Related Study Of LRP16 In Endometrial Endometrioid Adenocarcinoma & Preparation And Application Of AntiLRP16 Monoclonal Antibody

LRP16 is a novel gene cloned from lymphocytic cells of peripheral blood. The gene is located on chromosome 11q11.2,estrogen positive,and highly expressed in lots of estrogen-dependent tumors.Over-expression of LRP16 promotes not only proliferation of MCF-7 cell,but also invasive growth.Cell proliferation might be induced by increased cyclinE and cyclin D1 protein,modified by LRP16,which are two important proteins for G1/S transformation;Inhibition of expression of LRP16 makes invasion capacity of MCF-7 cell decrease,and its mechanism is related to down-regulation expression of E-Cadherin.In study of molecular mechanism,we found that LRP16 is a novel ERαco-activator,which increased the ERα-mediated transcriptional activity by the interaction with each other.To sum up, it may be an important role in the occurrence and development of breast cancer. So anti-LRP16 treatment will benefit patients of ERα(+)breast cancer.Both endometrial endometrioid adenocarcinoma(EEC)and breast cancer belong to estrogen-dependent tumor,and over-expression of LRP16 increases the invasion capacity of Ishikawa cell,whose effect is induced by down-regulation expression of E-Cadherin.Therefore,SectionⅠexplored that LRP16 is related to the occurrence,development and prognosis of carcinoma through analyses of EEC specimensWith deep insight of LRP16 research,it is increasingly significant in the occurrence and development of estrogen-dependent tumor.Accordingly,it is very necessary to produce and identify anti-LRP16 monoclonal antibody of high activity and sensitivity.SectionⅡcompromises preparation and application of anti-LRP16 monoclonal antibody.LRP16 McAb benefits the study of tissue distribution and functions of LRP16 protein,and will provide chance in tumor vaccine and clinical research.SectionⅠRelated study of LRP16 in Endometrial Endometrioid Adenocarcinoma.Objective:To explore the distribution and expression of LRP16 in endometrial endometrioid adenocarcinoma(EEC)and evaluate the correlation between the exp ression of LRP16 and clinical pathology character.Method:The expressions of LRP16,estradiol receptor alpha(ERα),Progesterone receptor(PR)and E-Cadherin expression were examined by immunohistochemistry in 76 cases of EEC. Results:In the nucleus,the cytoplasm,both nucleus and cytoplasm,positive rates of LRP16 were 67.11%(51/76),96.05%(73/76),64.47%(49/76), respectively.Among 76 cases,the positive rate of ERαwas 51.32%(39 /76).negative rate was 48.68(37/76).In ERαnegative cases LRP16 expression in the nucleus(32/37,86.49%)was obviously higher than that in ERαpositive cases(19/39,48.71%)(P＜0.01).and LRP16 expression in the nucleus was related with grade and E-Cadherin expression(P＜0.05)....There was no significantly difference between ERαexpression and LRP16 expression in the cytoplasm(P＞0.05);LRP16 expression in the cytoplasm was related with staging(P＜0.05)and PR expression(P＜0.05),but It was not correlated with grade(P＞0.05).Among cases of LRP16 expression in both nucleus and cytoplasm,ERαnegative rate(81.08%,30/37)was obviously higher than that of ERαpositive(48.72%,19/39)(P＜0.01),there were no relationship between LRP16 expression in the nucleus and staging(P＞0.05),but LRP16 expression in the nucleus was related with grade(P＜0.05).In EEC,LRP16 expression in the nucleus was negatively correlated with in the cytoplasm(P=0.0239＜0. 05).Conclusion:The LRP16 expression almost exists in all cells of EEC,and there are an intimate relationship between its sub-celluar Location and ERα expression.LRP16 expression in the cytoplasm may be correlated with good prognosis;the effect that LRP16 is expressed in nucleus is associated with carcinomatic invasiveness.So changes of sub-celluar Location of LRP16 in cytoplasm and nucleus indicates tumoric progression,which predicts that LRP16 gene may be a remarkable monitoring factor in EEC.SectionⅡPreparation and application of antiLRP16 monoclonal antibodyObjective:To produce and identify antiLRP16 monoclonal antibody.Methods: Lymphocytes from the spleen of mice being immunonized LRP16 antigen were fused with the myeloma cell line(SP2/0)using PEG4000.Hybridoma cells were established by selective growth of the fusion ceils in the HAT and HT medium, and the presence of anti LRP16 antibody was screened by indirect ELISA,and the clonality was achieved by limiting dilution.We have incubated cloning cells into mouse abdominal cavity to produce ascitic fluid contained monoclonal antibody. Chromatographies with SPA-Sepharose-6BFF affinity column were emploied to isolate the monoclonal antibody from ascitic fluid.Finally,the antibody were tested the activities and sentivities,isoforms and titer through Western blot immunochistochemisty and ELISA.Results:Only one hybridoma cell line, secreting McAb against LRP16,had been established.The modal number of chromosome is 95.The results of identifications showed that the antibodies kept high activitis and sensitivitis in detecting sample.The titer of ascitic fluid and the McAb purified are 1:3.2×10~7equally.The immunoglobulin of the McAb is classified as IgG1.Conclusion:Anti-LRP16 monoclonal antibody has been produced successfully with high sensitive and active and was named 14-2-1C2H10.