Objective To study the biological behavior of osteoblast and vascular endothelial cell culture. Methods The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortox of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C(co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibilitywere observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase(ALP) activity were measured. Results The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells .The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B(P＜0.01), and it was higher in group A than in group B(P＜0.05). In group C, the cellproliferation were increased slowly early, but fast later. Conclusion Thecellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity,and the combined cultured cells have greater proliferation ability.
Objective To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92(NK-92). Methods By use of liposomes pack, the pcDNA3.0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by51Cr-release assays. Results HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein andRNA. It also could be found that the survival rate of transfected PECs was muchhigher than that of non-transfected PECs, when both of them faced the lysismediated by human PBMC and NK-92.After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (Plt;0.05). Conclusion The PECs- transfected by HLAG1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.
Coronary atherosclerotic heart disease is a serious threat to human life and health. In recent years, the main treatment for it is to implant the intravascular stent into the lesion to support blood vessels and reconstruct blood supply. However, a large number of experimental results showed that mechanical injury and anti-proliferative drugs caused great damage after stent implantation, and increased in-stent restenosis and late thrombosis risk. Thus, maintaining the integrity and normal function of the endothelium can significantly reduce the rate of thrombosis and restenosis. Stem cell mobilization, homing, differentiation and proliferation are the main mechanisms of endothelial repair after vascular stent implantation. Vascular factor and mechanical microenvironmental changes in implanted sites have a certain effect on re-endothelialization. In this paper, the process of injury caused by stent implantation, the repair mechanism after injury and its influencing factors are expounded in detail. And repairing strategies are analyzed and summarized. This review provides a reference for overcoming the in-stent restenosis, endothelialization delay and late thrombosis during the interventional treatment, as well as for designing drug-eluting and biodegradation stents.
ObjectiveTo observe the effects of four prostaglandin E2 (PGE2) receptors (EP1-4R) on the activation of inflammasomes and cell damage in human retinal microvascular endothelial cells (hRMEC) in a high glucose environment.MethodsThe hRMEC were divided into normal group and high glucose group, and they were cultured in Dulbecco modified Eagle medium containing 5.5 and 30.0 mmol/L glucose, respectively. Flow cytometry was used to observe the apoptosis rate of the high glucose group and the normal group; enzyme chain immunosorbent assay (ELISA) was used to detect the level of PGE2 in the culture supernatant of hRMEC cells. Western blot was used to detect the protein expression of cyclooxyganese (COX2) and EP1-4R in hRMEC. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of EP1-4R mRNA in hRMEC. After 72 h of culture, the cells in the high glucose group were divided into control group, PGE2 group, EP1-4R agonist group, PGE2+EP1-4R inhibitor group, and dimethylsulfoxide group. According to the group, each group was given the corresponding agonist or inhibitor to continue the culture for 24 h. QRT-PCR was used to detect the expression of nucleotide-binding oligomerization structure-like receptor protein (NLRP3) and pro-interleukin (IL)-1β mRNA in each group of cells. ELISA was used to detect the content of IL-1β and lactic dehydrogenase (LDH) in the cell culture supernatant. Western blot was used to detect the expression of cleaved Caspase-1 in each group of cells. At the same time, hRMEC in a high glucose environment was given IL-1β stimulation for 24 