性的结直肠细胞系中表达水平高。有关RegⅣ功能机制的研究只有一篇有关它参与EGFR/AKT/AP-1信号通路的报道。RegⅣ作为一个从结直肠腺瘤的cDNA文库中筛选出来的高表达基因,该基因的编码产物在结直肠组织中的定位及表达情况是未知的;作为一个在癌前病变中高表达的基因,RegⅣ是否可作为结直肠癌早期诊断工具是未知的;作为一个较新的基因,RegⅣ的表达谱从未有人做详尽的报道。
为更多地了解RegⅣ,并为深入研究RegⅣ在恶性肿瘤发生发展中的作用提供数据支持,本课题拟制备RegⅣ蛋白的多克隆抗体及单克隆抗体;研究RegⅣ基因在结直肠癌中的表达情况;利用自制抗体,通过免疫组化的方法,分析RegⅣ在全身各器官正常及病变组织中的表达谱,以期为其功能的阐释及应用前景的描述提供新的视角。
我们利用原核系统诱导表达RegⅣ全长重组蛋白,将其纯化后与RegⅣ肽段蛋白同时免疫新西兰大白兔,制备多克隆抗体;用RegⅣ全长重组蛋白免疫BALB/c小鼠,与小鼠骨髓瘤SP2/0细胞体外融合,通过筛选、克隆化稀释及鉴定,获得稳定分泌RegⅣ单克隆抗体的杂交瘤细胞株。将自制的兔抗RegⅣ肽段多抗、自制兔抗RegⅣ重组蛋白多抗及商业化羊抗RegⅣ重组蛋白多抗的检验效能进行比较。利用自制兔抗RegⅣ重组蛋白多抗进行免疫组织化学分析RegⅣ在结直肠正常粘膜,腺瘤及癌配对组织中的表达定位情况,并用实时荧光定量PCR及Western-blot方法进行验证。我们通过对65例结直肠正常粘膜及匹配癌组织的免疫组化分析,研究RegⅣ的表达情况及其表达水平与结直肠癌临床病理参数之间的关系。随后,利用自制的单克隆抗体检测全身24种正常器官组织及40种良恶性病变组织中RegⅣ的表达分布。另外,检测18种神经内分泌肿瘤中RegⅣ的表达情况。
研究结果显示我们制备的多克隆抗体及单克隆抗体,效价好,特异性强,可用于ELISA、Western-blot、及免疫组化检测。相比于肽段蛋白作为抗原制备的多抗,全长重组蛋白的多抗作为检测工具更为可靠。免疫组化,Real-time PCR及Western-blot结果显示RegⅣ在结直肠腺瘤中的表达显著高于配对的正常粘膜与癌组织(p＜0.05),而在正常粘膜与癌组织中的表达未达到统计学差异(p＞0.05)。RegⅣ在结直肠腺瘤中的阳性染色率为87.5%,在癌旁粘膜中为100%,而在癌组织中为30.8%。RegⅣ的表达与结直肠癌组织学类型(粘液腺癌/印戒细胞癌,8/11,72.7%,p=0.004)及分化程度(低分化,7/11,63.6%,p=0.007)有关。在全身正常组织中,RegⅣ除较特异地表达于胃肠道外,还可在肾上腺与乳腺中被检测到。在所有被检恶性疾病组织中,RegⅣ倾向于在组织学类型为腺癌的恶性肿瘤中表达,其中在胰腺、胃、结肠、前列腺及肝脏转移性腺癌中呈中度或强阳性表达,但是在肺腺癌中RegⅣ表达为阴性。在18种类型的神经内分泌肿瘤中,RegⅣ主要表达于胃肠道神经内分泌肿瘤包括胰腺、胃、结直肠及阑尾,很少见于胃肠道外的神经内分泌肿瘤,除嗜铬细胞瘤(大多为中强阳性染色)及甲状腺髓样癌(一般为弱阳性)。CgA与RegⅣ染色比较发现,CgA表达阴性的胃肠道神经内分泌肿瘤中,RegⅣ可呈阳性表达。
通过以上研究,我们得出以下结论:
我们成功制备了RegⅣ的多克隆抗体及单克隆抗体,从而为RegⅣ表达谱及应用前景的研究奠定了良好的基础;
RegⅣ在结直肠腺瘤中高表达,可能参与了结直肠癌早期阶段的分子改变,而RegⅣ在结直肠癌中的表达与低分化及粘液腺癌有关,说明其可能在结直肠癌的发生发展过程中有多重作用;
在正常组织中,RegⅣ的表达部位主要局限于消化系统;恶性病变中,RegⅣ富集表达于腺癌中,可见,RegⅣ的表达具有一定的组织特异性,它可能在腺上皮肿瘤尤其是消化系统腺上皮肿瘤的发生发展中发挥作用。另外,RegⅣ在结直肠腺癌及其他多种组织来源的腺癌中呈阳性表达而在肺腺癌中完全阴性表达,使其有可能成为一个鉴别转移性腺癌原发灶为肺或肺外器官的辅助指标;
在神经内分泌肿瘤中,除嗜铬细胞瘤外,RegⅣ特异性地表达于胃肠道神经内分泌肿瘤,并可在CgA阴性表达的胃肠道神经内分泌肿瘤中阳性表达,表明RegⅣ可能具有辅助诊断胃肠道神经内分泌肿瘤的应用前景,为该类型肿瘤的明确诊断增加筹码。
Colorectal Cancer(CRC) is one of the commonest malignancies which threaten the health of human.Understanding of the mechanism of colorectal carcinogenesis has been gaining momentum for some years on account of its high incidence.Tumors carinogenesis is a process of multigene and multistep change.The adenoma-carcinoma sequence is now widely accepted as the major pathway for the colorectal cancer development in the general population.Colorectal adenoma is recognized as the precancerous lesion for most cases of colorectal cancer.Removal of these adenomas resulted in a markedly lower colorectal cancer occurrence than that without polypectomy.A 30%-46%recurrence rate of adenomas has been reported in researches with a follow-up period of 2.5-7 years.There is no effective criterion in evaluating whether adenoma will develop cancer or recur.A better understanding of the underlying mechanism of