Protective effects of icariin on human umbilical vein endothelial cell injured by angiotensin Ⅱ

To investigate the effects of icariin （ICA） on angiotensin Ⅱ（Ang Ⅱ）-induced injury in human umbilical vein endothelial cells line （ECV-304）. The ECV-304 cells were cultured in vitro. After 24 h incubating with icariin, the model of AngⅡ-induced injury in ECV-304 was established. The cell viability （MTT method）, Lactate dehydrogenase （LDH） release and Nitric oxide （NO） production in the medium, the capacity of scavenging superoxide anion radicals （O2^-） and hydroxyl radicals （.OH） were measured. The activities of superoxide dismutase （SOD）, total nitric oxide synthase （T-NOS）, inducible nitric oxide synthase （iNOS） and constitutive nitric oxide synthase （cNOS） in the cells were determined. Compared with the Ang Ⅱ-treated group, ICA can significantly raise the viability of EC, increase the activities of SOD, T-NOS and cNOS, increase the production of NO, enhance the capacity of scavenging superoxide anion radicals （ O2^- ） and hydroxyl radicals（.OH）, and lower LDH leakage and iNOS activity. The results suggest that ICA can protect endothelial cells （ECV-304） from Ang II-induced injury.

A MicroRNA Catalog of Swine Umbilical Vein Endothelial Cells Identified by Deep Sequencing

MicroRNAs （miRNAs） are endogenous -22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, little is known about miRNAs from pig. In this study, we sought to extend the repertoire of porcine small regulatory RNAs using Solexa sequencing. We sequenced a library of small RNAs prepared from immortalized swine umbilical vein endothelial cells （SUVECs）. We produced over 13.6 million short sequence reads, of which 8 547 658 perfectly mapped to the pig genome. A bioinformatics pipeline was used to identify authentic mature miRNA sequences. We identified 154 porcine miRNA genes, among which 146 were conserved across species, and 8 were pig-specific miRNA genes. The 146 miRNA genes encoded 116 conserved mature miRNAs and 66 miRNA^＊. The 8 pig-specific miRNA genes encoded 4 mature miRNAs. Four potential novel miRNAs were identified. The secondary structures of the 154 miRNA genes were predicted; 13 miRNAs have 2 structures, and miR-9 and miR-199 have 4 and 3 structures, respectively. 36 miRNAs were organized into 19 compact clusters, miR-206, miR-21 and miR-378 were the relatively highly expressed miRNAs. In conclusion, Solexa sequencing allowed the successful discovery of known and novel porcine miRNAs with high accuracy and efficiency. Furthermore, our results supply new data to the somewhat insufficient pig miRBase, and are useful for investigating features of the blood-brain barrier, vascular diseases and inflammation.

Martentoxin, a large-conductance Ca^(2+)-activated K^+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2＋-activated K＋ （BKca） channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-a in human umbilical vein endothelial cells （HU- VECs）. We found that, 1, 10 and 100 ~tmol/L martentoxin decreased nitric oxide production by HUVECs ex- posed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-a. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.

The effects of microRNA-34a regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide-induced human umbilical vein endothelial cells

BACKGROUND: Notch-1/NF-κB signaling plays a key role in the cecal ligation and puncture(CLP)-induced sepsis. This study aims to investigate the intervention effects of microRNA-34a(miR-34a) lentivirus regulating Notch-1/NF-κB signaling pathway on lipopolysaccharide(LPS)-induced human umbilical vein endothelial cells(HUVEC).METHODS: HUVEC were divided into four groups as the following: they were infected with negative control lentivirus(NC group) or miR-34a lentivirus(OE group); LPS(1 g/mL) was added on the third day on the basis of NC group and OE group for 24 hours(NC+LPS group or OE+LPS group). The levels of TNF-α, IL-1β, IL-6, and IL-10 in the cell supernatants, and the mRNA and protein expression of Notch-1 and NF-κB in the HUVEC were evaluated.RESULTS: After 24 hours, the levels of TNF-α, IL-1β, IL-6 in the cell supernatants and the protein expression of NF-κB from NC+LPS group were significantly higher than those of NC group, but IL-10 level and the protein expression of Notch-1 in NC+LPS group were the opposite. After intervention of miR-34a lentivirus, the cell supernatants TNF-α and the protein expression of NF-κB in OE+LPS group after 24 hours markedly decreased compared to NC+LPS group. While the cell supernatants IL-1β and IL-6 and the mRNA expression of NF-κB slightly decreased in OE+LPS group, IL-10 and the mRNA and protein expression of Notch-1 were the opposite.CONCLUSION: miR-34a regulating Notch-1/NF-κB signaling pathway can reduce the HUVEC damage caused by LPS stimulation.

Damaging Effect of Cigarette Smoke Extract on PrimaryCultured Human Umbilical Vein Endothelial Cells and Its Mechanism

Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Kinase domain insert containing receptor promoter controlled suicide gene system selectively kills human umbilical vein endothelial cells

AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.

Knockdown of Ezrin Suppresses the Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells In Vitro

Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells（ECs） are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin（ERM） family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells（HUVECs） in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.