h, and the activity of LDH in the supernatant of the cell culture medium was detected.ResultsThe apoptotic rate, COX2 protein expression, and PGE2 protein content in hRMEC in the high glucose group were significantly higher than those in the normal group, and they were time-dependent. Compared with the normal group, the expression levels of EP1R, EP2R, EP4R protein and mRNA in hRMEC in the high glucose group were higher than those in the normal group (P＜0.05). Compared with the control group, PGE2 group (t=4.627, P＜0.01), EP1-4R agonist group (t=3.889, 3.583, 2.445, 3.216; P＜0.05) hRMEC NLRP3 mRNA expression level was significantly increased; the expression level of pro-IL-1β mRNA increased, however the difference was not statistically significant (PGE2 group: t=1.807, P＞0.05; EP1-4R agonist group: t=1.807, 1.477, 0.302, 1.926, P＞0.05). Compared with the PGE2 group, the expression of NLRP3 mRNA in hRMEC in the PGE2+EP2R inhibitor group was significantly reduced (t=2.812, P＜0.05); the expression of pro-IL-1β mRNA in hRMEC in the PGE2+EP3R inhibitor group was significantly increased (t=4.113, P＜0.01). The protein content of IL-1β in the cell culture supernatant of the PGE2 group, EP1R agonist group and EP2R agonist group was significantly higher than that of the control group (t=5.155, 4.136, 4.817; P＜0.01). Compared with PGE2 group, the protein content of IL-1β in the cell culture supernatant of the PGE2+EP2R inhibitor group and the PGE2+EP4R inhibitor group were significantly lower than that of the PGE2 group (t=1.964, 4.765; P＜0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2 group and EP2R agonist group was significantly higher than that in the control group (t=5.332, 4.889; P＜0.05). The expression of cleaved Caspase-1 in hRMEC in the PGE2+EP2R inhibitor group was significantly lower than that of the PGE2 group (t=6.699, P＜0.01). The LDH activity in the cell culture supernatant of the PGE2 group and the EP2R agonist group was significantly higher than that of the control group (t=4.908, 4.225; P＜0.05). The activity of LDH in the cell culture supernatant of the PGE2+EP2R inhibitor group was significantly lower than that of the PGE2 group (t=5.301, P＜0.01). Compared with the control group, the LDH activity in the culture supernatant of hRMEC cells in the high glucose environment was significantly increased (t=3.499, P＜0.05).ConclusionsThe four receptors of PGE2 can activate NLRP3 and its effector molecules to varying degrees. EP2R mainly mediates hRMEC damage under high glucose environment.
【摘要】 目的 通过比较两种原代人脐静脉内皮细胞的分离培养方法并对细胞特异性抗原进行鉴定,探索提高原代内皮细胞体外培养存活率及纯化率的方法。 方法 采用一次性无菌注射器向人脐静脉灌注消化液,消化液的浓度和消化时间分别025%(质量体积比)胰蛋白酶,10 min和01%(质量体积比)胶原酶Ⅱ,15 min。通过在倒置显微镜下观察细胞的形态特点和用免疫荧光染色的方法对细胞进行鉴定,比较两种消化方法的优劣。 结果 01%胶原酶Ⅱ,15 min的消化方法较025%胰蛋白酶,10 min对原代人脐静脉内皮细胞有更好的分离效果,活细胞数量多且细胞纯度较高。免疫荧光染色结果表明细胞内有Ⅷ因子相关抗原表达。结论 胶原酶Ⅱ可以有效分离脐静脉内皮细胞,最佳消化条件是01%胶原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
ObjectiveTo observe the effect of interleukin-8 (IL-8) on the adhesion and migration of retinal vascular endothelial cells (RCEC). MethodsA cell experiment. Human RCEC (hRCEC) was divided into normal control group (N group), advanced glycation end product (AGE) treatment group (AGE group), and AGE-induced combined IL-8 antagonist SB225002 treatment group (AGE+SB group). The effect of AGE on IL-8 expression in hRCEC was observed by Western blot. The effect of SB225002 on hRCEC migration was observed by cell scratch assay. The effects of SB225002 on leukocyte adhesion and reactive oxygen species (ROS) on hRCEC were detected by flow cytometry. Student-t test was performed between the two groups. One-way analysis of variance was performed among the three groups. ResultsCompared with group N, the expression level of IL-8 in cells of AGE group was significantly increased, with statistical significance (t=25.661, P＜0.001). Compared with N group and AGE+SB group, cell mobility in AGE group was significantly increased (F=29.776), leukocyte adhesion number was significantly increased (F=38.159, 38.556), ROS expression level was significantly increased (F=22.336), and the differences were statistically significant (P＜0.05). ConclusionIL-8 antagonist SB225002 may down-regulate hRCEC adhesion and migration by inhibiting ROS expression.
