Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
To isolate and culture adi pose-derived stem cells (ADSCs), and to study the effects of the conditioned medium of ADSCs (ADSC-CM) treated with insul in on HaCaT cells. Methods ADSCs were isolated from adipose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5 × 104 cells/mL. The cells were divided into 2 groups: group A in which the cells were incubated in 1 × 10-7 mol/ Linsul in for 3 days, and group B in which the cells were not treated with insul in. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor (HGF). HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups: group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1 × 10-7 mol/ L insul in; group D1, 1 mL 2%FBS. Prol iferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration abil ity was measured by in vitro wound-heal ing assay 0, 12, 24, 36 and 48 hours after culture. Results The level of VEGF in groups A and B was (643.28 ± 63.57) and (286.52 ± 46.68) pg/mL, respectively, and the level of HGF in groups A and B was (929.95 ± 67.52) and (576.61 ± 84.29) pg/mL, respectively, suggesting differences were significant between two groups (Plt; 0.05). Cell prol iferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881 ± 0.039, 0.804 ± 0.041, 0.663 ± 0.027 and 0.652 ± 0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05). The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23% ± 1.98%, 8.82% ± 2.59%, 31.70% ± 8.85% and 29.60% ± 8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 (P lt; 0.05), group B1 was significantly higher than group A1 (P lt; 0.05). The migration distance of HaCaT cells in groups A1, B1,C1 and D1 at 36 hours was (0.184 6 ± 0.019 2), (0.159 8 ± 0.029 4), (0.059 2 ± 0.017 6) and (0.058 2 ± 0.012 3) mm, respectively, whereas at 48 hours, it was (0.231 8 ± 0.174 0), (0.205 1 ± 0.012 1), (0.079 2 ± 0.008 1) and (0.078 4 ± 0.011 7) mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours (P lt; 0.01), group A1 was significantly higher than group B1 (P lt; 0.05) at 36 and 48 hours, no significant difference was evident at other time points(P gt; 0.05). Conclusion ADSCs treated with insul in can significantly promote the prol iferation and the migration of HaCaT cells and inhibit their apoptosis.
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.
Objective In vivo, the microenvironment of epidermal stem cells (ESCs) is complex, and estrogen might be involved in the micro environment. To investigate the biological effects of estrogen on the prol iferation and migration of ESCs in vitro. Methods hESCs were isolated from normal human foreskin and cultured. The second generation of hESCs were identified with flow cytometry after being marked with integrin β1, cytokeratin 19 (CK19), CK14, and CK10 antigens.hESCs of 2 × 106 cell density were cultured with ESCs special medium supplemented with 0.1 nmol/L Diethylstilbestrol in group A (estrogen group), with ESCs special medium supplemented with 10 nmol/L Raloxifene hydrochloride in group B (ER blocking agent group), and with ESCs special medium in group C (control group), respectively. The 100 μm “scratch” wounds were created by scraping confluent hESCs plated on Petri dishes with a sterile pipette tip in vitro. The migrating cells from the wound edge were quantified at 24, 48, and 72 hours after incubation. The rates of wound heal ing were calculated by SigmaScan Pro 5.0 software at 72 hours. The prol iferating effect of estrogen on hESCs was determined with MTT method at 24, 48, 72, 96, and 120 hours. Results Cultured primary hESCs could adhere to the wall showing ovoid in shape and grew into colonies. Flow cytometry showed the positive results for integrin β1, CK19, and CK14 (with positive rate of 96.63%, 95.47%, and 94.27%, respectively) and the negative result for CK10 (with positive rate of 1.32%). In group A, the number of cells crossing the wound edge was more than those of group B and group C at 24, 48, and 72 hours. The rates of wound heal ing were 69.00% ± 0.05% in group A, 35.00% ± 0.05% in group B, and 48.00% ± 0.06% in group C at 72 hours, showing significant differences among groups (P lt; 0.05). The prol iferating speed of hESCs was significantly higher in group A than in groups B and C (P lt; 0.01), and significantly higher in group C than in group B (P lt; 0.01) at 24, 48, 72, 96, and 120 hours. Conclusion The estrogen can promote the prol iferation and migration of hESCs in vitro. It may be involved in many biological activity of skin.