adenoma development is crucial for prevention and early diagnosis of colorectal cancer.Although many genes have been reported to be in close association with the progression of colorectal carcinogenesis,they are far from enough to explain the malignant transformation.A lot of other genes,known or unknown,remain to be discovered.Identification and characterization of genes expressed exclusively or preferentially in human tumor tissue will,hopefully,shed light on the mechanism of colorectal carcinogenesis and provide useful biological markers for early diagnosis and therapy.
In 1999,we constructed three subtracted cDNA libraries by SSH.They were colonic adenocarcinoma-normal mucosa cDNA subtracted library(subtracted T-N library),colonic adenoma-normal mucosa cDNA subtracted library(subtracted A-N library),and colonic adenocarcinoma-adenoma cDNA subtracted library(subtracted T-A library).RegⅣgene was found in A-N library,the expression level of which was higher in adenoma than in normal mucosa.
Reg gene family,namely regenerating gene family,belongs to C-type lectin superfamily.The coding product of Reg gene family is a group of small secertory proteins which share similar C-type lectin domain structurally.Studies on Reg gene family revealed that they may serve as tissue mitogen,which are active in injury response and inflammation,and play a role in diabetes and tumors.Up to now,there are four human Reg gene family members,namely,RegⅠα,RegⅠβ,RegⅢand RegⅣ. Regenerating geneⅣ,the most recently discovered member of the family,was also named as regenerating protein-like protein(RELP) and gastrointesinal protein(GISP). Different from other Reg gene family members,which locate in chromosome 2,RegⅣlocates in chromosome 1.However,RegⅣshares common features with other members structurally and functionally.Current data show that RegⅣwas detected as a differentially expressed gene in drug-resistant colorectal cancer cell lines compared with the sensitive ones.Similar with other members of Reg gene family,RegⅣis overexpressed in inflammatory bowel disease and tumors of stomach,colorectum, prostate and pancreas.Till now,little has been revealed about underlying mechanism that how RegⅣfunctions,except that it might be involved in EGFR signal pathway.To have a better knowledge,we explored the expression profile of RegⅣby IHC analysis in normal and benign/malignant human tissues,which covered a much broader range than that in existing reports.After that,we demonstrated RegⅣexpression in colorectal tumors and analyzed its association with clinical features.Since RegⅣmay be associated with neuroendocrine differentiation,we studied its distribution in various neuroendocrine tumors.
