The generation of the endothelial specific cdc42-deficient mice and the effect of cdc42 deletion on the angiogenesis and embryonic development / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>High microvascular permeability plays an essential role in pathological process of multiple diseases such as septic shock, acute lung injury and acute respiratory distress syndrome, and burns. Inhibiting hyperpermeability is significant for controlling these conditions. Cdc42, as a main member of the small Rho GTPase family, plays a critical role in controlling and regulating the endothelial junctional permeability. We aimed to generate and identify endothelial specific cdc42-deficient mice by the Cre/loxp recombination approach, for examination in an animal model of the contribution of the cdc42 gene in the microvascular barrier function.</p><p><b>METHODS</b>We crossed cdc42(Flox/Flox) mice with mice expressing endothelial cell-specific Cre recombinase, and the offspring with the genotype cdc42(Flox/+)Tie2Cre(+/-) were back-crossed with the cdc42(Flox/Flox) mice. The cdc42(Flox/Flox)Tie2Cre(+/-) mice in the F2 generation were the target mice. If the cdc42 deficient mice did not survive, we would observe the cdc42 deficient mice embryos, and compare them with wild-type mice embryos.</p><p><b>RESULTS</b>Cdc42(flox/+)Cre(+/-) mice were mated with the cdc42(Flox/Flox) mice and among the living offspring there were no cdc42(Flox/Flox)Cre(+/-) target mice. We found the endothelial special cdc42 deficient embryos at the E7.5-E16.5 stage. We observed that cdc42 deficient embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos.</p><p><b>CONCLUSIONS</b>Endothelial specific knockout of cdc42 caused embryonic lethality and the mice did not survive to birth. The target embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. These results demonstrated that the cdc42 plays an important role in development of embryos and in development of microvessels as well as microvascular permeability.</p>

Alteration of PTEN gene expression affects the migration of human airway smooth muscle cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in human airway smooth muscle cell (HASMC) migration and related signaling pathway after interference with PTEN gene expression.</p><p><b>METHOD</b>HASMCs were infected with an adenovirus vector and RNA interference vector of human PTEN gene to establish the cell model with PTEN gene over-expression (Ad-GFP-PTEN-HASMC) and one with PTEN gene silencing (Ad-shPTEN-HASMC), using Ad-GFP-infected and a blank cells as the negative controls and LY294002 as the positive control. Fluorescence microscopy and flow cytometric analysis were used to evaluate the transfection efficiency, and Western blotting was performed to examine the expression of PTEN and the activation of AKT and ERK1/2 signal pathway. Transwell assay and wound healing assay were used to assess the migration of HASMCs.</p><p><b>RESULTS</b>The adenovirus over-expression vector and RNA interference vector significantly affected the expression of human PTEN gene. Up-regulation of PTEN gene resulted in a slow-down of the HASMC migration, an inhibition of PI3K/AKT signal pathway at the protein level but no changes in Ras-Raf-MEK1/2-ERK1/2 signal pathway. Down-regulated PTEN gene expression, however, was not associated with an enhancement of HASMC migration, but activated PI3K/AKT signal pathway and inhibited Ras-Raf-MEK1/2-ERK1/2 signal pathway.</p><p><b>CONCLUSION</b>Upregulation of PTEN gene can effectively inhibit airway smooth muscle cell migration, the effect of which is probably mediated by the PI3K/AKT pathway.</p>

Application of flexirigid thoracoscopy in the diagnosis of pleural disease with unknown etiology / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the diagnostic accuracy of flexirigid thoracoscopy for pleural diseases and the patients' compliance.</p><p><b>METHODS</b>Forty-seven patients with pleural effusion and thickening of unknown etiology underwent examinations with flexirigid thoracoscopy with subsequent pathological examination, and the diagnostic accuracy and the patients' compliance were observed.</p><p><b>RESULTS</b>Thoracoscopy identified lesions in the pleural and/or diaphragm in 42 patients and no lesions in 5 patients. Malignancy was confirmed in 21 (44.7%), tuberculosis in 17 (36.2%), idiopathic hypereosinophilic syndrome in 1 (2.1%), nocardiasis in 1 (2.1%), constrictive pericarditis in 1 (2.1%), chronic empyema in 2 (4.3%), splenic artery embolization in 1 (2.1%), and negative result in 3 (6.4%) of the cases. The diagnostic accuracy rate of flexirigid thoracoscopy reached 93.6%, and no serious complications in relation to the examination was found.</p><p><b>CONCLUSION</b>Flexirigid thoracoscopy is efficient and relatively safe for diagnosis of pleural diseases with or without hydrothorax.</p>