ObjectiveTo observe the changes of follistatin-like protein 1 (FSTL1) in serum of patients with proliferative diabetic retinopathy (PDR).MethodsTwenty PDR patients confirmed by clinical examination and 20 normal people were included in the study. Human retinal vascular endothelial cells (HRCEC) were divided into HRCEC blank control group, 3 h hypoxia group, 6 h hypoxia group. Human umbilical vein endothelial cell (HUVEC) were divided into HUVEC blank control group, 3h hypoxia group, 6h hypoxia group. Real-time quantitative PCR (RT-PCR) and ELISA were used to determine the expression of FSTL1, TGF-β, VEGF, connective tissue growth factor (CTGF) mRNA and protein in peripheral blood and cells of all groups from all subjects.ResultsThe expressions of FSTL1, TGF-β1, CTGF, VEGF mRNA in blood samples of patients with PDR were 1.79±0.58, 0.97±0.21, 1.85±0.69 and 1.38±0.44. The expressions of FSTL1, TGF-β1 protein were 1.19±0.50, 0.71±0.24 ng/ml and 734.03±116.45, 649.36±44.23 ng/L. Compared with normal people, the differences were statistically significant (tmRNA=0.90, 0.21, 2.85, 1.77; P=0.00, 0.00, 0.04, 0.02. tprotein=1.88, 7.68; P=0.00, 0.02). The cell viability of HRCEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.66±0.05 and 0.64±0.04, respectively. Compared with the blank control group, the difference was statistically significant (F=13.02, P=0.00). The cell viability of HUVEC cells in the 3 h hypoxia group and the 6 h hypoxia group were 0.63±0.06 and 0.68±0.06, respectively. Compared with the blank control group, the difference was statistically significant (F=26.52, P=0.00). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HRCEC blank control group and 3 h hypoxia group, the differences were statistically significant (F=14.75, 44.93, 85.54, 6.23; P=0.01, 0.00, 0.00, 0.03). Compared with the HRCEC blank control and 3 h hypoxia group, the expressions of FSTL1 and TGF-β1 protein were statistically significant (P＜0.05). There was a statistically significant difference in TGF-β1 protein expression in the hypoxic 6 h group (P=0.03) and no significant difference in FSTL1 protein expression (P=0.68). Comparison of FSTL1, TGF-β1, CTGF, and VEGF mRNA expression in HUVEC blank control group and 3h hypoxia group, the differences were statistically significant (F=19.08, 25.12, 22.89, 13.07; P=0.00, 0.00, 0.00, 0.01). Immunofluorescence staining results showed that FSTL1, TGF-β1, CTGF, and VEGF proteins were positively expressed in cells in the 3h hypoxia and 6h hypoxia groups.ConclusionThe expression of FSTL1 gene and protein in serum of PDR patients was significantly higher than that of normal people.
ObjectiveTo explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.MethodsRabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression.ResultsHDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a " honeycomb” network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant (P>0.05).ConclusionThree-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.
Objective To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism. MethodsThe HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. ResultsThe number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups (t=12.311, 15.643, 17.346; P＜0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups (t=16.311, 14.833, 18.442; P＜0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences (t=17.342, 16.813, 18.794; P＜0.001). ConclusionPSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.
Objective To investigate the expression of transcription factors including nuclear factor-κB （NF-κB） and activator protein-1 （AP-1） in vascular endothelial cells （ECs） in different flow fields， and provide experimental evidence for mechanical signal effects on gene regulation pattern of ECs. Methods Cultured human umbilical vein ECs were loaded into steady flow chambers of laminar flow or turbulent flow and observed at 6 time points （0.5 h， 1 h， 2 h， 3 h， 4 h and 5 h） based on different load time. Spacial and temporal characteristics of NF-κB and AP-1 expression in ECs in different flow chambers were detected at a protein level by laser confocal microscope. Results In laminar flow， NF-κB expression rose to peak at 1 hour （26.49±1.63， P＜0.05）and then declined. In turbulent flow， NF-κB expression rose to peak at 3 hours （34.41±6.43， P＜0.05）. In laminar flow， c-Jun/AP-1 expression was transiently elevated， reached its peak at 0.5 hour （18.95±5.38，P＜0.05）and then fell to its baseline level. In turbulent flow， c-Jun/AP-1 expression rose slowly but steady to peak（P＜0.05） . Conclusion The effects of turbulent flow on NF-κB and AP-1 expression in ECs are different from those of laminar flow. Up-regulation and activation of NF-κB and AP-1 expression in ECs induced by turbulent flow may cause pathological changes in morphological structure and functional behavior of ECs.