Effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 in vitro and its molecular mechanism / 中华烧伤杂志

Objective: To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 (HMEC-1) in vitro and the potential molecular mechanism. Methods: The experimental research method was used. HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table (the same grouping method below), which were transfected correspondingly for 48 h. The cell proliferation activity was detected using the cell counting kit 8 on 1, 3, and 5 days of culture. The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test, and the percentage of the residual scratch area was calculated. The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The protein expressions of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated VEGFR2 (p-VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in cells were detected by Western blotting. HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA (siRNA) negative control group, empty adenovirus+siRNA negative control group, P311 adenovirus+siRNA-VEGFR2 group, and empty adenovirus+siRNA-VEGFG2 group, which were treated correspondingly. The protein expressions of VEGFR2, p-VEGFR2, ERK1/2, and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection. The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide (DMSO) group, empty adenovirus+DMSO group, P311 adenovirus+ERK1/2 inhibitor group, and empty adenovirus+ERK1/2 inhibitor group, which were treated correspondingly. The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment. The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The sample number at each time point in each group was 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: Compared with that of empty adenovirus group, the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1, 3, and 5 days of culture (with t values of -0.23, -1.30, and -1.52, respectively, P>0.05). The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group (with t values of -2.47 and -2.62, respectively, P<0.05). At 8 h of culture, compared with those of empty adenovirus group, the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased (with t values of 4.49 and 4.78, respectively, P<0.01). At 48 h of transfection, compared with those of empty adenovirus group, the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes (P>0.05), and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased (with t values of 17.27 and 16.08, P<0.01). At 24 h of transfection, the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group (P<0.01). The protein expressions of VEGFR2, p-VEGFR2, and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group (P<0.01). The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group (P<0.05 or P<0.01). At 24 h of transfection, the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62, which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) μm in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than (11 600±2 776) μm in empty adenovirus+siRNA-VEGFR2 group (P<0.01). At 2 h of treatment, the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01), and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group (P<0.05). At 2 h of treatment, the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72, which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+DMSO group was (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) μm in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than (13 188±2 306) μm in empty adenovirus+ERK1/2 inhibitor group (P<0.01). Conclusions: P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.

Quantitative comparison of lung pathological processing and its diagnostics significance / 解放军医学杂志

Objective To qualitatively and quantitatively compare the effect of tracheal inflation method, vascular perfusion method and combined trachea-vascular perfusion method on alveolar structure and cell antigen preservation. Methods Fifteen healthy Sprague Dawley rats were randomly divided into three groups (5 each): tracheal inflation group, vascular perfusion group and combined trachea-vascular perfusion group. After lungs were respectively fixed with the methods mentioned above, tissue blocks were paraffin embedded and sectioned. HE staining and immunofluorescence staining were then performed for qualitative observation and stereological analysis of the fixation effect. Results Qualitative study showed that the fixation effect of cytoplasm and nuclear antigen was better in vascular perfusion group and combined trachea-vascular perfusion group than in tracheal inflation group. Stereological analysis showed that the alveolar linear intercept in combined trachea-vascular perfusion group was longer than that in tracheal inflation and vascular perfusion group (P<0.05). The lung parenchyma volume density and alveolar septum thickness of combined trachea-vascular perfusion group were smallest among all the three groups, followed by tracheal inflation group and vascular perfusion group in turn (P<0.05). Conclusions The combined trachea-vascular perfusion method could not only preserve better alveolar structure but also display perfect cellular antigen. Therefore, it is suitable for the morphometry-based researches on lung pathological and clinical diagnosis.

Alendronate treatment does not inhibit bone formation within biphasic calcium phosphate ceramics in posterolateral spinal fusion: an experimental study in porcine model / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Biphasic calcium phosphate (BCP) ceramics has a potential advantage as an osteoconductive matrix and has an optimal resorption rate for bone formation. Using BCP ceramics as a bone graft during spinal fusion requires osteogenesis within the material and subsequent bridging between adjacent vertebrae to provide long-term support. Bisphosphonates have been reported to prolong the process of bone healing. The influence of bisphosphonate treatment on bone formation within BCP ceramics in spinal fusion remains unknown. The aim of this study was to evaluate the influence of alendronate on BCP osteogenesis in posterolateral spinal fusion.</p><p><b>METHODS</b>Posterolateral spinal fusion with pedicle screw fixation was performed at the lumbar spine in twenty-two pigs. BCP ceramics were applied as a bone graft to obtain bone fusion between adjacent transverse processes. Eleven pigs in the treatment group received oral alendronate 10 mg/d for three months postoperatively. Eleven pigs in the control group did not receive treatment with alendronate. All animals underwent posterolateral spinal fusion with BCP ceramics. The fusion rate was evaluated three months after the operation.</p><p><b>RESULTS</b>The fusion rates evaluated by X-ray were 27.3% in the treatment group and 20% in the control group. The fusion rates using histological evaluation were 18.2% in the treatment group and 20% in the control group. The mean volumes of fusion mass were (3.64 +/- 0.86) cm(3) in the treatment group and (4.26 +/- 0.63) cm(3) in the control group. No significant differences were found in either trabecular bone volume or residual BCP volume between treatment and control groups using histological evaluation. The new bone formation within BCP ceramics was greater in the area adjacent to transverse process (P < 0.01).</p><p><b>CONCLUSION</b>Oral alendronate with a dose of 10 mg daily do not inhibit bone formation within BCP ceramics or affect the fusion rate in posterolateral spinal fusion from porcine models.</p>

