Objective To clarify that the vascular endothelial cell injury caused by obstructive sleep apnoea hypopnea syndrome (OSAHS) is partly mediated by miRNA-92a. Methods Serum miRNA-92a level was measured in patients who underwent polysomnography between January 2018 and December 2018. The correlation between miRNA-92a and OSAHS was analyzed. Meanwhile, endothelial cells were cultured in vitro, and morphological changes and JC-1 staining results of endothelial cells were observed after OSAHS serum stimulation, so as to further clarify the injury of endothelial cells. The changes of miRNA-92a target gene were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to further clarify the mechanism of endothelial cell injury. Results Seventy-two patients received polysomnography, including 22 cases in the non-OSAHS group, 18 in the mild OSAHS group, 10 in the moderate OSAHS group, and 22 in the severe OSAHS group. Serum miRNA-92a level was significantly increased in the OSAHS patients, and it also increased with the aggravation of OSAHS severity. OSAHS serum significantly damaged endothelial cells. Endothelial cells were swollen, disordered arrangement, and unclear boundaries. JC-1 staining showed that green fluorescence was significantly enhanced compared with the control group. RT-PCR and Western blot showed that the expressions of Krüppel-like factor-2 (KLF-2), Krüppel-like factor-4 (KLF-4) and endothelial nitric oxide synthase (eNOS) were significantly decreased under OSAHS serum stimulation. Conclusion Serum miRNA-92a of OSAHS patients is significantly increased, and reduces the expression of target genes KLF-2, KLF-4 and eNOS, affects the mitochondrial function of endothelial cells, and injures endothelial cells.
Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
The autograft and non-autograft cannot meet the needs of clinical vascular surgery. Since there are possibilities of thrombus formation in artificial vascular grafts, the methods for deposing the graft using physical and chemical ways or simply seeding with endothelial cells cannot produce satisfactory grafts for vascular operations until now. In order to increase the anticoagulative capacity of artificial vascular graft, it is rational to use genetic engineering methods modifying the endothelial cells to make it express anticoagulative factors stably. Although seeding artificial graft with the genetically engineered endothelial cells can possibly produce a satisfactory graft for vascular surgery, some problems still need to be solved.
Objective To explore an optional condition to induce mouse embryonic stem cell(ESC) to differentiate into endothelial cells so as to provide seedcells for tissue engineered vascular. Methods The embryos from one pregnant 12.5days mouse was harvested to culture the mouse embryonic fibroblasts(MEF). The ESC was reanimated by common method, and used to cultured into embryoid body(EB) in vitro. The EB which was used to induce into endothelial cells was divided into two groups. The EB was cultured in the EB medium with 3ng/ml transforming growth factor β1, 50 ng/ml vascular endothelial cell growth factor and 1 μmol/L potent and selective inhibitor of activin receptorlike kinase receptors in experimental group. The EB was cultured in the EB medium in the control group. After 14 days, RTPCR and immunohistochemistry were used to detect vWF and CD34, to analyze the morphology and type of the differentiated cells fromESC. Results The primary MEF had a high proliferation activity. At the 3rdday, the fusion rate of MEF was about 90% with a fusiform shape. The cells was fusiform shape and arranged compactly with fullness of nucleus and 2-3 entoblasts. The 3rd5th generations EB was polygonal with fullness of cytoplasm and 3-4 entoblasts. ESC could maintain undifferentiated state, and the cells unit lookedlike bird nest with smooth margin; the cells was small at size and b refractivity with high rate of nuclein and rapid proliferation. At 3 days of dropculture, EB can seen grossly and at 3 days of suspension, large and transparent EBformed. EB was spread radiately with an intensive adhesion at the 2nd day. In experimental group, many round cells was differentiated around EB from the 4thday to the 7th day, and form tubular structures from the 10th day to the 14th day. The vWF and CD34 were expressed. In control group, EB could not form tubularstructures, and the vWF and CD34 were not expressed. Conclusion ESC can differentiate into endothelial cells under some conditions, and form vessellike structure under condition culture, which can provide sources of seed cells for tissue engineered vessel.
ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
Objective To study the feasibility of transplanting autologous venous endothelial cells, as the liner, to the allogenic vein and to investigate the patency rate after such transplantation. Methods Autologous endothelial cells were gained after the administration of 0.2% collagenase and the centrifugalization of the enzyme liquid. The cells were not cultivated in a 60 ml plastic culture until the presence of the second generation. The cultivated cells were confirmed as endothelial cells by factor Ⅷ related antigen. The multiplied cells were lined in vitro onto the luminal surface of allogenic vein that was disposed by freeze-drying and radiation. The orthotopic transplantation of autologous venous endothelial cells was performed after the 9-day incubation. Results (9.47±0.35)×106 endothelial cells were obtained after the cultivation. Three hours after cell seeding, the luminal surface of allogenic vein was covered with vast endothelial cells but still had not formed an intact endomembrane. On day 9, the luminal surface was covered with a continuous endothelial monolayer and the arrangement and the shape of the cells all showed the perfect condition of endothelial cells. Eight weeks later, all the transplanted veins kept unobstructed. Conclusion The approach of lining allogenic vein with autologous endothelial cells in vitro may keep the vein unobstructed in the long term.
Objective To observe the vascular endthelial cellular apoptosis induced by transpupillary thermotherapy (TTT). Methods Vascular endothelial cells (VEC) cultured in vitro were treated with TTT, hyperthermia and TTT combined with indocyanine green (ICG) pretreatment. The cellular apoptosis was detected by doublelabelled flow cytometer (annexin Vfluroescein isothiocyanate and propidium iodide) analysis, fluorescent microscopy, nucleolus stainned with DNA dye hoechst 33258, DNA ladder detection and electron microscopy. Results Without significant rising of the temperature, TTT couldnprime;t increase the apoptosis of VEC. Pure hyperthermia and TTT combined with ICG pretreatment could increase apoptosis of VEC significantly, and the effect of the latter method was more obvious. The higher power of TTT was used and the longer duration the cells were cultured, the higher apoptosis rate of VEC was. Conclusion The induction of apoptosis of VEC might play an important role in the mechanism of the occlusion of CNV by TTT, and combining with ICG may obviously enhance the apoptosis rate at the same temperature, which may supply a theoretical basis for promoting the clinical effect of TTT.
OBJECTIVE: To explore an ideal way of small vessel anastomosis for microsurgery. METHODS: Anastomosis of both carotid arteries were performed in 20 rabbits. One side of the arteries were anastomosed with anastomotic clips, the other side of the arteries, as comparison, were anastomosed with suture. The vessels were harvested at first and 14th day after operation and were evaluated using operating microscope, light microscope and electronic microscope. RESULTS: The average anastomotic time for suture was about 15 minutes, while for the clips was 2 to 5 minutes. There were no difference in patency between the two techniques. Endothelialization at the anastomotic sites were both completed 14 days postoperatively. However, for the anastomotic clips, there were no endothelia damage and foreign bodies formation inside the vessels. CONCLUSION: This experiment has confirmed that the anastomotic clip’s procedure provides a very safe and easy way to perform anastomosis and reduce the incidence of thromboses.
Objective To investigate the effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.