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Objective To observe the repair effect of human ?-defensin 2 (hBD 2)-modified rat dermal multipotent stem cells (dMSCs) transplantation on infected wound. Methods Thirty Wistar rats were excised a piece of whole-layer back skin, 3 mm in diameter, then infected the wound with 1?10 8/ml pseudomonas aeruginosa 1 ml, then the rats were injected on the wounded back respectively with dMSCs modified by hBD 2 (n=10), or pure dMSCs (n=10) or none as control (n=10). The repair effect was evaluated by observing the amount of bacteria under the scar, wound healing time and the percentage of remaining wound area. Results The amount of bacteria under the scar in rats that were transplanted with dMSCs modified by hBD 2 was less than that in rats transplanted with dMSCs or controls (P
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Objective To explore the roles of NF-?B in expression of human ?-defensin-2(hBD-2) mRNA induced by TNF-? in the human airway primary epithelial cells.Methods After the human bronchial primary epithelial cells were stimulated with TNF? or first with NF-?B inhibitor PDTC,then TNF-?,the expression of hBD-2 mRNA was detected by RT-PCR.The I?B-? protein level in the cytoplasm was detected by Western blotting and the nuclear factor-kappa B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assays.Results The hBD-2 mRNA could be detected after 2.5 h of TNF-? stimulation and expressed in a dose-dependent manner.The NF-?B could be activated after 0.5 h of TNF-? stimulation.The supershifts assays indicated that the p65-p50 heterodimer formed complexes of NF-?B were involved in the activated of NF-?B.Conclusion TNF-? can induce the expression of hBD-2 mRNA in a dose-dependent manner.The p65-p50 heterodimer formed complexes of NF-?B play an important role in the regulation of hBD-2 gene expression in response to TNF-?.
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Objective To examine the expression of human ?-defensin 2 (hBD_ 2 ) recombinant adenovirus expression vector in rat dermal multipotent stem cells (dMSCs) and to observe the antiseptic activity of recombinant hBD_ 2 . Methods The expression of hBD_ 2 in dMSCs was examined by RT-PR, fluorescent immunochemistry and Western blotting, and the concentration of recombinant hBD_ 2 in supernate was measured by ELISA. The antiseptic activity of recombinant hBD_ 2 was assessed by K-B disc agar diffusion test. Results hBD_ 2 could be effectively expressed in dMSCs, and the concentration of recombinant hBD_ 2 in supernate was about 743.6 ng/ml . Recombinant hBD_ 2 in supernate showed antiseptic activity. Conclusion Recombinant adenovirus expression vector of hBD_ 2 could be effectively expressed in dMSCs, and the recombinant hBD_ 2 in supernate showed obvious antiseptic effects toward some standard bacteria lines.
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Objective To construct high expression system for obtaining human ?-defensin 2 (h?D2) in human lung cancer cell line A549. Methods A recombinant retrovirus expression vector pLNCX2-CEA (signal peptide)-h?D2 (mature peptide) was constructed, and then the retrovirus vector was transfected into PT67 cells by DOTAP. After screening and amplification of single clones by G418, the virus was used to infect A549 cells. A549 cells were also screened by G418, and the cell clones resistant to G418 were obtained. Expression of h?D2 was detected by Western blot analysis. Results A recombinant retrovirus expression vector pLNCX2-CEA-h?D2 was constructed successfully. h?D2 with high level of expression was obtained in A549 cells transfected with the expression vector. Conclusion Human lung cancer cell line A549 can be used for high expression of h?D2 with gene engineering technology.