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.

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.

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.

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-CdglyTK, 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 purifi ed. 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 ? uorescent protein (GFP) expression. Infected cells were cultured in culture media containing different concentrations of 5-? uorocytosine (5-FC) and ganciclovir (GCV), and the killing effects were measured. RESULTS: The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfullyconstructed 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 AdCMV-CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but signifi cantly 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. ? 2006 The WJG Press. All rights reserved.

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

瞄准：在煽动性的肠疾病(IBD ) 学习内皮和可诱导的氮的氧化物 synthases (eNOS 和 i NOS ) 和他们的角色的表示。方法：我们检验了重量的单位的效果一在人的脐的静脉 endothelial (HUVEC ) 的功能和生存能力上与活跃 Crohn 的疾病(CD ) 和 ulcerative (UC ) 从病人获得了。HUVEC 面对与活跃 CD 或 UC 从病人包含健康控制，或浆液的分享的浆液的媒介为 0-48 h 是有教养的。eNOS 和 i NOS 的表示被免疫荧光设想，并且由西方的污点的测密度术确定了。增长活动被 Ki-67 免疫的计算机化的图象分析估计反应房间，并且也面对 NOS 禁止者测试了， 10 (-4) mol/L L 名字。Apoptosis 和坏死被 annexin-V-biotin 方法并且由分别地染色的 propidium 碘化物检验。结果：在 HUVEC 立即在到 UC 的暴露以后，浆液 eNOS 显著地被导致，在 12 h 到达一座山峰。相反，在 eNOS 的减少与 CD 重量的单位在孵化以后被观察一 eNOS 铺平的 and 在与控制(18%+/-16% 对 23%+/-15% P【0.01 ) 相比的 20 h 是最小的。UC 或 CD 浆液与控制相比在 i NOS 引起了重要增加(UC：300%+/-21% ；CD：275%+/-27% 对 108%+/-14% ， P【0.01 ) 。Apoptosis/necrosis 特征没在任何一个实验显著地不同。增加的增长活动与 L 名字面对 CD 浆液或术后疗法被检测。文化与 CD 浆液在 24 h 处理以后显示出像试管的形成。结论：IBD 重量的单位一在 eNOS/iNOS 的比率的唤起的变化，而没影响 HUVEC 的生存能力。这些同时包含了 eNOS 的下面规定和 i NOS 的起来规定，导致增加的增长活动并且可能 endothelial 的减少的反煽动性的保护。

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.

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.

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.

Vein transplantation using human umbilical cord blood stem cells in the treatment of stroke sequela

BACKGROUND: Transplanted mononuclear cell (MNC) of umbilical blood can survive in central nervous system (CNS) of host through blood brain barrier, differentiate into nerve cells, migrate to damaged site and integrate morphological structure and function with nerve cells of host so as to improve deficiencies of sensatory function, motor function and cognitive function and influence on stroke sequela. OBJECTIVE: To observe the vein transplantation of human umbilical cord blood stem cells (HUCBSC) for improving neurological function, limb function and activity of daily living of patients with stroke and evaluate the reliability. DESIGN: Self-controlled study. SETTING: Department of Neurosurgery, the Second People's Hospital of Zhengzhou City; Red-crossed Blood Center of Henan Province; Department of Neurosurgery, the Fist Affiliated Hospital of Zhengzhou University. PARTICIPANTS: A total of 10 patients with stroke sequela were selected from Department of Cerebral Surgery, the Second People's Hospital of Zhengzhou City from April to December 2005. There were 9 males and 1 female aged from 35 to 75 years with the mean age of 56 years. All of them were diagnosed with CT and MRI examination and coincidence with diagnostic criteria of stroke established by the Fourth National Academic Meeting for Cerebrovascular Disease. All patients provided informed consent. METHODS: 80-140 mL umbilical blood of term birth of newborn was selected hermetically and maintained in sterile plastic bag. And then, the blood was centrifugated at the speed of 1 500 r/min for 30 minutes at 22 ℃ in order to separate MNC, i.e., HUCBSC. In addition, after final diagnosis during hospitalization, stroke patients were perfused with HUCBSC through superficial vein of back of the hand. Each patient was averagely perfused with 6 portions of HUCBSC (cellular numbers ≥ 1×108/portion) and the interval between each portion was 1-7 days with the mean interval of 4 days. MAIN OUTCOME MEASURES: ① Neurological function of stroke patients was evaluated with neurological function deficiency (NFD) before treatment and at 3 months after treatment. The scale includes consciousness, level fix function, facial paralysis, language, muscle force of upper limbs, muscle force of lower limb and step function. The total scores ranged from 0 to 45; meanwhile, the lower the scores were, the better the neurological function was. ② Motor function of injured limbs was evaluated with Fugl-Meyer Assessment (FMA), including motor function of upper limbs, motor function of lower limbs, balance ability, sensory function and motion of joint. The total scores ranged from 0 to 226; meanwhile, the higher the scores were, the better the motor function of limbs was. ③ Activities of daily living (ADL) was evaluated with Barthel Index (BI), including having meals, taking a bath, dressing oneself, putting on clothes, walking in balance and stair activity. The total scores ranged from 0 to 100; meanwhile, the higher the scores were, the stronger the ADL was. RESULTS: A total of 10 patients were involved in the final analysis. After treatment, NFD of stroke patients was (10.9±5.09) points, which was lower than that before treatment [(25.4±6.09) points, t =8.213, P < 0.01]. In addition, after treatment, FMA and BI of stroke patients were (80.9±25.00) points and (81.1±15.93) points, respectively, which were higher than those before treatment [(31.9±21.85) points, (36.2±19.41) points, t =13.024, 13.670, P < 0.01]. Immuno-suppressive drugs were not used during the whole therapeutic procedure; moreover, immunological rejection and allergic reaction were not observed during the same period. CONCLUSION: Transplanting HUCBSC through superficial vein of back of the hand is regarded as a simple and safe method for the treatment of stroke sequela.

