| [Objective]In view of the close relationship between Ras gene mutation,protein expression and tumor occurrence,development,invasion,metastasis and prognosis, foreign researchers have developed many kinds of monoclonal antibody hybridoma cell lines against the p21Ras(Y13-258,YA6-172,Y13-128,NCC001/004,Ras10/11, RAP-1/5) since the first strain anti-p21Ras monoclonal antibody hybridoma cell line(Y13-159) set up in 1982.Based on genetic engineering technology,the small molecule anti-p21Ras antibody(ScFv) was established showed good activity against tumor.However,the studies about Ras gene,p21Ras protein structure and function are rare and no research about the p21Ras antibody is found in domestic.The therapy targeting p21Ras signaling pathway has been a hot research area.With the support by the National Nature Science Funds of China and the Research Funds of Yunnan Province,our laboratory have obtained successfully anti-p21Ras monoclonal antibody hybridoma cell lines which proved that these antibodies could cross-reativate with p21Ras-H,N,K proteins.In this study,we focused on the expression of p21Ras in colorectal benign and malignant lesions by immunohistochemical method,detected the specificity and sensitivity of the antibody and the expression spectrum in tumors.By this study,we aim to the orentical and practical foundation for the clinical application of our antibody and to further confirm the relationship between p21Ras expression and the carcinogenesis,development and metastasis in cancer.[Method]1.Antibody(preparation by our laboratory ):Baseing on the 85% homology of three kinds of N-Ras protein amino acid sequence and 1-80 amino acid residues highly conserved in theory,our laboratory applied H-Ras gene CDS sequence expression product-p21Ras-H protein after renaturation to immune repeatedly Balb/c mice 4 times.Then obtaining its spleen B lymphocytes and integnating them with myeloma cells SP2/0;selective culture by HAT selective culture medium;cloning and subcloning of hybridoma calls and screening by indirect ELISA method.Eventually setting up anti-p21Ras monoclonal antibody hybridoma cell line KGH-R1.2.Specimen collection:formalin fixed paraffin blocks of 45 cases of normal colorectal tissue(with a 5-cm distance above from the tumor margin),73 cases of colorectal inflammatory polyps,48 cases of colorectal low-grade intraepithelial neoplasia,83 cases of colorectal high-grade intraepithelial neoplasia,94 cases of invasive colorectal adenocarcinoma were collected in Kunming General Hospital of Chengdu Military Region,.3.IHC staining:the expression of p21Ras were examined by immunohistochemical SP method and evaluated by semi-quantitative analysis(percentage of positive cells and Hscore score system).The statistical analysis was performed using the SSPS sofeware to determine the expression of p21Ras in benign and malignant lesions of the colon tissue and analysis of cancer and adjacent lesions of p21Ras expression,as well as the relationship of p21Ras expression and clinicopathnologic factors in colorectal adenocarcinomas and adenomas respectively.[Results]1.The expressionof p21Ras protein in colorectal epithelial cells localized in the cytoplasm and cell membrane.There is almost no p21Ras expression in normal colorectal epithelium.p21Ras expression in normal colorectal epithelium,colon common inflammatory polyps,colorectal low-grade intraepithelial neoplasia, high-grade colorectal intraepithelial neoplasia and invasive colorectal adenocarcinoma with Hscore score(0-300 points) in turn with the mean value of 1.23±2.98,7.14±16.74,66.54±59.91,94.20±87.46,116.14±103.30 respectively,and the percentages of positive cells(0-100%) score were 1.23±2.98,7.06±15.64,53.33±42.36,57.28±48.73,63.11±47.11 among five lesions in the percentage of positive cells,there was significant statistical difference(P＜0.01)and Hscore score(P＜0.01). Compare positive cell percentage values between 2 groups:there was no significant difference normal mucosa and inflammatory polyp(P＞0.05);colorectal polyps and colorectal low-grade inflammatory intraepithelial neoplasia there was a significant difference(P＜0.01);colonic low grade intraepithelial high-grade colorectal intraepithelial neoplasia was no significant difference(P＞0.05);colorectal high-grade intraepithelial neoplasia with invasive colorectal cancer,no significant difference(P＞0.05).When comparing the Hscore score,we found the similar results:in normal mucosa and inflammatory polyp there were no significant difference(P＞0.05);and in colorectal polyps and colorectal low-grade inflammatory intraepithelial neoplasia there was a significant difference(P＜0.01);in colon low grade intraepithelial neoplasia and high-grade colorectal intraepithelial neoplasia no significant difference (P＞0.05)were found;also in colorectal high-grade intraepithelial neoplasia and invasive colon cancer there were no significant differences(P＞0.05).2.There was a significant difference among different histological grades in colorectal tissues by Hscore(P＜0.01) and positive cell percentage value(P＜0.01).Also between in colorectal adenocarcinoma and mucinous adenocarcinoma, Hscore score means were 121.43±101.70 and 35.40±66.99 respectively, significantly