The recombinant RegⅣprotein was expressed in prokaryotic system,after that it was purified by affinity chromatograph beads.The purified recombinant protein and chemically synthesized RegⅣpolypeptide were both used to immunize New Zealand Rabbits to produce antiserum.Meanwhile,BALB/c mice were immunized by RegⅣrecombinant protein,the spleen cells of which were fused with mouse myeloma cell SP2/0 to produce RegⅣmonoclonal antibody.Self-prepared rabbit anti-popypeptide RegⅣantibody,self-prepared rabbit anti-recombinant human RegⅣantibody and commercial goat anti-recombinant human RegⅣantibody were compared on their detection efficacy of RegⅣpositive cases.Self-prepared rabbit anti-recombinant human RegⅣantibody was chosen to analyse RegⅣexpression in colorectal normal mucosa,adenoma and colorectal carcinoma tissue by immunohistochemistry. Quantitative PCR and Western-blot were conducted to verify RegⅣexpression level. An additional set of 65 CRC cases was used to evaluate RegⅣexpression and its association with clinicopathological parameters.Then,24 types of human normal tissues and 40 types benign and malignant tissues were involved in the study to detect RegⅣexpression profile with the application of RegⅣmonoclonal antibody.Besides that,immunohistochemistry was also done on 18 types of neuroendocrine tumors to analyze RegⅣdistribution in neuroendocrine tumors.
Our data showed that the self-prepared RegⅣpolyclonal and monoclonal antibody were qualified by their specificity and sensitivity for ELISA,Western-blot and immunohistochemistry assays.High expression level of RegⅣin colorectal adenoma was demonstrated by Q-PCR,Western-blot and IHC analysis(p<0.05),while RegⅣexpression in normal mucosa and carcinoma tissue were of no significant difference(P>0.05).RegⅣpositive staining was found in evident association with poor differentiated tumors(7/11,63.6%,p=0.007) and tumors with histological type of mucinous adenocarcinoma/signet-cell carcinoma(8/11,72.7%,p=0.004).In human normal tissues,besides its relative specificity to gastrointestinal tract,RegⅣcould also be detected in normal adrenal gland and mammary gland.Among all the malignancy of various histology types under evaluation,RegⅣshowed predominance in adenocarcinomas,which was intensively positive in adenocarcinoma of pancreas, stomach,colon,and prostate,but negative in that of lung.Of the 18 types neuroendocrine disorders,RegⅣexpression was positive in gastrointestinal neuroendocrine tumors including stomach,colon,rectum and pancreas but not in non-digestive system except for medullary thyroid cancer(usually weak) and pheochromocytoma(usually moderate/strong).Comparasion between Chromogranin A(CgA) and RegⅣstaining in neuroendocrine tumors revealed that RegⅣcould detect neuroendocrine tumors that CgA could not.
Conclusion:
We successfully prepared RegⅣpolyclonal and monoclonal antibody,which are basic tools for RegⅣ's expression profile and for the assessment of its application potential;
RegⅣis overexpressed in colorectal adenoma.Therefore,RegⅣmay play an important role in early stage of colorectal carcinogensis.However,interestingly,RegⅣexpression in CRCs was in association with poor differentiation,suggesting that RegⅣmay have multiple roles in colorectal carcinogenesis.
In normal tissues,RegⅣexpression is mainly restricted to digestive system; among different types of malignant lesion,RegⅣis preferentially expressed in adenocarcinoma,which suggests that RegⅣexpression was of organ and histology specificity,and it may be involved in tumorogenesis of glandular epithelial carcinoma especially in that of digestive system epithelial carcinoma.
The adenocarcinoma of stomach,colorectum,pancreas,and prostate are RegⅣstrong positive,but lung adenocarcinoma is definitely negative,which indicates RegⅣ is a possibly good marker in determining the origin of metastatic adenocarcinoma from lung or outside lung organs.
RegⅣmay widen the arsenal for diagnosis of neuroendocrine tumors with a restricted expression pattern in digestive tract neuroendocrine tumors and pheochromocytoma.
引文
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