Influence of histatin 1 on the proliferation and migration of HaCaT cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the influence of histatin 1 (Hst1) on the proliferation and migration of human epidermal cell line HaCaT.</p><p><b>METHODS</b>(1) HaCaT cells were routinely cultured and divided into control group, 100, 30, and 3 µg/mL Hst1 groups, 10 ng/mL recombinant human epidermal growth factor (rhEGF) group, and 30 µg/mL Hst1 + 10 ng/mL rhEGF group, according to the random number table (the same dividing method used for following grouping), with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cell proliferation was assayed by cell counting method at post culture hour (PCH) 24, 48, and 72. (2) HaCaT cells were divided into control group and 100, 30, and 3 µg/mL Hst1 groups, with 27 samples in each group. NO stimulating factor was added in control group, while Hst1 in corresponding concentration was added in the latter 3 groups. Cell cycle was assayed with flow cytometry at PCH 24, 48, and 72, and PI was calculated. (3) HaCaT cells were divided into control group, 30 µg/mL Hst1 group, 10 ng/mL rhEGF group, 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group, with 10 samples in each group. NO stimulating factor was added in control group, while Hst1 and(or) rhEGF in corresponding concentration(s) was (were) added in the latter 5 groups. Cells in each group were divided into two portions: cells in one portion were treated by mitomycin C for 2 hours, while cells in the other portion were not. Scratching assay was conducted in both portions of cells. Cell migration was measured at post scratching hour (PSH) 0, 16, and 24, and the wound-area healing rate was calculated. Data were processed with analysis of variance, and LSD- t test or Dunnett t test was applied in paired comparison among groups.</p><p><b>RESULTS</b>(1) At PCH 24, the cell numbers in 10 ng/mL rhEGF group and 30 µg/mL Hst1 + 10 ng/mL rhEGF group were significantly higher than that in control group (with t values respectively 3.813, 5.410, P < 0.05 or P < 0.01). Except for cell numbers in 30 µg/mL Hst1 group and 3 µg/mL Hst1 group at PCH 48, cell numbers in the other groups as treated by Hst1 and (or) rhEGF were significantly higher than those in control group at PCH 48 and 72 (with t values from 7.754 to 24.979, P values all below 0.01). At PCH 72, the cell number was obviously higher in 100 µg/mL Hst1 group [(19.21 ± 0.59)×10⁴] than in 30 µg/mL Hst1 group [(16.19 ± 0.53)×10⁴)] and 3 µg/mL Hst1 group [(15.38 ± 0.13)×10⁴], with t values respectively 11.391, 19.017, P values all below 0.01. The cell number was higher in 30 µg/mL Hst1 + 10 ng/mL rhEGF group than in 30 µg/mL Hst1 group, 3 µg/mL Hst1 group, and 10 ng/mL rhEGF group (with t values from 4.579 to 34.884, P < 0.05 or P < 0.01). Cell numbers in all groups increased with prolongation of time. (2) Compared with those in control group at PCH 24 and 48, the percentage of cells in G0/G1 phase was decreased, the percentage of cells in S phase was increased (except for cell percentage of 30 µg/mL Hst1 group at PCH 24), and PI value was significantly increased in 100 µg/mL Hst1 group and 30 µg/mL Hst1 group (with t values from 4.752 to 16.104, P values all below 0.01). The PI value in 3 µg/mL Hst1 group was obviously higher than that in control group only at PCH 48 (t = 4.609, P < 0.01). At PCH 72, only the PI value in 100 µg/mL Hst1 group was higher than that in control group (t = 8.005, P < 0.01). Compared among the groups treated by Hst1, the percentage of cells in G0/G1 phase showed an elevating trend, and the percentage of cells in S phase and the PI value showed a declining trend along with the decrease in Hst1 concentration at each time point. Compared within each group treated by Hst1, the percentage of cells in G0/G1 phase declined first and then elevated, while the percentage of cells in S phase and the PI value elevated first and then declined along with prolongation of time. (3) Without treatment of mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group (75.9 ± 3.9)% at PSH 16 was significantly higher than those in control group and 10 ng/mL rhEGF group [(53.0 ± 3.5)%, (61.7 ± 2.5)%, with t values respectively 12.241, 7.598, P values all below 0.01], but lower than those in 30 µg/mL Hst1 + 10 ng/mL rhEGF group, 15 µg/mL Hst1 + 5 ng/mL rhEGF group, and 15 µg/mL Hst1 + 10 ng/mL rhEGF group [(95.0 ± 4.1)%, (97.0 ± 3.7)%, (80.5 ± 5.9)%, with t values from -11.324 to -2.502, P < 0.05 or P < 0.01]. After being treated by mitomycin C, the wound-area healing rate in 30 µg/mL Hst1 group at PSH 16 [(54.1 ± 4.5)%] was higher than that in control group [(35.8 ± 5.7)%, t = 7.790, P < 0.01], but lower than that in the same Hst1 concentration but without mitomycin C treatment group (t = -10.863, P < 0.01). There was no statistically significant difference in the wound-area healing rate between 30 µg/mL Hst1 group and other groups treated by Hst1 and rhEGF at PSH 16 (with t values from 0.061 to 2.030, P values all above 0.05). Compared within each group with or without treatment of mitomycin C, the wound-area healing rate at PSH 16 was not significantly different from that at PSH 24 (with F values from 0.856 to 3.062, P values all above 0.05).</p><p><b>CONCLUSIONS</b>Hst1 can promote the proliferation and migration of HaCaT cells. It has synergic effect with rhEGF on the promotion of cell proliferation, but their synergic effect on cell migration is not obvious.</p>