Establishment of the sweep pattern visual evoked potential system and its application / 法医学杂志

OBJECTIVE@#To establish an acuity inspection system with sweep pattern visual evoked potential (SPVEP) so as to provide the evidence for acuity objective inspection.@*METHODS@#Based on the domestic sweep pattern visual evoked apparatus, sections of hardware were reformed and a manipulation program possessing false random control software was compiled. The SPVEP acuity for the 78 eyes (10 normal eyes, 10 ametropia eyes, 48 prevalence eyes, 10 false ametropia eyes) was estimated with our acuity objective inspection system, then compared with the E visual acuity of those eyes by statistical procedure.@*RESULTS@#There was a close correlation between the SPVEP acuity and E visual acuity for 78 eyes (r2 = 0.946).@*CONCLUSION@#SPVEP acuity inspection system can be applied to estimate objective acuity.

Effect of hypoxia on the gene profile of human bone marrow-derived mesenchymal stem cells / 生理学报

Our previous study demonstrates that hypoxia promotes human bone marrow-derived mesenchymal stem cell (hMSC) proliferation. The aim of the present study was to investigate the gene profile involved in this process by using cDNA microarray. Cultured hMSCs were treated with hypoxia (3% O(2)) for 4 h, 12 h, 24 h, 36 h, 48 h and 72 h, respectively. Then these cells were collected to prepare total RNA. Hypoxia-induced gene expression profile was examined and analyzed by GenePix Pro 4.0 software. Some of cDNA microarray results were confirmed by RT-PCR. Microarray analysis identified that 282 genes expressed differentially, of which most were involved in metabolism. The number of differentially expressed genes at different hypoxia time points was different, and most genes were regulated after 24-hour hypoxia. Among the 282 differentially expressed genes, 4 hypoxia-inducible factor 1 (HIF-1) targeted genes and 10 genes that changed at 3 continuous time points were found. The results obtained indicated that 4 HIF-1 targeted genes, i.e., transforming growth factor beta3 (TGFbeta3), phospho-glycerate kinase 1 (PGK1), insulin-like growth factor binding protein 3 (IGFBP3) and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3), displayed up-regulated pattern at 36 h under hypoxia. BNIP3 displayed a dynamically up-regulated pattern at 12, 36 and 72 h under hypoxia. However, TGFbeta3 and PGK1 were down-regulated at 72 h. In addition, the gene expressions of adenylate kinase 3-like 1 (HAC), neurofilament light polypeptide 68 kDa (NEFL), N-myc downstream regultated gene 1 (NDRG1), discoidin domain receptor family member 1 (DDR1), tribbles homolog 3 (TRIB3), nucleoprotein (AHNAK) and eukaryotic elongation factor selenocyteine-tRNA-specific (EESTS) were up-regulated. Moreover, the gene expressions of EESTS, NEFL were up-regulated at 5 different time points under hypoxia. Furthermore, it was found that the gene expressions of histone cluster 1 (HIS1) and transferring receptor (TFRC) were down-regulated. These results suggest that the proliferation of hMSCs induced by hypoxia is a complex process in which a number of genes may be involved.

Effects of oxygen-glucose deprivation on cultured rat hippocampal neurons / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effects of oxygen-glucose deprivation on cultured rat hippocampal neurons.</p><p><b>METHODS</b>The hippocampal neurons cultured for 12 d were exposed to combined oxygen-glucose deprivation for 0.5 - 4 h and then cultured with original medium in normoxia for 28 h. Necrotic neurons were identified by 0.4% trypan blue staining and apoptotic neurons were detected by a TUNEL technique. Meanwhile, the area, perimeter and circle diameter of cell bodies were measured respectively by a photography analysis system.</p><p><b>RESULTS</b>The percentage of necrotic cells in cultured hippocampal neurons increased significantly during oxygen-glucose deprivation, but the percentage of apoptotic cells increased significantly after 28 h oxygen-glucose recovery. Photography analysis showed that area, perimeter and circle diameter of the necrotic cell bodies were larger than those of the apoptotic ones.</p><p><b>CONCLUSION</b>Oxygen-glucose deprivation can lead to severe damage of cultured hippocampal neurons. The necrosis is major during acute oxygen-glucose deprivation, while the apoptosis is major 28 h after oxygen-glucose recovery.</p>