OBJECTIVE: To elongate the proliferation life-span of human umbilicus vein endothelial cell (HUVEC). METHODS: We synthesized the human telomerase reverse transcriptase mRNA (hTERT mRNA) by in vitro transcription, then transferred the hTERT mRNA into HUVEC in quicent stage by lipofect introduction. RESULTS: Telomerase expressed transiently in HUVEC, and the cell life-span was elongated for 7 population doublings. CONCLUSION: Telomerase can be reconstructed controllably and transiently in HUVEC by hTERT mRNA introduction, this method has the potential to be used to elongate the lifespan of cells cultured in vitro.
OBJECTIVE To compare the permeability and incidence rate of complication of arteriovenous internal fistula made by autogenous, homologous, and artificial Teflon blood vessels. METHODS Two hundred and forty one cases with arteriovenous internal fistula made by autogenous, homologous, and artificial Teflon blood vessels were followed up to compare the permeability and incidence rate of complication at 6 months, 1 year, 3 years, and 5 years. RESULTS The incidence rate of complication of autogenous blood vessels was lowest, it had no statistical differences compared with arteriovenous internal fistula made by homologous blood vessels. The permeability of arteriovenous internal fistula made by homologous blood vessels was highest, and it had no statistical differences compared with autogenous blood vessels. The permeability of arteriovenous internal fistula made by artificial Teflon blood vessels was lowest, but the incidence rate of complication was highest, and it had significantly statistical differences compared with arteriovenous internal fistula made by autogenous blood vessels (P lt; 0.01). CONCLUSION Arteriovenous internal fistulas made by autogenous and homologous blood vessels have high permeability and low incidence rate of complication, they are superior to the arteriovenous internal fistula made by artificial Teflon blood vessels.
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.
Objective To investigate the biological response and chemotaxis of endothel ial cells on template materials with different protein concentrations on the same surface, to provide the evidence for deep understanding of chemical induced cell motil ity. Methods Microcontact printing technique was employed to fabricate template materials with four different concentrations of collagen (50, 100, 200, 300 μg/mL) on the same substrate. Scanning electron microscopy was employed to characterize the qual ity of polydimethylsiloxane (PDMS) stamp. Confocal laser scanning microscopy (CLSM) was util ized to characterize the absorption of different concentrations of FITC conjugated collagen (50, 100, 200, 300 μg/mL) on the substrates surfaces. Software was used to analyze the fluorescence intensity of adsorbed protein on the substrates. Albumin was then used to block the substrates for cell culture of human umbil ical vein endothel ial cells (hUVEC). Substrates with no collagen adsorption were used as control samples. The influence of different concentrations of collagen on the prol iferation of hUVEC was investigated via MTT assay at 6, 24, 48 and 72 hours of culture. The cytoskeletal structures of cells were characterized by CLSM. The cell’ s migration speed and absolute displacement were measured by path measurement of single cell after 24 hours of culture. Results Fabricated PDMS stamps with complete pattern were flat. Template substrates were fully covered with evenly distributed collagen protein. The fluorescence intensities were 38.51 ± 1.63, 55.21 ± 3.88, 73.17 ± 3.59, and 80.95 ± 1.12 in adsorbed FTIC conjugated collagen with 50, 100, 200 and 300 μg/mL, respectively. Endothel ial cells spread better on various substrates coated with collagen than those of control samples. The prol iferation of endothel ial cells on collagen coated substrateswas significantly higher than that of control group (P lt; 0.05). With collagen concentration increasing from 50 µg/mL to 300µg/mL, the prol iferation abil ities and absolute displacements of endothel ial cells significantly increased (P lt; 0.05). Except for the group with 300 μg/mL, the migration speed of endothel ial cells on collagen coated substrates was significantly lower (P lt; 0.05) than that of control group. However, the migration speed of endothel ial cells on collagen coated substrates significantly increased (P lt; 0.05) along with collagen concentration increasing from 50 µg/mL to 300 µg/mL. Conclusion It is feasible to acquire domains with different protein concentrations on the same substrate using microcontact printing technique for investigating cell’s chemotaxis.
ObjectiveTo explore the value of ultrasound evaluation and marking before arteriovenous internal fistula in end-stage renal disease hemodialysis patients. MethodsTwenty-five uremia end-stage patients were admitted into our nephrology department from January 2012 to July 2012. All of the patients had encountered several times of fistula failure or had difficulty in establishing the forearm arteriovenous fistula. We focused on observing the brachial artery, radial artery, cephalic vein, the basilica vein and great saphenous vein. We measured the diameter of the vessels and marked the trend of arteries and veins in the body surface under the ultrasonic navigation. Our goal was to look for appropriate bypass vessels in the elbow and the upper arm. ResultsFourteen patients had endured several times of fistula failure. Among the 14 patients, 9 patients completed the surgery of reengineering fistula and autogenous great saphenous vein transplantation, 2 accepted artificial vascular transplantation, 1 completed the removal of blood clots in the left upper limb artificial blood vessels and arteriovenous internal fistula molding, and 2 gave up surgery. Eleven patients could not complete the arteriovenous fistula operation for the fine forearm superficial vein. Of them, 2 patients accepted artificial vascular operation, 6 underwent autogenous great saphenous vein transplantation, 1 with slender radial artery in diameter completed higher position fistula between the brachial artery and median cubital vein, and 2 gave up surgery. ConclusionArteriovenous internal fistula preoperative ultrasound assessment and marking have very important value in improving the success rate of operation in end-stage uremia patients.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.
At present, the whole lifecycle management of vascular access for hemodialysis in China is still in its early stages. Faced with a large group of chronic kidney disease patients, hospitals at all levels lack systematic and continuous nursing management models. To address the issue of lacking continuous and effective nursing management of vascular access for dialysis during the period from hospitalization for autologous arteriovenous fistula surgery to outpatient maintenance hemodialysis treatment, this article introduces the background, specific implementation methods, and preliminary results of the new model of integrated medical and nursing follow-up management of vascular access for patients with hemodialysis during hospitalization and outpatient period constructed by the Wenjiang Hemodialysis Center of West China Hospital, Sichuan University. The purpose is to explore a new model for continuous and effective management of vascular access for hemodialysis patients.