Effect of substrate stiffness on biological behavior of fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of substrate stiffness on proliferation, migration of fibroblast and integrin β(1) expression in fibroblast.</p><p><b>METHODS</b>Fibroblasts were inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa. After being cultured for 5 days or 6 days, cells were counted and cell proliferative activities (recorded as absorbance value) were assessed with methyl thiazolyl blue (MTT). After being cultured for 3 days, cell cycle was detected and proliferation index (PI) was calculated. The cell scratch test was used for determination of cell migration rate on post scratch day (PSD) 0 (the day of scratch), 1, 2, and 3. After being cultured for 2 days, the expression of integrin β(1) was determined by flow cytometry with fluorescence. Data were processed with one-way analysis of variance.</p><p><b>RESULTS</b>(1) The proliferative speed and proliferative activity of fibroblasts were all increased along with the increase in substrate stiffness. PI of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa was respectively 24.8%, 27.4%, 32.4%. On PSD 2, migration rate of fibroblasts inoculated on silicon substrate with stiffness of (19.8 ± 1.1) and (200.1 ± 2.6) kPa was respectively (91.4 ± 5.1)%, (100.0 ± 1.3)%, which were higher than that of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa [(55.8 ± 6.8)%, with F value respectively 3.5, 4.0, P values all below 0.01]. (3) The expression rate of integrin β(1) in fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa was the lowest (43.22%), and that in fibroblast inoculated on silicon substrate with stiffness of (200.1 ± 2.6) kPa was the highest (81.26%).</p><p><b>CONCLUSIONS</b>Substrate stiffness may have a great effect on proliferation and migration of fibroblast during the process of wound healing and scar formation, which can be related to regulation of integrin β(1) expression.</p>