Objective To study the migration of Schwann cells from the nerve autograft in the acellular nerve allograft of the rats in vivo. Mehtods The sciatic nerves (20 mm long) of the SD rats were harvested and prepared for the acellular nerve grafts by the chemical extraction. Then, they were observed by the gross view, HE staining, and Antilamininstaining, respectively. Another 32 female SD rats weighing 250-300 g were obtained for the study. A 2-mm-long nerve autograft was interposed between the two 10-mm-long nerve allografts to form a 22-mm-long composite. Then, the composite was placed in the muscle space, together with a sole 22-mm-long nerve allograftas a control. They were harvested at 5，10，15 and 20 days, respectively, and were then given the HE staining and the S-100 staining. Results The acellular nerve graft was semitransparent under the gross view. HE staining showed that no cell was observed within the nerve graft. Anti-laminin staining showed that the basal membrane was partially interrupted, with a positive result (dark brown). All the nerve grafts in both the groups exhibited the existenceof the cells. The S-100 positive cells were observed from the 15th day at the far ends of the two allografts of the composite; however, there were no suchcells observed within the sole nerve allograft. Conclusion Schwann cells from the sciatic nerves (2 mm- long) of the rats can migrate in the acellular nerve allograft to the far ends of the neighboring 10-mm-long nerve allografts at 15 days after operation, which offers the theoretical basis forthe repair of the longrange nerve defect by the composite of the acellular nerve allografts with the interposed nerve autograft.
ObjectiveTo establish human bladder cancer cell line with silenced Fibulin-5 gene and observe the effects and mechanism of Fibulin-5 gene silencing on the proliferation activity and migration of the bladder cancer cells.MethodsThe human bladder cancer cells 5637 were divided into group F5 and group NC, and the cells in group F5 were infected with Fibulin-5 RNA interference (RNAi) lentivirus while the cells in group NC were infected with negative-control virus. Then the expression of Fibulin-5 mRNA was detected by real-time quantitative polymerase chain reaction, the cell proliferation activity was detected by MTT, the migration rate was detected by wound healing method, and the expression levels of proteins in receptor tyrosine kinase (RTK) pathway were detected by PathScan RTK Signaling Antibody Array Kit.ResultsThe Fibulin-5 mRNA expression decreased significantly by Fibulin-5 RNAi lentivirus (0.067±0.013 in group F5 vs. 1.001±0.000 in group NC), and the gene silencing efficiency reached 93.3%, so the Fibulin-5 silencing cell line was established successfully. Comparing with group NC, the relative absorbance value and migration rate of cell 5637 in group F5 decreased significantly (P<0.01); in addition, the expression levels of anaplastic lymphoma kinase, Axl, p44/42 mitogen activated protein kinase, and Src protein were up-regulated in group F5 (P<0.05).ConclusionFibulin-5 may play a role in the proliferation and migration of bladder cancer cells, and may have an inhibitory effect on extracellular signal-regulated kinase and its signaling pathway proteins.
ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation.
MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro.
ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group.
ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
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 investigate the influence of hypoxia on pro-metastasis induced by epithelial-mesenchymal transition (EMT) of human lung adenocarcinoma.
MethodsThe human lung cancer cell line H460 was cultured in hypoxic condition and the morphologic changes of the cells were observed under the microscope. The EMT-related markers including E-cadherin and vimentin were detected by Western blot. Transwell migration assay and transwell invasion assay were employed to detect the migratory and invasive activity of cancer cells.
ResultsHypoxic induced morphological changes were consistent with the mesenchymal phenotype, such as an elongated fibroblastic morphology, and conversion from a tightly packed epithelial cobblestone pattern to a loosely packed scattered phenotype. Compared with the control group, hypoxic attenuated the quantity of E-cadhenrin, but increased vimentin, which resulted in promotion of migration and invasion of H460.
ConclusionHypoxia induces EMT in H460 and enhances the invasive and migratory abilities of lung cancer cells.
