Regâ…£ Antibody Preparation And Its Expression Profile And Application

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.