Objective To establ ish an efficient and stable culture method of human umbil ical vein endothel ial cells (HUVECs) in vitro so as to provide good source of seed cells for tissue engineered vascular grafts and for precl inical research. Methods The umbil ical cords were harvested from full-term normal delivered neonates, which were perfused with0.1% collagenase II by self-made needle and were digested at 37 and 5% CO2 humidified incubator. The HUVECs were cultured in endothel ial culture medium (ECM) containing 5% fetal bovine serum (FBS) and 1% endothel ial cell growth factor (ECGS). HE staining of the umbil ical cords before and after digestion was used to observe the detachment of HUVECs, flow cytometry to detect the purity of primary HUVECs, and inverted phase contrast microscope to observe the morphology of the cultured HUVECs. The growth of the 3rd passage cells was measured by MTT assay; immunocytochemical technique and matrigelbased capillary-l ike tube formation assay were carried out to identify the function of HUVECs. Results After digestion of 0.1% collagenase II, marked HUVECs detachment was observed with complete digestion. The purity of the HUVECs was 99.56% by digestion of 0.1% collagenase II at 37 and 5% CO2 humidified incubator for 15 minutes. Primary HUVECs showed a cobblestone or pitching stone-l ike appearance in vitro, forming a confluent monolayer cells after 2-3 days of culture. MTT assay demonstrated that HUVECs showed the fastest growth speed at 3 to 4 days, and showed growth of cell fusion at about 5 days. Immunocytochemistry showed that HUVECs highly expressed endothel ial marker factor VIII. Matrigel based capillary-l ike tube formation assay showed that it could form endothel ial-l ike tube structures after 24 hours of culture. Conclusion Using improved method and ECM could obtain high quantity and high qual ity primary HUVECs, which might be a kind of promising seed cells for tissue engineering and precl inical research.
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.
Objective To evaluate the effect of copper-ion on the prol iferation and differentiation of human umbil ical vein endothel ial cell (HUVEC). Methods HUVEC were cultured and passaged in vitro. HUVEC were inoculated into 96-well plate with density of 5 × 103/well. All the cells were divided into 3 groups randomly according to different culture mediums: group A (5 μmol/L CuSO4), group B (25 μmol/L CuSO4), group C (control group). Every group had 4 wells, and the basic culture medium was MCDB131. The cell growth curves of 3 groups were drawn by using MTT. HUVEC were inoculated into 6-well plate with density of 2 × 105/well. Grouping of the cells was the same as the above. The gene expressions of endothel ial nitric oxide synthase (eNOS) and tyrosine kinase with immunoglobul in-l ike and EGF-l ike domain 1 (Tie-1) were detected by real-time RT-PCR. Results The growth curves revealed that the exponential growth time was the first 3 days, plateau growth time begun on the 4th day. The prol iferation of group A was ber than that of groups B and C from the 3rd day, within 2 days, the prol iferation of group B was ber than that of group C; however, it decreased and was weaker than group C from the 4th day, all showing statistically significant difference (P lt; 0.05). The results of real-time RT-PCR revealed that the expressions of eNOS in groups A, B and C were 7.294 ± 1.488, 0.149 ± 0.044 and 1.000 ± 0.253; and the expressions of Tie-1 in groups A, B and C were 1.481 ± 0.137, 1.131 ± 0.191 and 1.000 ± 0.177. Group A compared with groups B and C, both of 2 genes were up-regulated (P lt; 0.05). Group B compared with group C, eNOS was down-regulated (P lt; 0.05) and the difference of Tie-1 expression was not statistically significant (P gt; 0.05). Conclusion 5 μmol/L copper-ion can promote the prol iferation and differentiation of HUVEC effectively.
Objective To investigate the effect of keratin 17 (K-17) on the migration, prol iferation and tube formation of human umbil ical vein endothel ial cell (HUVEC), and to real ize the role of K-17 in angiogenesis. Methods After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and Ncontrol-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell prol iferation abil ity was detected by cell counting. After 30-hour culture, the cell’s abil ities of migration and differentiation to tube were detected by 24-well Mill icell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. Results After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the prol iferation, compared with both control and negative control groups (P gt; 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Mill icell wells within 24 hours (3719.0 ± 319.0) was smaller than both control (7 437.5 ± 212.0) and negative control (7 356.3 ± 795.7) groups, with significant difference (P lt; 0.01). However, there was no significant difference between the control group and the negative control group (P gt; 0.05). After HUVEC were transfected with K-17- siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 ± 0.5), (3.6 ± 0.5) and (3.2 ± 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P lt; 0.01), and there was no significant difference between the negative control group and the control group (P gt; 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 ± 0.02, 0.08 ± 0.01 and 0.72 ± 0.03, respectively. There was significant difference among these three groups (P lt; 0.01). Conclusion K-17 has no impact on cell prol iferation, but may augment endothel ial cell migration, which may facil itate angiogenesis.