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。

Transplantation of human umbilical cord blood mesenchymal stem cells to treat a rat model of traumatic brain injury

In the present study, human umbilical cord blood mesenchymal stem cells were injected into a rat model of traumatic brain injury via the tail vein. Results showed that 5-bromodeoxyuridine-labeled cells aggregated around the injury site, surviving up to 4 weeks post-transplantation. In addition, transplantation-related death did not occur, and neurological functions significantly improved. Histological detection revealed attenuated pathological injury in rat brain tissues following human umbilical cord blood mesenchymal stem cell transplantation. In addition, the number of apoptotic cells decreased. Immunohistochemistry and in situ hybridization showed increased expression of brain-derived neurotrophic factor, nerve growth factor, basic fibroblast growth factor, and vascular endothelial growth factor, along with increased microvessel density in surrounding areas of brain injury. Results demonstrated migration of transplanted human umbilical cord blood mesenchymal stem cells into the lesioned boundary zone of rats, as well as increased angiogenesis and expression of related neurotrophic factors in the lesioned boundary zone.

大蒜素改善PM_(2.5)暴露诱导的HUVEC自噬损伤

目的研究大蒜素改善细颗粒物(PM_(2.5))暴露损伤人脐静脉内皮细胞(HUVEC)自噬的机制。方法将HUVEC分为对照组、PM_(2.5)组(5 mg/L)、大蒜素组(10 mg/L)和联合组(10 mg/L大蒜素与5 mg/L PM_(2.5))。采用透射电镜观察各组自噬小体数;采用细胞免疫荧光、免疫印迹方法检测LC3、p62针点数和p62、p38、Beclin 1、LC3BⅡ/Ⅰ蛋白表达及p38蛋白的磷酸化水平。结果与对照组比较,PM_(2.5)组细胞活力明显降低,自噬小体数增加,Beclin 1、LC3BⅡ/Ⅰ和p62蛋白表达升高(P<0.05)。与PM_(2.5)组比较,联合组细胞活力升高,自噬小体数减少,Beclin 1、LC3BⅡ/Ⅰ和p62蛋白表达及p38蛋白磷酸化水平降低(P<0.05)。结论大蒜素改善PM_(2.5)对HUVEC的损伤,可能与抑制p38蛋白磷酸化,促进PM_(2.5)抑制的自噬流有关。

3D生物打印构建HGFs和HUVECs共培养体系促进HUVECs成血管

目的:构建人牙龈成纤维细胞(human gingival fibroblasts,HGFs)和人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)共培养体系,促进HUVECs成血管。方法:取HGFs和HUVECs培养传代至3~5代进行实验。通过慢病毒转染用红色荧光蛋白标记HUVECs。构建HGFs和HUVECs二维共培养体系,将HGFs与HUVECs以不同比例(4∶1、1∶1、1∶4)共培养,荧光显微镜下观察血管网络形成情况。配制5%甲基丙烯酰化明胶(GelMA)30胶,构建HGFs和HUVECs三维共培养体系,激光共聚焦显微镜下观察血管网络的形成情况。对HUVECs形成的血管网络进行定量分析。将HUVECs从三维共培养系统中分离出来,评估单独培养和共培养一定时间后HUVECs的增殖、迁移和小管形成效果以及相关血管生成基因的表达水平。结果:二维共培养时,当HGFs和HUVECs以比例1∶1共培养时血管网络形成的效果最佳。三维共培养时,HGFs可以促进HUVECs血管生成。HGFs和HUVECs共培养组的增殖、迁移和小管形成效果更好,相关血管生成基因的表达水平远远高于HUVECs单独三维培养组。结论:HGFs和HUVECs共培养能够引导和促进HUVECs出芽及迁移。