Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.     

Effects of the grub extract on apoptosis of MCF-7 human breast cancer cell line

AIM: To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS: MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope, light microscope, AO/EB double fluorescent staining under fluorescent microscope. FCM was used to assay the change of apoptotic rate. The expression of Bcl-2, Fas, caspase-9, caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored.RESULTS: (1) The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners. The inhibitory rate in exposure group was significantly different from that in control group (P<0.01). (2) Morphological changes of apoptosis including nuclear condensation, fragment and apoptosis body formation were observed by invert microscope. (3) The MCF-7 human breast cancer cells in experimental group by HE staining showed nuclear condensation and blue-black, cytoplasm slight red, nuclear chromatin condensation and fragment shape, apoptosis body formations. (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope. (5) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. (6) The expression of Bcl-2 was down-regulated, while that of Fas, caspase-3, caspase-9 was up-regulated, compared with control group (P<0.01).CONCLUSION: (1) The proliferation of MCF-7 human breast cancer cell line can be inhibited significantly by the grub extract in vitro. (2)The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be mediated by down-regulation of Bcl-2 and up-regulation of Fas, caspase-3, caspase-9. This type of apoptosis starting and performing is through death receptor pathway and mitochondrial pathway.     

Role of autophagy in ursolic acid-induced injury of human umbilical vein endothelial cells

AIM: To investigate the role of autophagy in the injury of human umbilical vein endothelial cells (HUVECs) induced by ursolic acid (UA). METHODS: HUVECs were cultured in vitro with UA at various concentrations for 36 h and the proliferation inhibitory rate of HUVECs was determined by MTT method. The change of ultrastructure was observed under transmission electronic microscope (TEM). The autophagy was observed using fluorescent microscope by monodansylcadaverin (MDC) staining. The protein level and mRNA expression of microtubule-associated protein light chain 3(LC3) and Beclin-1 were detected by Western blotting and RT-PCR, respectively. Cell apoptotic rate was measured by flow cytometry analysis. RESULTS: UA at various concentrations showed significantly dose-dependent inhibitory effect on the proliferation of HUVECs. Autophagy was induced in HUVECs treated with UA as detected by MDC staining and TEM. The protein level and mRNA expression of LC3 and Beclin-1 in HUVECs were significantly increased following the treatment with UA, which was also in a time-dependent manner. Compared with UA group, addition of 3-methyladenine(3-MA) inhibited the increase in autophagic vacuoles and exacerbated the apoptosis. CONCLUSION: Autophagy shows protective effect on the proliferation inhibition of HUVECs induced by UA and the proliferation inhibition can be enhanced by the autophagy inhibitor 3-MA. 3-MA may enhance the apoptotic rate of HUVECs induced by UA.     

Cholesterol induces endothelial cells injury by increasing production of reactive oxygen species and activating NF-κB

AIM: To study the increased level of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and their correlation with the injury caused by cholesterol on HUVECs, and to clarify the original source of intracellular ROS. METHODS: The cells of HUVECs-12 were cultured in F12 medium with 10% FBS and divided into normal control group (without any treatment), solvent group (treated with 0.25% dehydrated alcohol), cholesterol group (treated with 50 mg/L cholesterol) and N-acetyl-L-cysteine(NAC) group(pretreated with 10 mmol/L NAC for 1 h and then treated with 50 mg/L cholesterol for 48 h). The intracellular ROS levels were determined by flow cytometry (FCM) with DCFH-DA as fluorescent probe. Nuclear translocation of NF-κB subunit p65 was detected by immunocytochemistry staining. LDH activity and concentration of nitric oxide in the supernatant of the cell culture were also determined. The concentration of MCP-1 protein in cultured supernatant was measured by ELISA. The intracellular levels of ROS and the changes after adding 4 kinds of enzyme inhibitors (NADPH oxidase inhibitor diphenyl iodide, mitochondrial respiratory chain enzyme complex inhibitor rotenone, NOS inhibitor L-NAME and xanthine oxidase inhibitor oxypurinol) were observed. RESULTS: (1)Compared to the normal control cells, 50 mg/L cholesterol increased intracellular ROS (P<0.01) and activated the nuclear translocation of NF-κB p65. A significant increases in LDH activity and the MCP-l protein were also observed. The NO level decreased in the cells. (2)Compared to the cholesterol control cells, diphenyl iodide decreased intracellular ROS significantly (P<0.01).Retenone also inhibited the generation of ROS partially (P<0.05). The other inhibitors almost did not affect the level of ROS caused by cholesterol (P>0.05). CONCLUSION: Free cholesterol increases ROS generation in endothelial cells, activates intracellular NF-κB, thus leading to endothelial cell injury. NADPH oxidase was the main source of ROS generation in HUVECs cultured with free cholesterol.     

Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

砂梨品种花粉形态的扫描电镜研究

运用扫描电镜对砂梨(P.pyrifolia Nakai)的79个品种的花粉粒形态进行观察,结果表明:1.砂梨品种花粉粒为近球形、扁球形、长球形,极面直径为22～34μm。2.花粉粒极面观多呈三角形或椭圆形,少数为近圆或方形(菱形);多数具3条萌发沟,少数有4条萌发沟。3.花粉表面雕纹(纹饰)多为条状纹,亦有网状纹、云片状纹、波浪状纹及瘤状纹,在条纹间有的无孔穴,有的有不同密度、不同大小的孔穴,条纹粗细亦存在着差别,证明品种间存在着差异。     

Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

ABSTRACT: BACKGROUND: Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. RESULTS: Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. CONCLUSION: Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.     

Primary culture,identification and in vitro angiogenesis of mouse pulmonary microvascular endothelial cells

AIM: To establish a simple and reliable method for primary culture of mouse pulmonary microvascular endothelial cells (PMVECs), and to investigate the ability of angiogenesis of the cells in vitro. METHODS: PMVECs from C57BL/6 new born mice were cultured with peripheral pulmonary tissue pasted method. The cells were observed under inverted phase-contrast microscope and identified by immunocytochemical and immunofluorescent methods. PMVECs were seeded at the density of 20 000 cells per well in 96-well plate on top of growth factor-reduced matrigel and incubated for 24 h at 37 ℃ in 5% CO₂. The plates were examined every 2 h for tube formation under an inverted microscope. RESULTS: The primary PMVECs showed fusiform shape or polygonal, regular cobblestone morphology after fusion to monolayer, and expressed factor Ⅷ -related antigen (ⅧF-Ag). Under fluorescence microscope, those cells demonstrated positive staining for uptaking acetylated low-density lipoprotein (DiI-Ac-LDL) and binding Bandeiraea simpli-cifolia lectin I (FITC-BSI). The purity of PMVECs was above 90%. PMVECs formed capillary-like structures within 4 h to 6 h after plated on matrigel and fully developed a "honeycomb" appearance at 10~12 h, and then the capillary tubes began to break apart. CONCLUSION: The tissue block pasted method for primary cultivation of PMVECs is easy to handle, with high yield and purity, and will further facilitate related studies of well-established tube-formation assay in vitro.     

Skeletal muscles of mdx mice were damaged after overload exercise

AIM:To observe the effects of overload exercise on skeletal muscles in X-linked muscular dystrophy(mdx) mice.METHODS:Mdx mice and C57 mice were carried out swimming and hanging tail movement tests (mdx mice as control did not exercise). It lasted for 13 minutes each time per day, and lasted 3 days. Evans blue was injected into tail vain. The mice were killed the next day, and the hind limbs were taken photographs after skins were flayed. The gastrocnemius muscles and diaphragms cryostat sections were made. Under a fluorescence microscope, Evans blue staining was seen. Then the sections were tested by routine HE staining, the histological change of muscles was analyzed under a light microscope.RESULTS:Many blue colored longitudinal lines were observed in skeletal muscles of mdx mice, whereas they were hardly seen in control mdx and C57 mice. Under a fluorescence microscope, some muscle fibers of mdx mice were stained with Evans blue, few muscle fibers of control mdx mice were stained, and C57 mice were not. Under a light microscope, HE staining of muscles showed some degenerated muscle fibers became round in shape and the myonuclei became condensed, or necrotic fibers had amorphous structures, most of them in the degenerated and necrotic fibers of diaphragms C57 mice did not have these changes.CONCLUSION:Overload exercise did harm to skeletal muscles of mdx mice; Vital staining with Evans blue is useful not only for distinguishing degenerating muscle fibers, but also for studying the degeneration process in dystrophin-deficient muscle.     