Change of IL-22R1 expression in human airway smooth muscle cells in response to different stimulating agents / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of serum of asthmatic patients, dexamethasone, interleukin-4 (IL-4), interferon-gamma (IFN-γ) and transforming growth factor-β (TGF-β) on the expression of interleukin-22 receptor 1 (IL-22R1) mRNA and protein in HASMCs in vitro.</p><p><b>METHODS</b>IL-22R1 mRNA and protein expressions in HASMCs treated with different stimulating agents were measured by real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>IL-22R1 mRNA and protein expressions in HASMCs were significantly increased after stimulation by serum from asthmatic patients, but decreased after co-stimulation with dexamethasone. IL-22R1 mRNA and protein expressions in the cells both increased after stimulation by IL-4, IFN-γ and TGF-β.</p><p><b>CONCLUSION</b>IL-22R1 in HASMCs might be involved in the pathogenesis of asthma, and the therapeutic effect of dexamethasone on asthma is mediated, at least partially, by IL-22R1. The effects of IFN-γ, IL-4, and TGF-β on asthma may also be attributed to their actions on HASMCs.</p>

Association of the C3 gene polymorphisms with susceptibility to adult asthma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association between single nucleotide polymorphisms (SNPs) of the complement component 3 gene (C3) and adult asthma of Hans in southern China.</p><p><b>METHODS</b>A case-control study was performed. Four hundred and eighty-four adult asthma patients diagnosed in Nanfang Hospital and Affiliated Hospital of Guangdong Medical College, and 553 healthy subjects were collected from 2006 to 2010 for the study. MassARRAY-IPLEX and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) techniques was used to determine the genotypes of the rs10402876 and rs366510 loci of C3 gene.</p><p><b>RESULTS</b>Genotypes GG, GT and TT in the rs366510 locus, and genotypes GG, GT and TT in the rs10402876 locus were detected. A total of 98.94 percent of samples were genotyped. There were no significant differences in genotype frequencies (chi-square =0.346,P=0.841) and allele frequencies (chi-square =0.101,P=0.751) of rs10402876 between the two groups. However, genotype and allele frequencies of the rs366510 locus were significantly different (chi-square =9.759, P=0.008, Bonferroni correction, P=0.016; chi-square =5.294,P=0.021, Bonferroni correction, P=0.042, respectively). Compared with genotypes GG+GT, genotype TT of rs366510 significantly increased the risk of asthma, with the odds ratio of 1.471 (95% confidence interval 1.125-1.923).</p><p><b>CONCLUSION</b>These results suggest that C3 gene could be associated with adult asthma of Han population in southern China.</p>

Construction of a recombinant adenovirus vector containing PTEN and its expression in airway smooth muscle cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector containing phosphatase and tensin homolog deleted on chromosome 10 (PTEN) using the pAdxsi system.</p><p><b>METHODS</b>PTEN cDNA from plasmid pcDNA3-PTEN was cloned into the shuttle plasmid pShuttle-GFP-CMV. The shuttle vector was transformed into competent DH5alpha strain with the vector pAdxsi to achieve the homologous recombination. The recombinant construct was subsequently linearized with PacI and transfected into HEK293 cells via Lipofectamine 2000. The recombinant adenovirus particles were collected, and after titration, the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. The expression of PTEN mRNA and protein in the recombinant adenovirus vector and airway smooth muscle cells were detected by PCR and Western blotting, respectively.</p><p><b>RESULTS</b>GFP was expressed in HEK293 cells infected by recombinant adenovirus, and the expression intensity increased gradually with the passage of time, with obvious cytopathic effect (CPE) noted in the cells. After 3 cycles of amplification, the titer of adenovirus containing PTEN reached an appropriate level. The viral titer of pAdxsi-GFP-PTEN was 2x10(10) pfu/ml, and PTEN mRNA expression was detected by PCR. The homologous protein expressed in the infected human airway smooth muscle cells significantly increased in comparison with that in the control cells.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing PTEN is constructed successfully, which provides an experimental basis for studying the role of PTEN gene in asthma therapy.</p>

