To meet the need of cultivating high-quality personnnel,enhance the medicalstudents' ability to analyze and solve problems,directing at the problems existed in pharmacology experiment,we have adjusted and reformed the content of the experiment teaching in pharmacology and conducted the survey and research among the students so as to lay a solid fundation to its reasonable implement.

Objective To evaluate the growth inhibition of human gastric carcinoma cell lines SGC 7901 in vitro and the expression of bcl-2, bcl 2l12 and bax with docosahexaenoic acid (DHA) and 5-fluorouracil (5-FU). Methods The effect of DHA and 5-FU was measured by trypan blue, and the interaction between two agents was judged by combination index (CI). Cells were observed by inverted microscope. Flow cytometry was used for analysis of apoptosis by PI staining and Annexin-V/PI. RT-PCR was used to analyze the levels of bcl-2, bcl 2l12 and bax mRNA. Results DHA significantly inhibited the growth of SGC 7901 cells in a dose- and time-dependent way ( P < 0. 05 ), the IC50 of 24 h and 48 h was 67. 81 μg/ml and 45.76 μg/ml, and a strong synergism was found in the combination of DHA and 5-FU (CI < 1 ,P <0. 01 ). Treated by DHA and 5-FU for 48 h, cells became sparse under inverted microscope. DHA or 5-FU was able to induce apoptosis and the effect became even more significant by the combination of DHA and 5-FU. Cells were holted in phase of G01/G1 and S. RT-PCR showed that DHA or 5-FU down-regulated the expression of bcl-2 and bcl 2l12 mRNA, while bax mRNA expression was not downregnlated. Conclusions DHA could inhibit the growth of gastric carcinoma cells, DHA and 5-FU had synergetic effect in the inhibition of the cells growth and blockage of the cell cycles possibly by down-regulating the expression of bcl-2 and bcl 2l12.

This study aimed to construct prokaryotic recombinant plasmids for expression of the extracellular domains of NMDAR1 protein, purify and characterize the immunoreactivity of the recombinant proteins. Based on the mRNA sequence of human NMDAR1 gene, we predicted the structure of the antigenic domains in the extracellular part of the protein using the "phyre2" software. Primers were designed to amplify the nucleic acid fragments encoding the NMDAR1 extracellular antigenic domains by RT-PCR. The amplified gene fragments were cloned into pCold-SUMO vector to construct the recombinant plasmids which were transformed into Escherichia coli DH5α. The positive colonies harboring the recombinant plasmids were picked and verified by PCR and DNA sequencing. Then, the recombinant plasmids were transformed into E. coli BL21(DE3) strain and induced by IPTG for protein expression. The recombinant proteins were purified by Ni-NTA affinity chromatography. The target proteins were further purified by removing the 6 His-SUMO tag using enzyme excision followed by gel filtration chromatography using AKTA purifier. The purity of the recombinant proteins were evaluated by SDS-PAGE and the immunoreactivity were characterized by Western blotting. Three DNA fragments encoding the extracellular domains of NMDAR1 protein, including NR1-M1 (encoding 19-393 aa), NR1-S1 (encoding 394-544 aa) and NR1-S2 (encoding 663-800 aa), were amplified by RT-PCR. The NR1-S1 and NR1-S2 were linked with G (arginine) and T (threonine) amino acid as a combined fragment. The NR1-M1 and NR1-S1-GT-S2 fragments were cloned into pCold-SUMO vector and two recombinant plasmids, pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2, were generated and expressed in E. coli. SDS-PAGE analysis showed that the recombinant plasmids expressed soluble NR1-M1 and NR1-S1-GT-S2 proteins in bacterial. After affinity chromatography and gel filtration chromatography, we obtained high purity target proteins. Western blotting assay showed that the recombinant proteins NR1-M1 and NR1-S1-GT-S2 can bind specially with their corresponding antibodies, suggesting the recombinant proteins retained antigenic reactivity. We constructed a prokaryotic expression system for expressing the NMDAR1 protein extracellular parts that had immunoreactivity successfully, and the purified proteins can be used for studying NMDAR1 function and testing the autoantibodies.