Effect of calcium on the activity and expression of integrin beta1 promoter in HaCaT cells and cell migration / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of calcium on the activity and protein expression of integrin beta1 promoter in human immortal keratinocyte colony HaCaT cell and cell migration.</p><p><b>METHODS</b>(1) HaCaT cells were cultured in vitro (12-slot plate) and divided into 5 groups according to the random number table, with 18 slots in each group: reporter plasmid pGL3 promoter (positive control group, PC), pGL3 empty vector (negative control group, NC), pGL3-1756 bp (total length promoter group, TL), pGL3-1442 bp (distal promoter group, D), and pGL3-261 bp (proximal promoter group, P) was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0.00, 0.03, 0.09, 0.30, 0.80, or 1.20 mmol/L calcium (3 slots in each group with each concentration). Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. (2) HaCaT cells steadily transfected with small interfering RNA-integrin beta1 vector (steadily transfected in brief) constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin beta1 protein was determined with Western blot. (3) Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media (divided according to the random number table, with 3 slots of cells for each concentration) for 12 hours after scratch test. Cell migration rate was observed and determined. (4) Data were processed with one-way analysis of variance and independent samples t test.</p><p><b>RESULTS</b>(1) The luciferase activity of cells in TL group increased from 0.16+/-0.09 to 0.39+/-0.09 and 0.35+/-0.05 (with t value respectively 3.143, 3.140, P values all below 0.05) as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0.30 mmol/L, and it decreased as calcium concentration increased to 0.80 and 1.20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity (0.56+/-0.32, 0.64+/-0.06) at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group (with t value respectively 0.887, 6.122, P values all below 0.05). There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. (2) The expression amount of integrin beta1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium (0.58+/-0.09, 1.40+/-0.29, 1.41+/-0.09, 0.99+/-0.10, 1.16+/-0.15) were all increased as compared with that cultured with 0.00 mmol/L calcium (0.53+/-0.10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6.199, P values all below 0.05). (3) Migration speed of normal HaCaT cells cultured with 0.09, 0.30 mmol/L calcium increased obviously as compared with that cultured with 0.00 mmol/L calcium, and it slowed down when cultured with 0.80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium.</p><p><b>CONCLUSIONS</b>Distal promoter region of integrin beta1 plays a vital role in regulating integrin beta1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin beta1 promoter and cell migration.</p>

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

<p><b>OBJECTIVE</b>To study the subcellular localization of human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) protein in endothelial cells.</p><p><b>METHODS</b>Human umbilical vein endothelial cell strain ECV304 were cultured in vitro. The fusion protein of enhanced green fluorescent protein (EGFP)-EOLA1 expressing plasmid was constructed. Empty plasmid with EGFP at N side (pEGFP-N2) and fusion protein expressing plasmid EGFP-EOLA1 was respectively transfected into ECV304 cells with liposome. After being cultured for 48 hours, the expression levels of EGFP and fusion protein EGFP-EOLA1 in cells were detected with Western blot. The subcellular localization of EOLA1 protein was detected by laser scanning confocal microscope and immunoelectron microscopy.</p><p><b>RESULTS</b>The EGFP-EOLA1 coexpression plasmid was verified to be successfully constructed by enzyme cutting and gene sequencing. The fusion protein of EGFP-EOLA1 was observed to express in transfected cells through Western blot. Green fluorescence scattered all over the ECV304 cells transfected with empty plasmid and cells transfected with fusion protein expressing plasmid, and it gathered obviously in the nuclei in the latter cells. Immune deposits were observed in the matrix of cells transfected with fusion protein expressing plasmid but not in the cells transfected with empty plasmid.</p><p><b>CONCLUSIONS</b>EOLA1 protein is localized in the nucleus and the matrix of ECV304 cell, and it plays its role as a signal transduction factor.</p>

Signal transduction mechanism in burn wound healing / 中华烧伤杂志

After 50 years of development in science of burns care in China, we have basically solved coverage of deep wounds of burn trauma, as well as role of multiple growth factors and stem cell in wound healing, making great contribution to improving the treatment of patients with large area of deep burns. Surgeons are paying close attention to problems of wound healing, especially in the fields of scarless healing and rehabilitation. To solve these problems, we need to do further investigation on multiple growth factors as well as proliferation/differentiation of stem cells in regulation of cell growth and differentiation in wound healing. Therefore, we are facing a even more serious challenge.

Influence of hypoxia on the proliferation and activity of human umbilical vein endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of hypoxia on the proliferation and activity of human umbilical vein vascular endothelial cells (EA. hy926).</p><p><b>METHODS</b>EA. hy926 cells were cultured in vitro and divided into normal control and hypoxia groups. The cells in hypoxia group were placed into hypoxic jar and treated with mixed gases(94% N2 +5% CO2 + 1% O2) for 1,3,6 and 12 hours. Then the total proteins were extracted for the determination of the expression of vascular endothelial growth factor (VEGF) and proliferation cell nuclear antigen (PCNA). The cell cycle and growth curve were determined with flow cytometry and MTT method, respectively.</p><p><b>RESULTS</b>The expression of PCNA protein began to increase at 3 post-hypoxia hour (PHH), peaked at 6 PHH, but without obvious difference compared with that at 12 PHH. The expression of VEGF began to increase at 1 PHH, peaked at 6 PHH, and decreased at 12 PHH, though it was still markedly higher than that of normoxia at 12 PHH. MTT results showed that the cell activity began to increase at 1 PHH, and it was still to increased at 3 PHH, then decreased at 6 PHH, and it was lower than that in control group at 12 PHH. The number of cells in G0/G1 phase was decreased, but the cells in S and G2/M phase was increased at 1, 3, 6 PHH when compared with those in normal controls. The proliferation index (PI) of cells in hypoxia group at 1PHH (43 +/- 9)%, 3PHH (39 +/- 11)%, 6 PHH (40 +/- 11))% were higher than that before hypoxia (32 +/- 9)% and 3 (39 +/- 11) % and 6 hours (40 +/- 11)% after hypoxia (P < 0.05). The PI was obviously lower at 12 PHH (27 +/- 4))% compared with that of cells under normoxic condition (P < 0.05).</p><p><b>CONCLUSION</b>Short-term hypoxia is beneficial to promote the proliferation of the cells, but this effect will be inhibited with the prolongation of hypoxia.</p>