Effect of recombinant adenovirus carrying PTEN gene on the proliferation of human airway smooth muscle cells in vitro and the mechanisms / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of exogenous phophatase and tensin homolog deleted on chromosone 10 (PTEN) gene transfer via recombinant adenoviruses on the proliferation of human airway smooth muscle cells (HASMCs) in vitro and investigate the possible mechanisms.</p><p><b>METHODS</b>With a recombinant adenovirus vector containing PTEN (Ad-PTEN) constructed using the pAdxsi system, PTEN gene was transiently transfected into HASMCs and the transfection efficiency was determined by fluorescence microscope. RT-PCR and Western blotting were performed to detect the expression of PTEN mRNA and protein in the infected cells. MTS/PMS assay was used to analyze the proliferation of HASMCs, and the cell cycle changes of the transfected cells were evaluated by flow cytometry with PI staining. The expression levels of Akt and p-A kt proteins were detected by Western blotting, and P21 mRNA expression determined by RT-PCR.</p><p><b>RESULTS</b>The recombinant adenovirus Ad-PTEN showed a wild-type PTEN gene transfer efficiency of 98% at the multiplicity of infection (MOI) of 100. RT-PCR and Western blotting showed that infection with the recombinant adenovirus resulted in PTEN overexpression in the HASMCs, causing also increased ratio of G(0)/G(1) cells and proliferation inhibition of the ASMCs. The overexpression of PTEN significantly decreased the expression level of p-Akt but increased P21 mRNA expression.</p><p><b>CONCLUSION</b>The recombinant adenovirus containing PTEN can be successfully transfected into HASMCs cultured in vitro, resulting in PTEN overexpression at both the mRNA and protein levels. PTEN overexpression can efficiently inhibit the proliferation of HASMCs possibly through the PI3K/PKB/AKt and P21 pathways.</p>

Effects of dexamethasone on intracellular expression of Th17 cytokine interleukin 17 in asthmatic mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of dexamethasone on intracellular expression of Th17 cytokine interleukin 17 and the mechanisms in asthmatic mice.</p><p><b>METHODS</b>Experimental asthma was induced by ovalbumin (OVA) sensitization in 20 in female Balb/c mice with (dexamethasone group, n=10) or without dexamethasone treatment (model group, n=10), with another 10 serving as the control group. The levels of IL-17 in the bronchoalveolar lavage fluid (BALF) and serum of the mice were measured by enzyme-linked immunosorbent assay (ELISA), and the airway inflammation was evaluated by HE staining. The expressions of IL-17 and RORgammat mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of RORgammat protein was measured by immunohistochemical staining.</p><p><b>RESULTS</b>The levels of RORgammat and IL-17 mRNA and protein in the asthmatic model group were significantly higher than those in the control group (P<0.01), and the increased expressions of RORgammat and IL-17 mRNA and protein in the asthmatic mice were significantly reduced by dexamethasone treatment (P<0.05).</p><p><b>CONCLUSION</b>Dexamethasone can inhibit the release of IL-17 probably by inhibiting RORgammat expression and blocking Th17 differentiation in asthmatic mice.</p>

Rho-kinase signaling pathway participates in endothelin-1-induced human airway smooth muscle cell migration and cytoskeletal reorganization / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell (ASMCs) migration and cytoskeletal reorganization induced by endothelin-1 (ET-1).</p><p><b>METHODS</b>Primary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-1 treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation of myosin-phosphatase target 1 (p-MYPT1) was examined using Western blot analysis.</p><p><b>RESULTS</b>At the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect (P<0.01). Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 micromol/L significantly blocked ASMC migration (P<0.01). ET-1 (10 nmol/L) exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPT1 protein expression after ET-1 exposure for 15 and 30 min (P<0.01), but prolonged exposure failed to result in the expression enhancement (P>0.05).</p><p><b>CONCLUSION</b>Rho-kinase signaling pathway may play an important role in ET-1-induced ASMC migration and reorganization of actin cytoskeleton.</p>

Peripheral natural killer cell counting and its clinical significance blood in patients with bronchial asthma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the changes in natural killer (NK) cell count in the peripheral blood of asthmatic patients.</p><p><b>METHODS</b>The number of NK cells in the peripheral blood was determined with flow cytometry in 63 asthmatic patients with acute episodes, 65 patients with stable asthma and 62 healthy nonatopic subjects.</p><p><b>RESULTS</b>A significant decrease in NK cell number was noted in asthmatic patients during acute exacerbation [(13.9-/+9.4) %] in comparison with patients with stable asthma [(22.5-/+12.3) %] and healthy subjects [(19.6-/+10.1)%] (P<0.05), and the NK cell number showed no significant difference between the latter two groups (P>0.05).</p><p><b>CONCLUSION</b>NK cell number is reduced in acute exacerbation of asthma, suggesting its important role in the asthmatic process.</p>

