ABSTRACT
Objective: To clone the 5＇-flanking region of the human CD34 gene containing transcriptional regulatory sequence (TRS). Methods: According to the registered 5＇-flanking region of CD34 gene, two pairs of primers were designed and net-PCR was used to amplify 661 bp long TRS of CD34 gene. The CD34 TRS fragment was cloned into reported plasmid pEGFP-1. The role of the regulating the specific expression of recombinant plasmid pCD34 EGFP in hematopoietic and non-hematopoietic cells was observed. Results: Restrictive endonuclease identification and DNA sequencing provedthat the CD34 promoter cloned was consistent with the sequence reported to a large extent. It could induce the EGFP gene to express in hematopoietic cell line K562 specifically, while has no effect on hepatocellular carcinoma cell HepG-2. Conclusion: The cloned CD34 gene TRS has the effect of regulating gene expression specifically. The study established the fundament for the construction of specific gene expression vector used in hematopoietic system cells.
ABSTRACT
Objective:To investigate the role of blockade of CD40 ligand-CD40 costimulatory pathway by anti-CD40L mAb on T lymphocytes typing and cytokines, and provide the experimental clues of inducing donor-specific T cell anergy.Methods:To monitor primary MLR, C57BL/6 H-2b spleen T cells were isolated as responder cells, and BALB/C H-2d spleen cell as stimulator cells. Anti-CD40L mAb was added to primary MLR cultures(termed anti-CD40L mAb group), and in control group(no anti-CD40L mAb). Incubated for 7 days, the cells responsiveness rates were detected by 3 H-TdR methods at the indicated time points, and supernatants were assayed for IFN-?,IL-2,IL-4,IL-10 cytokines levels by commercial ELISA kits. Day 5 MLR-cultured cells were assessed for the expressions of CD4, CD8, CD25, CD69, CD40L and CD45RA with flow cytometry(FCM).Cells proliferation for 5 days and cytokines in secondary MLR were assayed according above-mentioned method.Results:Blockade of the CD40-CD40L pathway by anti-CD40L mAb can induce the hyporesponsiveness to alloantigen in primary and secondary MLR culture. In primary MLR culture, the expressions of CD4 +T and CD8 +T cells and CD4 +CD25 +T, CD4 +CD69 +T, CD4 +CD40L +T, CD8 +CD25 +T and CD8 +CD69 +T cells in anti-CD40L mAb group were lower than that in control group ( P 0.05).The expression of CD4 +CD45RA + T in anti-CD40L mAb-cultured group was higher than that in control group( P