Up-regulation interleukin-6 and interleukin-8 by activated protein C in lipopolysaccharide-treated human umbilical vein endothelial cells

Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.

Effect of IBD sera on expression of inducible and endothelial nitric oxide synthase in human umbilical vein endothelial cells

AIM： To study the expression of endothelial and inducible nitric oxide synthases （eNOS and iNOS） and their role in inflammatory bowel disease （IBD）. METHODS： We examined the effect of sera obtained from patients with active Crohn＇s disease （CD） and ulcerative colitis （UC） on the function and viability of human umbilical vein endothelial cells （HUVEC）. HUVECs were cultured for 0-48 h in the presence of a medium containing pooled serum of healthy controls, or serum from patients with active CD or UC. Expression of eNOS and iNOS was visualized by immunofluorescence, and quantified by the densitometry of Western blots. Proliferation activity was assessed by computerized image analyses of Ki-67 immunoreactive cells, and also tested in the presence of the NOS inhibitor, 10^-4 mol/L L-NAME. Apoptosis and necrosis was examined by the annexin-V-biotin method and by propidium iodide staining, respectively. RESULTS： In HUVEC immediately after exposure to UC, serum eNOS was markedly induced, reaching a peak at 12 h. In contrast, a decrease in eNOS was observed after incubation with CD sera and the eNOS level was minimal at 20 h compared to control （18%±16% vs 23%± 15% P〈0.01）. UC or CD serum caused a significant increase in iNOS compared to control （UC： 300%±21%; CD： 275% ± 27% vs 108% ± 14%, P〈0.01）. Apoptosis/necrosis characteristics did not differ significantly in either experiment. Increased proliferation activity was detected in the presence of CD serum or after treatment with L-NAME. Cultures showed tube-like formations after 24 h treatment with CD serum. CONCLUSION： IBD sera evoked changes in the ratio of eNOS/iNOS, whereas did not influence the viability of HUVEC. These involved down-regulation of eNOS and up-regulation of iNOS simultaneously, leading to increased proliferation activity and possibly a reduced antiinflammatory protection of endothelial cells.

Effects of Fumonisin B1 on Biomechanics and Cytoskeleton of Human Umbilical Vein Endothelial Cells

Objective Fumonisin B1(FB1)is an important mycotoxin in nature worldwide.The biomechanical properties of cells are closely related to their structure and function,and the cytoskeleton is the structural and functional basis of cells motility,and therefore,from a biomechanical point of view,the purpose of this study is to investigate the effects of FB1 on the biomechanical properties,migration capacity and cytoskeletal structure of human umbilical vein endothelial cells(HUVECs),which may lay an experimental foundation for further exploration of the toxicity mechanism of fumonisin.Methods HUVECs were cultured and treated with different concentrations of FB1.Then,CCK-8 kit was used to detect the effect of FB1 on the survival rate.The osmotic fragility of the cells was measured after treatment with different osmotic pressures for30 min.The cell membrane fluidity was measured by fluorescence polarization method.The cell electrophoretic mobility was measured by cell electrophoretic apparatus.The migration capacity of the cells was observed by scratch repair assay.The changes of reactive oxygen species and cytoskeletal structure were observed by confocal laser scanning microscopy.Finally,the mRNA and protein relative expression levels of cytoskeletal binding proteins were detected by real-time PCR,Western blotting and confocal laser scanning.Results The results of CCK-8 showed that FB1 could significantly inhibit the proliferation of HUVECs in a dose-and time-dependent manner.After treatment of HUVECs with FB1,the hypotonic resistance of the cell,cell surface charge,cell membrane fluidity and migration capacity were all weakened,while reactive oxygen species were significantly increased and the cytoskeletal structure was significantly reorganized.Furthermore,RTPCR results showed that the mRNA relative expression levels of cytoskeletal binding proteins,exception of actin,were down-regulated after treated with FB1.Besides,Western blotting and statistical analysis based on fluorescence intensity of laser confocal microscopy confirmed theses changes in protein level.Conclusions FB1 can significantly affect the biomechanical properties and motility of HUVECs,which may be directly correlated to the remodel of F-actin cytoskeleton,as well as the relative expression changes of cytoskeletal binding proteins.It is significant for further exploring the toxicity mechanism of fumonisin.

Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells

AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Effects of miR-21 antisense oligonucleotides on proliferation,migration and autophagy of human umbilical vein endothelial cells

Objective:To investigate the effects of microRNA-21 antisense nucleotide(AS-miR-21)on the proliferation,migration and autophagy of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with1,000 nmol/L rapamycin for 6 h(rapamycin group)or ASmiR-21 transfection followed by 1,000 nmol/L rapamycin for6 h(AS-miR-21+rapamycin group).HUVECs without any treatment were defined as control group.The proliferation and migration abilities of HUVECs were detected by methyl thiazolyl tetrazolium(MTT)assay,scratch wound healing assay and transwell test,respectively.The expressions of microtubule-associated protein light chain 3 Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ)and Becline-1 were determined by western blotting.Results:The rapamycin group showed decreased OD value and migration rate,an increased ratio of LC3 Ⅱ/Ⅰ and up-regulated expression of Beclin-1 compared with the control group(P<0.05).The AS-miR-21+rapamycin group demonstrated lower OD value,migration rate,the number of migrated cells,and significantly higher ratio of LC3 Ⅱ/Ⅰ and Beclin-1 protein expression level than the control group and the rapamycin group(P<0.05).Conclusion:AS-miR-21 suppressed the autophagy,proliferation and migration in the HUVECs model of autophagy induced by rapamycin.