different between the two groups(P＜0.05);positive cell percentage values were 66.40±46.20 and 25.40±42.77 respectively,there are significant differences between the two groups(P＜0.05).Expression of p21Ras in the groups of Lymph node metastasis,Hscore P21ras score mean 85.52±95.06,Expression of p21Ras in the groups of non-lymph-node metastasis,Hscore score mean 131.21±104.57,significantly different between groups(P＜0.05);the percentage of positive cells values were 68.97±45.88 and 51.19±48.12 respectively,significantly different between the two groups(P＜0.05).But the expression was not correlated with patient's sex,ages,tumor sizes and invasive depth for Hscore score(P＞0.05).By evaluating percentage of positive cells,we found the same results.3.When analyzing the expression of p21Ras in colorectal cancer and adjacent tissues(including normal epithelium,low grade intraepithelial neoplasia,high grade intraepithelial neoplasia),we found the expression of p21Ras increased with malignant grades whether by Hscore or by percentage of positive cells method.Hscore score means were 10.27±24.28,59.45±69.18,71.89±98.39 and 116.14±103.30 respectively,there were statistically significant differences in four groups(P＜0.01), results of the comparision between groups:there were a significant difference between normal mucosa and colorectal low-grade intraepithelial neoplasia(P＜0.01), there was no significant difference between colon low grade intraepithelial neoplasia and high-grade intraepithelial neoplasia(P＞0.05),between colorectal high-grade intraepithelial neoplasia and invasive colon cancer there were significantly different (P＜0.05).Percentage of positive cells values were 8.11±17.58,41.87±43.48,39.72±48.84 and64.18±46.80 respectivelyy,there were statistically significantly different four groups(P＜0.01).Comparing positive cell percentage values between 2 groups: there was a significant difference normal mucosa and colorectal low-level intraepithelial neoplasia(P＜0.01),colonic low grade intraepithelial neoplasia and high-grade intraepithelial neoplasia was no significant difference(P＞0.05),colorectal high-grade intraepithelial neoplasia and invasive colon cancer were significantly different(P＜0.05).4.A high incidence of male to female morbidity in mucinous carcinoma, comparing with ordinary adenocarcinoma,there was a significant difference(P＜0.05); The average age of onset of mucinous adenocarcinoma is lower than normal,but there was not statistically significant(P＞0.05);mucinous adenocarcinoma of the incidence of the main parts of the left colon,but there was no significant difference compared to ordinary adenocarcinoma(P＞0.05);mucinous adenocarcinoma of lymph node metastasis was significantly higher than ordinary adenocarcinoma,there was a significant statistical difference(P＜0.01).5.Expression of p21Ras in colorectal adenomas:Hscore score mean of men group was 60.25±75.11,of women group was 59.33±77.69,there was not significant between the two groups(P＞0.05);the group of≤50 years was 56.89±76.88,the group of＞50 years was 60.11±74.01,there was not significant(P＞0.05); the group of left colon was 60.36±81.84,the group of the right half of the large intestine was 60.83±79.77,there was no significant(P＞0.05);the group of tube was 63.22±82.18,tubular-villous was 74.48±83.37,villous was 73.25±81.72, between three groups there was nosignificant(P＞0.05);the group of tumor diameter ＜1cm was 59.33±75.49,1-2cm was 63.24±82.52,＞2cm was 60.87±89.34, between three groups three was no significant(P＞0.05);the group of low-grade intraepithelial neoptasia was 59.45±69.18,high-grade intraepithelial neoplasia was 71.89±98.39,between three groups there was no significant(P＞0.05);Positive cell percentage values in male group were 40.99±46.58,in women group were 41.56±46.87,there was not significant(P＞0.05);the group of≤50 years were 38.74±45.21,the group of＞50 years were 40.45±49.69,there was no statistically significant between groups(P＞0.05);left-side colon group was 40.58±49.34, right-side colon was 40.62±46.88,between the two groups there was not significant (P＞0.05);the group of tube was 40.63±42.11,tube-villous to 39.85±46.96, villous to 40.55±49.97,there was no significant difference between groups(P＞0.05); tumor diameter＜1cm group were 42.54±50.45,1-2cm group were 39.98±41.23,＞2cm group were 41.22±43.51,between groups there was no statistically significant (P＞0.05);low grade intraepithelial neoplasia group were 41.87±43.48,high-grade intraepithelial neoplasia group were 39.72±48.84,between the two groups there was no significant(P＞0.05).6.The expression of p21Ras in the same disease of independent group and the adjacent group was no statistically significant(p＞0.05).[Conclusion]1.We observed the expression of p21Ras in the early stages of colorectal cancer(adenomas),suggesting that the relationship of Ras gene and the occurrence and development of colorectal cancer are closely.2.There is no expression of p21Ras protein in normal colon tissue and benign lesions,but in the precancerous lesions and cancer tissues the expression were increased,so we suggest that KGH-R1 monoclonal antibody can be explored in tumor gene therapy.
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