Résumé
Objective To construct and confirm the JC virus(JCV) T antigen expression plasmid using mouse keratin 19 (K19) promoter specific for the gastric epithelial cells.Methods The Ndel site was mutated by FCR with Bell insertion at both sides.The DNA fragment digested by Bcl Ⅰ was ligated with the plasmid containing K19 promoter via Bam Ⅰ site.The DNA sequence was confirmed by restriction enzyme digestion and direct DNA sequencing.Cytokeratin 19 protein was examined to screen gastric carcinoma cell for transfection of K19-JCV T antigen expression plasmid by immunohistochemistry.The Western blot was employed to detect the JCV T antigen expression in the gastric carcinoma transfectant.Results K19-JCV T antigen expressing plasmid was successfully constructed.The ACS strongly expressed cytokeratin 19 protein and was selected for the transfection of K19-JCV T antigen expressing plasmid.JCV T antigen was positively expressed in the AGS transfectant.Conclusion The synonymous mutation and compatible ligation are useful in the plasmid construction.The methy lation of restriction enzyme should be considered.It is meaning for the transgenic animal model of gastric carcinoma to successfully construct the JC virus T antigen expression plasmid in gastric mucosa.
Résumé
This study was purposed to investigate the effect of ligustrazine on the expression of bFGF in bone marrow stromal cells (BMSC) and to explore the mechanism of hematopoietic reconstitution after bone marrow transplantation (BMT). The mice were randomly divided into 3 groups: normal group, saline group and ligustrazine group. BMT mouse models were established. The mice of normal group were not treated, the mice of saline group were given normal saline (0.2 ml/mouse, twice a day) through gastric tube, while the mice of ligustrazine group were given ligustrazine (0.2 ml/mouse, twice a day) through gastric tube. On day 7, 14, 21 and 28 after BMT, the femora were taken and the bone marrow mononuclear cell (BMMNC) suspensions were used for the cultivation of bone marrow stromal cells according to Dexter's culture method. The mRNA and protein expressions of bFGF in BMSC were assayed by RT-PCR and Western blot respectively. The results showed that the expression of bFGF in BMSC on the level of mRNA and protein were all reduced significantly after BMT, and increased slowly with the time. On day 7, 14 and 21 after BMT, the expressions of bFGF mRNA and protein in bone marrow stromal cells of ligustrazine group and saline group were lower than that in bone marrow stromal cells of normal group, but the expressions of bFGF mRNA and protein in ligustrazine group were obviously higher than that in saline group (P < 0.01 or P < 0.05). On day 28 after BMT, the expressions of bFGF mRNA and protein in ligustrazine group returned to normal level, while the expressions of bFGF mRNA and protein in saline group not returned to normal level, there was significant difference between these two groups. It is concluded that ligustrazine can enhance bFGF expression level in bone marrow stromal cells after syngeneic bone marrow transplantation in mice, which confirms that ligustrazine can enhance the repair of bone marrow microvessels, improve bone marrow microenvironment and promote hematopoietic reconstitution.