Relationship between Pst-Ⅰ polymorphism of mucin 4 gene and primary hepatolithiasis / 中华肝胆外科杂志

Objective To investigate the relationship between Pst-Ⅰ polymorphism of mucin 4 gene and primary hepatolithiasis in the Chinese population.Methods Venous blood of 96 healthy controls and 56 patients with hepatolithiasis were collected,and DNA was extracted.Polymerase chain reaction combined with restriction enzyme (PCR-RFLP) digestion was used to detect Pst-Ⅰ polymorphism of mucin 4 gene in the two groups.The genotype and gene frequency between the two groups were then compared.Results The genotype frequencies of GC,GT,TT in the control and the hepatolithiasis groups were 21.3％,12.7％,55.6％ and 53.2％,41.2％,19.8％,respectively.The alleles C and T gene frequencies in the control and the hepatolithiasis groups were 21.5％,72.7％ and 66.3％,30.2％,respectively.There were significant differences in Pst-Ⅰ polymorphism of mucin 4 genotype frequency and gene frequency between the two groups.Conclusion The data showed Pst-Ⅰ polymorphism of mucin 4 gene was associated with primary hepatolithiasis in Chinese patients.

In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4 / 中国病理生理杂志

AIM:Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor.During our previous research,4 HLA-A2-restricted peptides had been identified as immunogenic in vivo.In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides.METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant(IFA)subcutaneously in combination with the epitope at the adjacent location.After the 3rd peptide vaccination for 10 d,the peptide-specific immune response was evaluated by ELISPOT and ELISA.The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of pep-tide-specific CD8+T cells capable of recognizing the MHC-peptide was detected by flow cytometry.RESULTS: Among the 4 candidate HLA-A2 restricted peptides,the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides.The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells.P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs.Intracellu-lar cytokine staining assay indicated the presence of P 2004-1Y9V specific CD8 +T cells in the P2004-1Y9V vaccinated mice.CONCLUSION:P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.

Construction ,Expression and Characteristic Analysis of Recombinant Human TIM-4-EGFP Fusion Protein / 华中科技大学学报(医学版)

Objective The present study aimed to explore the expression and purification of a fusion protein of human TIM-4 and EGFP in Escherichiacoli(E.coli)and evaluate its bioactivity.Methods The cDNA fragments of human TIM-4 and EG-FP genes were respectively amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a. The constructed re-combinant plasmid pET-28a-TIM-4-EGFP was transformed to E. coli BL21 (DE3)for the expression under the induction of IPTG.The expressed protein was purified by Ni-NTA resin.Recombinant protein was analyzed by SDS-PAGE and Western blotting ,and its binding activity to the apoptotic cells was detected under the fluorescence microscope.Results The TIM-4-EG-FP vector was constructed and expressed in E. coli. The TIM-4-EGFP fusion protein was identified and verified by SDS-PAGE and Western blotting.Our results demonstrated that all the TIM-4-EGFP fusion proteins recognize and bind directly to apoptot-ic cells ,but not to viable cells.We further verified that the interactions of TIM-4-EGFP with apoptotic cells were blocked by TIM-4-Ig fusion proteins.Conclusion We successfully constructed a fusion protein encoding human TIM-4 and EGFP ,and ex-pressed it in E.coli. The fusion protein shows a readily obtainable source of biologically active TIM-4 ,which has considerable potential for further studies on human TIM-4 and its receptor.

Function of mucin 2, mucin 4 on lithogenic bile of calculus of intrahepatic duct / 中华肝胆外科杂志

Objective To investigate the function of mucin 2,mucin 4 on formation of lithogenic bile in patients with calculus of intrahepatic duct.Methods Bile duct mucosa,bile duct wall,bile and plasma were collected from 56 patients with calculus of intrahepatic duct (CID group) and 17 individuals without calculus of intrahepatic duct (control group).The bile duct wall was stained with mucin 2 (MUC2) and mucin 4 (MUC4).Reverse-transcription polymerase chain reaction (RT-PCR) was applied to study the mRNA expressions of MUC2 and MUC4 in the bile duct mucosa.The correlation of the bile duct and serum lipid index was analyzed.Results Serum lipid index in the CID group was significantly higher than control group (all P＜0.05).Biliary total bile acids and bile acids mol percentage were lower in the CID group than control group (both P＜0.05).The expressions of MUC2 was not increased significantly in CID group than the control group (all P＞0.05).The expressions of MUC4 were more significantly increased in CID group than the control group (P＜0.05).The mRNA of MUC4 in the CID group was also more significantly increased than in control group (P＜0.01).There were no correlations between MUC4 expression and the level of biliary total bile acid in the CID group (r=-0.374,P＜0.05).Conclusion The expression of MUC4 in patients with CID was enhanced,which promoted the absorption of bile acid by the mucosal epithelium of the bile duct,and caused a large amount of mucin to be secreted into bile,which may be related to the formation of stony bile.

Identification and molecular modification of HLA-A2-restricted cytotoxic T-lymphocyte epitopes from pancreatic tumor antigen MUC4 / 中国病理生理杂志

AIM:To observe whether modified epitopes from pancreatic tumor antigen mucin 4 (MUC4) have HLA-A2-restricted antitumor ability.METHODS:RT-PCR and Western blot were used to identify the expression of MUC4 in the pancreatic tumor cell lines CAPAN-2 and ASPC-1.HLA-A2 epitopes from MUC4 protein were predicted by the software of NetCTL 1.2, BIMAS, SYFPEITHI and IEDB.The modified peptides from MUC4 containing HLA-A2 were obtained by replacing anchor residues of the binding anchor motifs.The peptides were synthesized by standard solid-phase methods.The binding affinity of the peptides to HLA-A2 molecule was evaluated by T2 binding assay.ELISPOT assay was used to investigate the ability of the peptide to induce specific restricted cytotoxic T-lymphocytes (CTLs) and release of IFN-γ.The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro.RESULTS:The expression of MUC4 was observed in the CAPAN-2 cells and ASPC-1 cells.The candidate peptides P1944-1Y, P1944-2L, P1944-1Y2L, P2004 and P2004-1Y9V showed moderate affinity toward HLA-A2 molecule.T2 binding assay showed that P1944-1Y2L and P2004-1Y9V had significantly higher affinity for HLA-A2 than the native peptides.ELISPOT assay showed P1944, P1944-1Y2L, P2004 and P2004-1Y9V were able to induce specific CTLs and more amounts of IFN-γ were released.ELISPOT assay showed that significantly more amounts of IFN-γ released by P1944-1Y2L and P2004-1Y9V were observed than the native peptides.The CTLs induced by P1944, P1944-1Y2L, P2004 and P2004-1Y9V lyzed the CAPAN-2 cells.P1944-1Y2L and P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell line CAPAN-2 than the native peptide-specific CTLs.CONCLUSION:Compared with the native peptides, modified epitopes P1944-1Y2L and P2004-1Y9V have higher binding affinity with HLA-A2 and retain immunogenecity.In addition, the anti-tumor immunity of modified epitopes P1944-1Y2L and P2004-1Y9V is stronger than that of the native peptides.The peptides P1944-1Y2L and P2004-1Y9V are excellent HLA-A2-restricted CTL epitopes from tumor antigen MUC4, which could serve as new candidates towards antitumor peptide vaccines.