Involvement of JNK signal pathway in hypoxia related upregulation of calcium/calmodulin-dependent serine protein kinase in endothelial cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the expression of calcium/calmodulin-dependent serine protein kinase (CASK) induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event.</p><p><b>METHODS</b>EA. hy926 cells were cultured in normoxic condition for 0, 12, 24, 48, 72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1, 3, 6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3, 6, 12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 (0, 10, 100 nmol/L and 1,10 micromol/L) 1h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot.</p><p><b>RESULTS</b>CASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia (0.010 +/- 0.003, P < 0.01), and it reached the peak 12 hrs after hypoxia (0.192 +/- 0.023, P < 0.01). The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 micromol/L SP600125 pretreatment.</p><p><b>CONCLUSION</b>JNK signal pathway is involved in short-term hypoxia related CASK upregulation.</p>

Screening of AGGF1 interacting with inhibitor of differentiation-1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the proteins interacting with inhibitor of differentiation 1(Id1) using yeast two-hybrid analysis in adult human lung cDNA libraries.</p><p><b>METHODS</b>The coding sequence of Id1 was amplified by PCR and cloned into the bait plasmid. The recombinant bait vector pHybLex/Zeo-Id1 was verified by restriction endonuclease digestion before transformation into the yeast strain EGY48/pSH18-34, which was tested subsequently for reporter genes Leu2 and LacZ activation. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into the yeast strains and screened to obtain Leu2(+) and Leu2(+)LacZ(+) clones, with the false positive clones excluded using positive and negative controls, and the plasmid of the true positive clone was sequenced and blasted for homological analysis.</p><p><b>RESULTS</b>Successful construction of pHybLex/Zeo-Id1 was confirmed by enzyme digestion. After transformation of pHybLex/Zeo-Id1 into EGY48/pSH18-34, no specific reporter genes Leu2 and LacZ activation was found. The pHybLex/Zeo-Id1 plasmid and the cDNA library plasmid were sequentially transformed into yeast strain, and 198 Leu(+) clones and 19 Leu(+)LacZ(+) double positive clones were obtained. After elimination of the false positive clones, one true positive clone was obtained, whose plasmid analysis by sequencing and blasting indicated high homology (99.5%, 556/559) to AGGF1 (an angiogenic factor with G-patch and FHA domains 1). AGGF1 expression was confirmed in the true positive yeast cells by Western blotting.</p><p><b>CONCLUSION</b>AGGF1 is confirmed to interact with Id1 by yeast two-hybrid analysis for screening adult human lung cDNA libraries.</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>

Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To amplify the full-length cDNA and characterize the structure and biological function of a novel expression sequence tag ST55 (GenBank Accession No. BM121646).</p><p><b>METHODS</b>Rapid amplification of cDNA ends was used to clone the full-length of cDNA of ST55 in this study. Then, its tissue distribution was checked by Northern blots, and the associated protein was screened by GAL 4-based yeast two-hybrid. The effect of stable transfection of the cDNA on cell proliferation was evaluated in ECV304 cells.</p><p><b>RESULTS</b>A full-length 1404 bp cDNA was cloned, and it was accepted as a novel human mRNA by GenBank (No. AY074889), named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1). Bioinformatic analysis found that the EOLA1 encoded 158 amino acids, 17.89 kDa protein, and mapped to chromosome Xq27.4 with 5 exons. EOLA1 expressed in different human normal tissues and cancer cell lines. Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein. The interaction between EOLA1 and MT2A was confirmed by co-immunoprecipitation experiments. Stable transfection of EOLA1 was noted to stimulate ECV304 cell proliferation (P < 0.05).</p><p><b>CONCLUSION</b>The findings suggest that EOLA1 is a novel gene and the interaction of EOLA1 and MT2A may play an important role in cell protection in inflammation reaction.</p>

