Using The MCM2 As A Novel Cell Replicotion Marker In Diagnosing Colorectal Tumor

[Background] MCM2 is a marker of the family of minichromosome maintenance ( MCM ) proteins , which play a critical role in DNA replication by helping to ensure that DNA is replicated once and only once per cell cycle . some studies show that MCM2 was a specific markers of the cell cycle state in tissues , its expression was seen during all phases of the cell cycle , However, MCM2 was lost after exiting from the cell cycle , with rapid loss after differentiation and slower loss in quiescent ( GO ) cells . As a result of the sensitivity of antibodies to MCM2 as a markers of cell cycle state and because of the aberrant entry into the cell cycle in dysplasia and malignancy , MCM2 protein has been proposed as a candidate marker for cancer screening surveillance , and as prognostic marker . Williams et al demonstrated that MCM2 surface positivity correlated with the severity of cell dysplasia . in 2002 R.Justin et al firstly revealed that MCM2 was confined to the proliferative compartmant in the basal third to half of the crypts of normal large bowel mucosa, By contrast , MCM2 was expressed throughout the epithelium including in surface colonocytes when adenocarcinomas are present . though retrieved from the faecal surface to detect MCM2 expressionin colonocytes . they detected MCM2-positive cell in all patients with colorectal cancer , and no one case was MCM2 expressed in all control individuals . As a result, in 2003' colorectal surgical conference of England and North Ireland , they advocated to early screen colonic cancer by detection of MCM2 expression in stool . but in recent years , MCM2-positive cells can also be detected in patients of colonic adenomas , therefore , those patients whose stool was detected with MClVb-positive cells can't be directly diagnosed as colonic adenocarcinomas because of exception of colonic adenomas . consequently , the difference of distribution and expression of MCM2 between colonic adenomas and colonic adenocarcinomas have become the frontline subjects and the most pressing task in the research field of early diagnosis of colonic carcinoma .[Objective] The objective of this study include : 1 Investigate the expression and distribution of the MCM2 in tissues between the normal large bowel mucosa and colonic adenomas or adenocarcinomas . 2 Using the technique of realtime quantitative PCR precisely detect the difference of MCN^'s expression between the normal large bowel mucosa and colonic adenomas or adenocarcinomas , to provide foundation for further research of MCIMb's expression in different phases of malignant cells .[Methods] 1 50 specimens were taken from operational patients or bronchoscopy at the west china hospital of Sichuan university from September 2004 to December 2004 including : 12 colonic carcinomas ; 33 colonic adenomas and 5 nomal colonic mucosa . all specimens were confirmed by pathological diagnosis and put in -150°C refrigerator in time .2 Immunohistochemistry( IHC ) : According to the principle of specificantigen-antibody combination , MCM2 protein of tissues ( nomal large bowel mucosa , colonic adenomas or adenocarcinomas ) were detected by specific anti-human MCM2 antibody with immunohistochemistry technique to reveal MCM2S expressional location . 3 Realtime quantitative PCR was applied to detect the expression of MCM2-mRNA . total RNA was isolated using Trizol Reagent from colonic adenomas or adenocarcinoma . Rt-PCR was performed by using the specific primers PI ( 5'-CACATCGAGTCCATGATCC-3' ) ; P2( 5'-CAAAAGTCTTGCGCATGCT-3' ) with 45 cycles of amplification to detect the difference of expression between the normal large bowel mucosa and colonic adenomas or adenocarcinoma from the horizontal of mRNA. [Results ] 1 Immunohistochemistry show that MCM2 expression in healthy colon is confined to basal third to half of colonic crypts with no expression in surface colonocytes , however , in colonic adenocarcinomas or adenomas , MCM2 expression is seen throughout epithelium , including in surface colonocytes . this result is in accordance with R.Justin etal . 2 Using the technique of realtime quantitative PCR , we can detect about 160 bp cDNA fragment from total RNA of the human's colonic adenomas or adenocarcinoma and find their difference quantity of expression in both tissues . in the group of colonic adenocarcinoma , their mean is about 31.5833 and their mean is about 16.6061 in the group of colonic adenomas , and their quantitive difference of expression in both tissues have significant statistic differences (P=0.004)[Conclusions ] : In summary , our results suggest that : 1 In normal large bowel mucosa , MCM2 expression is confined to the proliferativecompartmant in the basal third to half of the crypts , By contrast, MCM2 is expressed throughout the epithelium including in surface colonocytes . their MCM2 expressional location is identical between colonic adenomas and colonic adenocarcinomas 2 Though MCM2 is expressed thoughout the epthelium in colonic adenomas and adenocarcinomas . their expression of quantities are different in both tissues to give us a chance to use quantitative detection to indentify colonic adenomas from MCM2-positive cells and enhance the realizability or utilizability of MCM2 as a marker of early detection for colonic cancer .