Regulation of proliferation and apoptosis of human vascular endothelial cell by Acheron / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell.</p><p><b>METHODS</b>(1) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4.1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)], blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 µg protein), CASK antibody precipitation group (100 µg protein), IgG antibody group (100 µg protein), Western blot group (20 µg protein). Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn), KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/L PBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group (transfected with pcDNA-Achn vector), and blank control group (treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test.</p><p><b>RESULTS</b>(1) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10.777, 6.112, P values all below 0.05). The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3.1 group (with t value respectively 5.367, 6.053, 9.831, P values all below 0.05). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group (with t value respectively 5.481, 9.517, P values all below 0.05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [(15.6 ± 0.5)%] as compared with that in LPS induction group [(32.8 ± 2.6)%, t = 10.083, P < 0.05], and that in cotransfection group showed further inhibition [(7.0 ± 2.0)%, t = 9.827, P < 0.01]. (4) Apoptosis rate in Achn inhibition group [(45.6 ± 10.9)%] was higher than that in blank control group [(13.2 ± 4.3) %, t = 7.043, P < 0.05]; while that in Achn induction group [(5.3 ± 2.9)%] was lower than that in blank control group (t = 6.499, P < 0.05).</p><p><b>CONCLUSIONS</b>Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.</p>

Inhibition of integrin beta1 expression in human keratinocyte stem cells by vector-1 based interfering RNA strategy / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of integrin beta1 on the proliferation and differentiation of human keratinocyte stem cells (KSCs).</p><p><b>METHODS</b>DNA oligonucleotides targeting integrin beta1 at different locations were synthesized and inserted into BamHI-1 HindIII linearized p Silencer 3.1/H1 plasmids. The inserted sequences were verified by DNA sequencing. The KSCs were divided into control (without transfection), T1 (with transfection of vacant vector), T2 (with transfection of si integrin beta(1-1) vector), T3 (with transfection of si integrin beta(1-1) vector), and T4 (with transfection of si Negative vector) groups. The change in the expression of integrin beta1, was determined with Western blotting. The positive vector with the highest expression of integrin beta1 was selected and named as integrin beta1, and semi-quantitative RT-PCR was employed to detect the change in the expression of integrin beta1 mRNA.</p><p><b>RESULTS</b>The protein expression of integrin beta1, was not suppressed in control and T1 group, but it was suppressed in T2 and T3 groups, especially in T3 group (the suppression rate was 60%-70%, which was named si integrin beta1). The expression of integrin beta1 mRNA was obviously decreased by integrin beta1, transfection (the suppression rate was 70%).</p><p><b>CONCLUSION</b>The expression of integrin beta1, mRNA and protein could be down-regulated with recombinant si integrin P, vector transfection.</p>

Purification of human endothelial overexpressed lipopolysaccharide-associated factor 1 protein / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of obtaining of a highly pure protein of human endothelial overexpressed lipopolysaccharide-associated factor 1 (EOLA1) with metal chelation chromatography.</p><p><b>METHODS</b>Inclusion bodies of the E. coli transformed with EOLA1 gene were extracted and washed with BugBuster Protein Extraction Reagent. The primary purified products were purified by His. Bind Resin Chromatography under denaturing condition and dialyzed for renaturation, and then were analyzed with SDS-PAGE, Western blotting and peptide mass fingerprinting (PMF).</p><p><b>RESULTS</b>EOLA1 was mainly expressed in E. coli as insoluble inclusion bodies. The protein content in the primary extracted inclusion bodies accounted for over 75%, and it accounted for more than 90% after chromatography and renaturation. It was indicated by PMF that the targeted protein peptide overlaid many of designed protein peptide.</p><p><b>CONCLUSION</b>The method of EOLA1 protein purification and renaturation was convenient and efficient, and by this method sufficient amount of highly pure EOLA1 protein could be obtained for the preparation of EOLA1 monoclonal antibody and for the study of its gene function.</p>