21 ± 10.08) and the control group (11.85 ± 13.53) (P < 0.05), but there were no difference in the long-pulse GES group and the control group (P > 0.05); In the tracer-positive neuron of short-pulse GES group, the number of TRPA1-positive neuron was 5.30 ± 4.49, significantly lower
than long -pulse GES group (7.84 ± 7.51) and the control group (9.69 ± 13.10) (P < 0.05), but there were no difference in the long-pulse GES group and the control group (P > 0.05). Conclusion: The study we have performed showed that the change of NK1R-positive neuron and TRPA1-positive neuron in jugular and nodose induced by short pulse GES, which suggested that NK1R and TRPA1 might involving in regulating the gastric sensory function in the case of the stimulation of short pulse gastric electrication. Key Word(s): 1. Short-pulse GES; MK-8669 Presenting Author: YUE YUAN Additional Authors: GUIYONG PENG, XIUFENG KANG, JIANHUA DAI, DONG MU, SHIXIANG XUE Corresponding Author: GUIYONG PENG
Affiliations: Third Military Medical University Objective: Construct a lentiviral vector containing the human tumor necrosis factor receptor associated death domain protein (TRADD) gene. Explore the combined effect of TRADD lentiviral vector and TNF-α on proliferation and collagen I synthesis of hypertrophic scar fibroblast (HSFb) and fetal fibroblast (FFb). learn more Methods: The TRADD specific fragment was amplified by polymerase chain reaction (PCR) and cloned into the EcoR I site of the lentiviral vector pLVX-EGFP-3FLAG-Puro. The recombinant plasmid was transformed into DH5α competent cells and identified by colony PCR, then the positive clones were detected by DNA sequencing analysis. TRADD lentiviral vector was produced after the 293FT packing cells were contransfected with pLVX-TRADD-EGFP-3FLAG-Puro and lentiviral packaging plasmids, while titer of virus was detected by Real-time PCR and expression of TRADD-GFP-FLag fusion protein was analyzed by
Western-blot. After transfected with the TRADD lentiviral vector and treated with 10 ng/ml TNF-α, the proliferation and collagen I synthesis of HSFb and FFb were measured by methyl thiazolyl tetrazolium (MTT) and enzyme linked immunosorbent assay (ELISA), find more respectively. Results: Positive clones of 1200 bp straps were obtained, and TRADD gene sequence of the cloned was consistent with that in Genbank. The green fluorescence and fusion protein were observed in 293FT cells after transfected with TRADD lentiviral vector. Real-time PCR showed the titer of the virus was 3.22 × 108 IU/ml. TRADD lentiviral vector could selectively prohibit proliferation of HSFb through up-regulating TRADD expression, while 10 ng/ml TNF-α showed no significant effects on growth of HSFb and FFb. The combined effect of TRADD lentiviral vector and 10 ng/ml TNF-α on inhibiting collagen I synthesis of HSFb was stronger than that of FFb. Conclusion: In this study, the TRADD lentiviral vector is constructed successfully.