Effects of different lighting on reproductive system in depressive female rats

AIM: To investigate the effects of different lighting on the reproductive system in depressive female rats. METHODS: Healthy adult female rats were randomly chosen as control group, and the depressive adult female rats in SPF grade were randomly divided into 5 groups(7 rats each):depressive model group, sulfur lamp group, heat radiation lamp group, fluorescent lamp group and LED lamp group. After 45 d of continuous illumination, the estrous cycle was observed by the vaginal exfoliated cells, and the organ indexes of ovary and uterus were calculated. The concentrations of estiadrol(E₂), prolactin(PRL), progesterone(PROG) and testosterone(T) in the serum were detected by ELISA, and the histopathological lesion of ovary was observed under microscope with HE staining. RESULTS: The estrous cycle exhibited serious disorder, the ovaries exhibited serious congestion, and the organ indexes of ovary and uterus and the concentrations of E₂, PRL, PROG and T decreased significantly in the rats in depressive model group compared with control group(P<0.05). The estrous cycle and histopathological damage of ovary were obviously improved, and the concentrations of E₂, PRL, PROG and T were significantly increased after the sulfur lamp lighting in the depressive female rats compared with depressive model group. No obvious change and improvement of the reproductive functions in the heat radiation lamp group, fluorescent lamp group and LED lamp group was observed. CONCLUSION: The reproductive functions of depressive female rats are improved by sulfur lamp lighting.     

Activating effect of astragalus saponin on mouse peritoneal macrophages in vitro

AIM: To investigate the immunomodulatory effect of astragalus saponin (AS) on macrophages and explore the mechanism of its immunomodulation. METHODS: By adding different concentrations of AS into cultured mouse peritoneal macrophages in vitro, the influence of AS on the synthesis of nitro oxide (NO) was detected by NO kit (enzymatic method). MTT assay was used to determine the cytotoxicity of macrophages induced by AS. The morphological changes of macrophages were observed under transmission electron microscope. LSCM and specificity fluorescent probe Fluo-3/AM were applied to observe the change of Ca2+ in macrophages induced by AS. RESULTS: AS significantly increased NO synthesis, enhanced the effects of mouse peritoneal macrophages on killing carcinoma cells. Cell surface projection exhibited multiplication, becoming thickening and growth longer via transmission electron microscope. Increase in intracellular Ca2+ in macrophages was also observed. CONCLUSION: AS enhances the immune function of macrophages by increasing NO synthesis and enhancing cytotoxicity. The increased intracellular Ca2+ may be the mechanism of immunomodulatory effect of AS.     

Anthocyanins attenuates hepatic fibrosis and inflammation in non-alcoho-lic fatty liver disease mice

AIM:To explore the therapeutic effect of anthocyanins from Fructus Acanthophorae on high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice and the potential mechanism. METHODS:NAFLD mouse model was established by high-fat diet, and interferred with anthocyanins. The liver weight, and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC) and low-density li-poprotein cholesterol (LDL-C) were measured. The liver tissues were staining with HE, Oil Red O and Masson's trichrome. The protein levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10 in the liver tissues were determined by Western blot. The liver macrophage, white blood cell and mononuclear cell infiltration was detected by immunohistochemical method. The chemokines CCL7 and MCP-1 were also measured by immunohistochemical method. RESULTS:Anthocyanins significantly inhibited the increases in the liver weight, ALT, AST, TG, TC and LDL-C induced by high-fat diet. Anthocyanins attenuated the liver fibrosis and inflammatory cell infiltration caused by high-fat diet, and reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-10 and inflammatory chemokines CCL7 and MCP-1 in the liver tissues. CONCLUSION:Anthocyanins significantly alleviate non-alcoholic fatty liver disease caused by high-fat diet though reducing inflammatory factors, inflammatory cell infiltration and inflammatory chemokines.     

Genotype evaluation,muscle histopathology and ultrastructure in a Duchenne muscular dystrophy animal model

AIM: To evaluate the genotype , muscle histopathology and ultrastructure in dko mice. METHODS: Dystrophin/Utrophin-deficient double knockouts (dko) mice were obtained from university of Oxford, UK. Genotype of filial generation of heterozygote was evaluated by PCR-SSP. HE staining and fluorescent immunohistochemistry by SABC-Cy3 were used to detect striated muscle of dko mouse, and the muscle ultrastructure was observed by transmission electron microscope(TEM). RESULTS: In 112 filial generation mice, there were 28 mdx (25.0%), 26 dko (23.2%) and 58 heterozygote (51.8%), which coincided with the law of Mendelian inheritance. HE staining showed that the myocytes were not very uniform, there were phenomenon of round outline, centrally nucleated fibers, widening interspace, inflammatory cell infiltration and connective tissue proliferation in dko mice. There were no any immunofluorescent expression of dystrophin and utrophin in sarcolemma in dko mice. TEM showed sarcolemma breakage, separation and edema, and loose myofibril texture, inflammatory cell infiltration and connective tissue proliferation in dko mice. CONCLUSION: PCR-SSP is a very quick and accurate way for genotype evaluation of filial generation. The pathophysiology of dko mouse was very similar to Duchenne muscular dystrophy (DMD), and dko mouse is an ideal animal model for study of DMD clinical therapy.     

