1.Effect of recombinant adenovirus vector expressing human endostatin on endothelial cell proliferation
Hui TANG ; Yongqing XU ; Chunxiao LI ; Xiuqiong ZHANG ; Tiane ZHENG ; Xusheng LIU ; Wanyi LIANG
Chinese Journal of Tissue Engineering Research 2008;12(50):9986-9989
BACKGROUND: Scar hypertrophy is always followed by the wound healing in burn and trauma. Endothelial cells play a key role in scar hypertrophy, so inhibitory growth of endothelial cells can relieve scar hypertrophy to a certain degree. OBJECTIVE: To construct a recombinant adenovirus vector expressing human endostatin (Ad/hEnd), and to investigate the cooperative effect of Ad/hEnd and keratinocyte on endothelial cell proliferation. DESIGN, TIME AND SETTING: Observational study, which was performed in the State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital of the Third Military Medical University of Chinese PLA between September 2006 and May 2007. MATERIALS: pAdTrack-CMV and pAdEasy-1 were obtained from Stratagene Company, USA; 293 cell and Ecoli.DH5α were stored in our laboratory. METHODS: The endostatin gene sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and polymerase chain reaction (PCR) based on mRNA of human fetal hepatic tissue and inserted into the adenovims shuttle plasmid pAdTrack-CMV to obtain recombinant plasmid pAdTrack-ES. After identification, positive recon was transformed into pAdeasy 1 recipient virus to screen positive clones. The adenovirus Ad/hEnd was generated from 293 cells and identified by PCR and fluorescence microscope. Then the keratinocytes were infected with Ad/hEnd, and co-cultured with endothelial cells by nest dish culture method. The content of endostatin was detected, and the non-transfection keratinocytes were used as the controls. MAIN OUTCOME MEASURES: Homologous recombination and identification of pAd/hEnd; generation and identification of Ad/hEnd; endostatin expression after 293 cell transfection; purification and titer measurement of Ad/hEnd; content of endostatin in culture solution; apoptotic percentage of endothelial cells; inhibitory ratio of endothelial cells. RESULTS: Ad/hEnd was constructed and the virus titer was generally up to 1.65×1012 PFU/L. Ad/hEnd-infected keratinocytes could effectively express and secrete endostatin of which the content reached 226 μg/L after 3 days of co-culture. The apoptotic percentage and inhibitory ratio of the endothelial cells co-cultured with Ad/hEnd-infected keratinocytes were significantly higher than those in control group (P<0.05). CONCLUSION: Ad/hEnd-infected keratinocytes co-cultured with endothelial cells can promote apoptosis and inhibit proliferation of endothelial cells through excretion of endostatin.
2.Effect of propofol on HMGB1/TLR4 signaling pathway during hepatic ischemia-reperfusion injury in rats
Shuzhen YU ; Weiwei ZHANG ; Junming REN ; Jianfeng WEI ; Yu ZHANG ; Lina ZHENG ; Lijun HAO ; Yuehong QI ; Tiane LUO ; Yongqing GUO
Chinese Journal of Anesthesiology 2019;39(7):870-872
Objective To evaluate the effect of propofol on high-mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4) signaling pathway during hepatic ischemia-reperfusion (I/R) injury in rats.Methods Thirty-six clean-grade healthy male Sprague-Dawley rats,aged 3 months,weighing 250 -300 g,were divided into 3 groups (n=12 each) using a random number table method:sham operation group (group S),hepatic I/R group (group I/R) and propofol group (group P).Hepatic I/R injury was induced by occluding the portal vein and hepatic artery supplying the left and middle lobes of the liver for 1 h followed by 6-h reperfusion in anesthetized rats.Propofol was infused via the tail vein at a rate of 12 mg ·kg-1 · h-1 starting from 20 min before ischemia until 6 h of reperfusion in group P.The rats were sacrificed at 6 h of reperfusion,and the left lobe of the liver was removed for microscopic examination of the pathological changes which were scored and for determination of the expression of HMGB1,TLR4,tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-6) in liver tissues (by Western blot).Results Compared with group S,pathological scores of liver tissues were significantly increased,and the expression of HMGB1,TLR4,TNF-α and IL-6 was up-regulated in I/R and P groups (P<0.05).Compared with group I/R,pathological scores of liver tissues were significantly decreased,and the expression of HMGB1,TLR4,TNF-α and IL-6 was down-regulated in group P (P< 0.05).Conclusion The mechanism by which propofol reduces liver I/R injury is associated with blocking HMGB-1/TLR4 signaling pathway and inhibiting inflammatory responses in rats.