Influence of human telomerase reverse transcriptase gene transfection on the proliferation of human embryonic fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of human telomerase reverse transcriptase (hTERT) gene transfection on the proliferation of human embryonic fibroblasts (hEF).</p><p><b>METHODS</b>hEFs were cultured in vitro. Sense recombinant eukaryotic plasmid (pIRES2-EGFP-hTERT) and pIRES2-EGFP vacant vector were transfected into hEF respectively with Lipofectin reagent, and were named as hEF-hTERT and hEF-EGFP. The hTERT, Id1, PCNA and I, III type collagen expression in these cells were detected by Western blot. Then the cell cycle and growth curve were measured and plotted with flow cytometry and MTT method, respectively.</p><p><b>RESULTS</b>1. The expression of hTERT, Id1, PCNA, type I and III collagen in hEF-hTERT were much higher than that in hEF and hEF-EGFP. 2. As shown in the growth curve, the OD value of hEF-hTERT at 4 to 6 days after culture was obviously higher than that of hEF and hEF-EGFP (P < 0.05), while no difference existed between hEF and hEF-EGFP from 1 to 6 days after culture (P > 0.05). 3. The cell number in G0/G1 phase in hEF-hTERT was less than that in hEF and hEF-EGFP. The cell number of hEF-hTERT in S and G2/M phase and its proliferation index (57.47%) increased when compared with that in hEF-EGFP (13.13%) and hEF (17.38%), but there was no difference between hEF and hEF-EGFP.</p><p><b>CONCLUSION</b>Exogenous hTERT gene transfection could promote the proliferative capacity of hEF.</p>

Direct evidence of the existence of specific LPS binding sites on vascular endothelial cells / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the binding characteristics of endothelial cell (EC) with LPS free from the participation of serum factors.</p><p><b>METHODS</b>Laser confocal microscope was employed in the observation of the binding of EC with FITC-LPS. The KD and the binding sites of each EC were calculated by radioligand binding assay of receptors (RBA) using [(3)H]-LPS.</p><p><b>RESULTS</b>The binding of EC with LPS was saturable, time and concentration dependent and it could be competed with overdosed LPS of the same type. The fluorescence mainly distributed in cytoplasm, especially near the nucleus, which could also be stained.</p><p><b>CONCLUSIONS</b>There might be some specific LPS binding sites existing on ECs and LPS could function intracellularily.</p>

Thermal stress can inhibit proliferation of ECV304 cells / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To observe the effects of thermal stress on proliferation of human vascular endothelial cells (VECs) and explore its significance.</p><p><b>METHODS</b>Changes of VECs proliferation were investigated with (3)H-TdR incorporation method after ECV304 was treated at 43 degrees for 2 hours, while expressions of intercellular adhesion molecule-1 (ICAM-1), inhibitor of differentiation-1 (ID1), and P16 and P21 proteins were determined by Western Blotting.</p><p><b>RESULTS</b>The effect of inhibition of VECs growth after thermal stress was detected by (3)H-TdR incorporation experiment. Western blotting showed ICAM-1, a marker of activated endothelial cells, was increased markedly after thermal stress. Expression of ID1 protein declined gradually with increasing expressions of its downstream genes, P16 and P21 following the thermal stress.</p><p><b>CONCLUSIONS</b>Thermal stress could strongly activate VECs and inhibit proliferation of VECs through ID1, thus down regulating cyclin-dependent kinase inhibitors, P16 and P21, which might be an essential pathway for recovery of VECs after thermal stress.</p>

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS).</p><p><b>METHODS</b>Two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced.</p><p><b>RESULTS</b>These analyses have identified both novel and known genes whose expression is influenced by LPS. The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction.</p><p><b>CONCLUSIONS</b>SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.</p>