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells

AIM and METHODS:To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION:The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression.     

Phosphorylation PKCδ participates in apoptosis of PC12 cells induced by 6-hydroxydopamine

AIM: To observe the effect of phosphorylation protein kinase C delta (PKCδ) on the procedure of PC12 cells apoptosis induced by 6-hydroxydopamine(6-OHDA) and to investigate the potential molecular pathogenesis of Parkinson disease.METHODS: TUNEL staining and transmission electron microscope were applied to measure apoptosis when dopaminergic PC12 cells exposed to the excitomotors and inhibitors of PKC before 6-OHDA for 18 hours. The expression of phosphorylation of PKCδ was detected by Western blotting. RESULTS: PMA, an activating agent of PKCδ, significantly increased PC12 cell apoptosis induced by 6-OHDA. Rottlerin, an inhibitor of PKCδ, protected PC12 cells apparently. As contrast, bisindolylmaleimide I, an inhibitor of general PKC and G6976, the inhibitor of calcium-dependent PKC, did not show any protective role. CONCLUSION: The phosphorylation PKCδ is one of the important links in the process of PC12 cell apoptosis induced by 6-OHDA. PKCδ may directly participate in neurodegeneration process in parkinsonian.     

The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Effects of asymmetric dimethylarginine on apoptosis and expression of p38 mitogen-activated protein kinase in endothelial outgrowth cells

AIM: To investigate the effects of asymmetric dimethylarginine (ADMA) on apoptosis, adhesion ability and phosphorylation of p38 mitogen-activated protein kinase in endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, and induced into EOCs and then expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were treated with different concentrations of ADMA (0, 1, 5, 10, 30 μmol/L) for 48 h. Apoptotic incidences of EOCs were quantitatively determined by flow cytometry. The morphologic changes of the apoptotic cells were visualized by DAPI staining and Annexin-V/PI staining. Adhesion ability of EOCs was measured by replacing the cells on dishes and adherent cells were counted under the inverted microscopy. The p38MAPK activity was evaluated by immunoblotting technique with phospho-p38MAPK antibody. RESULTS: EOCs possessed many endothelial characteristics. Immunostaining showed that the surface antigen factor VIII, CD34 and Flk-1 were positive. The fluorescent staining revealed that EOCs were double positive for Dil-ac-LDL-uptaking and FITC-UEA-I-lectin binding. ADMA (1-30 μmol/L) induced apoptosis of EOCs in a dose-dependent manner (P<0.01). Obvious change of apoptotic morphology in EOCs incubated with ADMA was observed with DAPI staining and Annexin-V/PI staining. ADMA (5-30 μmol/L) inhibited the adhesion ability of EOCs, whereas ADMA at concentration of 1 μmol/L had no effect. ADMA (5-30 μmol/L) induced phosphorylation of p38MAPK in a dose-dependent manner (P<0.01). CONCLUSION: ADMA induces apoptosis and inhibits adhesion ability in EOCs. Activated p38MAPK might be involved in the course of apoptotic effects of ADMA.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

Preliminary study of bone marrow mesenchymal stem cell-derived exosomes in repair of spinal cord injury in rats

AIM:To study the influence of bone marrow mesenchymol stem cell-drived exosomes (BMSC-exosomes) on hindlimb activity, and the numbers of reactive astrocytes and residual neurons in spinal cord injury (SCI) rats. METHODS:BMSCs were cultured using the whole bone marrow adherent culture method and surface markers CD90 and CD34 were verified by flow cytometry. Exosomes were isolated by ultracentrifugation and the morphology of exosomes was observed under transmission electron microscope. The protein markers CD63 and CD9 were verified by Western blot. After exosomes were applied to SCI rats, the Basso, Beattie and Bresnahan locomotor rating scale score, the Nissl staining of the lesion site, and the numbers of reactive astrocytes and residual neurons were assessed at various time points. RESULTS:Transmission electron microscopic observation revealed the presence of saucer-shaped vesicles. BMSC-exosomes were found to express high levels of CD63 and CD9. Compared with injury group, significant improvement of hindlimb activity scores from day 14 after injury in treatment group was observed (P<0.05), and less reactive astrocytes and more residual neurons from day 7 after injury were also observed (P<0.05). CONCLUSION:BMSC-exosomes inhibit reactive astrocytes and death of neurons, and improve hindlimb activity in the rats after SCI.