Shikonin inhibits the proliferation of human airway smooth muscle cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the inhibitory effect of shikonin on the proliferation of human airway smooth muscle cells (HASMCs) in vitro.</p><p><b>METHODS</b>HASMCs from the trachea were obtained by primary culture of the tissue explants and adherent culture. The HASMCs were exposed to shikonin at 0 (control group), 0.5, 1, 2, 5, 10, 20, and 40 micromol/L for 12, 24, and 48 h, after which the cell proliferation was assessed by 3-(4,5-carboxymethoxypheny1)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay. Flow cytometry was used for cell cycle analysis of the HASMCs exposed to shikonin at 40, 20, 10, 5 micromol/L and 0 micromol/L (control group) for 24 h. Immunocytochemistry with SP method was performed to detect the expression of proliferating cell nuclear antigen (PCNA) in the HASMCs treated with shikonin at 20 micromol/L and 0 micromol/L (control group) for 24 h.</p><p><b>RESULTS</b>Shikonin at the concentrations of 20 and 40 micromol/L significantly inhibited the proliferation of the cells (P<0.05), and the effect was especially obvious after 48 h of cell exposure, with inhibition rates of 30.1% and 42.9%, respectively. No significant difference was found between the two concentrations for their cell growth inhibition effects (P>0.05). Shikonin at the concentrations of 20 and 40 micromol/L caused significant cell cycle arrest in G(0)/G(1) phase (P<0.05), the effect of which, however, was not concentration-dependent (P>0.05). Shikohin at 20 micromol/L significantly down-regulated the expression of PCNA in the cells (P<0.05).</p><p><b>CONCLUSION</b>Shikonin can inhibit the proliferation of HASMCs in vitro.</p>

Association between ADAM33 gene polymorphism and bronchial asthma in South China Han population / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the association between the polymorphism of T(1) locus allele in ADAM33 gene and bronchial asthma in South China Han population.</p><p><b>METHODS</b>Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing were used to determine the polymorphism of T(1) locus allele in ADAM33 gene in 160 unrelated patients with asthma and 95 unrelated healthy controls from South China Han population.</p><p><b>RESULTS</b>No significant difference was found in T(1) locus allele distribution frequency in populations of UK, US, Germany, Korea, and South China (Chi(2)=9.085, P=0.109). The frequencies of the genotypes (TT, TC, CC) were 80.6% (n=129), 16.9% (n=27) and 2.5% (n=4) in the 160 asthmatic patients and 94.7% (n=90), 3.2% (n=3) and 2.1% (n= 2) in the 95 controls, respectively, showing a significant difference in the distribution of the genotypes (TT, TC, CC ) between asthmatic patients and healthy controls (Chi(2)=10.955, P<0.05). The frequencies of the alleles (T, C) were 0.891 and 0.109 in the asthmatic patients and 0.963 and 0.037 in the controls, respectively, showing also a significant difference in the allele frequency between them (Chi square =8.299, P<0.05). The presence of C allele of ADAM33 gene T1 locus was found to be a greater risk factor in asthmatic patients than in the healthy controls. The odds ratio (OR) of TC and TC+CC were 6.279 (1.849-21.328) and 4.326 (1.620-11.550), respectively, with P value of 0.001 and 0.002, respectively, in comparison with TT genotype.</p><p><b>CONCLUSION</b>The polymorphism of T(1) locus allele in ADAM33 gene is associated with the susceptibility to asthma in South China Han population.</p>

Expression of eosinophil major basic protein mRNA in bronchial asthma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of eosinophil major basic protein (MBP) mRNA in bronchial asthma and explore its significance.</p><p><b>METHODS</b>Peripheral blood eosinophil MBP mRNA levels were measured in 40 patients with asthma and 20 normal controls by semi-quantitative RT-PCR. The association of MBP mRNA levels with eosinophil count and pulmonary function was also analyzed.</p><p><b>RESULTS</b>Compared with the normal control group, MBP mRNA level were significantly increased in asthma patients (0.37-/+0.11 vs 0.17-/+0.04, P<0.001), so was the eosinophil count (0.86-/+0.52 vs 0.21-/+0.10, P<0.001). MBP mRNA levels in patients with moderate persistent asthma (0.42-/+0.05) and those with severe persistent asthma (0.47-/+0.05) were significantly higher than those in patients with mild persistent asthma (0.25-/+0.06, P<0.001), and the difference in MBP mRNA levels between moderate persistent asthma patients and severe ones was also significant (P<0.05). Among the asthma patients, MBP mRNA levels showed an inverse correlation with pulmonary function (r=-0.7490, P<0.001).</p><p><b>CONCLUSION</b>Increased MBP mRNA expression level may correlate with the severity of asthma. MBP may play an important role in the development of asthma.</p>