The Effects and Mechanism of GSA on Expression of MCP-1 in Cultured Human Umbilical Vein Endothelial Cells

Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.

通过调控VEGF信号通路探讨补肾通络方对子宫内膜损伤后HUVECs促增殖、血管生成和迁移作用

目的探析补肾通络方对子宫内膜损伤后人脐静脉内皮细胞模型(human umbilical vein endothelial cells,HUVECs)促增殖、血管生成和迁移作用,及对调控血管内皮生长因子(vascular endothelial growth factor,VEGF)信号通路的影响。方法选择2020年1月—2021年12月,购自武汉大学典型培养物保藏中心的12份HUVECs,给予50 ng/mL VEGF处理后,分别实施20%、10%、5%补肾通络方含药血清和无补肾通络方含药血清干预处理。比较补肾通络方对子宫内膜损伤后,VEGF信号通路的影响及对HUVECs增殖、迁移、血管生成的影响。结果比较20%、10%、5%补肾通络方含药血清和无补肾通络方含药血清干预后24 h的HUVECs活性和24 h细胞生长检测所得OD值,差异无统计学意义(P>0.05);20%补肾通络方含药血清组48 h、72 h的HUVECs活性、细胞生长检测所得OD值及VEGF表达量、HUVECs细胞迁移数均高于10%、5%补肾通络方含药血清组和无补肾通络方血清组,差异有统计学意义(P<0.05)。20%补肾通络方含药血清干预组的血管网状结构完整度优于10%、5%补肾通络方含药血清和无补肾通络方血清,无补肾通络方血清干预组的血管网状结构完整度最差,补肾通络方可逆转VEGF抑制细胞活性的趋势,促进HUVECs的血管形成。结论将补肾通络方应用到子宫内膜损伤中,可逆转VEGF抑制细胞活性的趋势,增强HUVECs的细胞活性,促进细胞增殖、迁移,促进血管新生,促进内皮细胞网络结构的重建,利于促进内皮细胞损伤的修复,利于子宫内膜功能的恢复,且与补肾通络方含药血清浓度呈一定量效关系。

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Expression of Soluble Vascular Endothelial Growth Factor Receptor-2 and Its Effect on Proliferation of Vascular Endothelial Cells

Vascular endothelial growth factors（VEGFs） respectively bind to each of three receptor tyrosine kinases（RTKs）,known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases（VEGFRs） are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor（sKDR） on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells（HUVEC）,and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-（4,5-dimethylthiazol-2-yl）2,5-diphenyltetrazolium bromide]（MTT）,and on angiogenesis by chick chorioallantoic membrane（CAM） experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction（RT-PCR）,and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.

苜蓿素对LPS诱导HUVECs炎症反应的消除作用

【目的】研究苜蓿素对脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)炎症损伤的保护作用。【方法】以HUVECs为研究对象,用MTT法确定苜蓿素的处理浓度,用酶联免疫分析法确定LPS建模浓度以及检测HUVECs炎症相关因子(TNF-α、iNOS、COX-2、IL-1、IL-6)表达,用Western-blot法测定NF-κB和MAPK信号通路蛋白(β-Actin、P65、IKB、P-P65、P-IKB、P38、JNK、ERK1/2、P-P38、P-JNK、P-ERK1/2)表达。【结果】不同浓度的LPS均能促进TNF-α的表达(P<0.01),其中LPS浓度在2.5~7.5μg/mL时TNF-α的表达量最高,选用2.5μg/mL作为建模浓度,在该浓度下LPS促进炎症相关因子和炎症相关通路蛋白表达(P<0.01),而作为阳性对照辛伐他汀能抑制LPS诱导的炎症相关因子和炎症相关通路蛋白表达(P<0.01);苜蓿素浓度在3μg/mL之内对HUVECs活性无显著差异,因而选用3、2、1μg/mL作为苜蓿素高、中、低剂量组;苜蓿素高、中、低剂量组均能下调LPS诱导的炎症相关因子和炎症相关通路蛋白表达(P<0.01),而且苜蓿素对炎症相关因子的抑制效果弱于辛伐他汀,而对炎症相关通路蛋白的抑制效果强于辛伐他汀,各炎症相关因子表达随苜蓿素浓度升高而降低(P<0.01),炎症相关通路蛋白表达情况也有这个下降趋势。【结论】苜蓿素能够通过抑制NF-κB和MAPK信号通路消除LPS对HUVECs诱导的炎症损伤。