Effect of substrate stiffness on biological behavior of fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of substrate stiffness on proliferation, migration of fibroblast and integrin β(1) expression in fibroblast.</p><p><b>METHODS</b>Fibroblasts were inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa. After being cultured for 5 days or 6 days, cells were counted and cell proliferative activities (recorded as absorbance value) were assessed with methyl thiazolyl blue (MTT). After being cultured for 3 days, cell cycle was detected and proliferation index (PI) was calculated. The cell scratch test was used for determination of cell migration rate on post scratch day (PSD) 0 (the day of scratch), 1, 2, and 3. After being cultured for 2 days, the expression of integrin β(1) was determined by flow cytometry with fluorescence. Data were processed with one-way analysis of variance.</p><p><b>RESULTS</b>(1) The proliferative speed and proliferative activity of fibroblasts were all increased along with the increase in substrate stiffness. PI of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5), (19.8 ± 1.1), and (200.1 ± 2.6) kPa was respectively 24.8%, 27.4%, 32.4%. On PSD 2, migration rate of fibroblasts inoculated on silicon substrate with stiffness of (19.8 ± 1.1) and (200.1 ± 2.6) kPa was respectively (91.4 ± 5.1)%, (100.0 ± 1.3)%, which were higher than that of fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa [(55.8 ± 6.8)%, with F value respectively 3.5, 4.0, P values all below 0.01]. (3) The expression rate of integrin β(1) in fibroblasts inoculated on silicon substrate with stiffness of (16.2 ± 0.5) kPa was the lowest (43.22%), and that in fibroblast inoculated on silicon substrate with stiffness of (200.1 ± 2.6) kPa was the highest (81.26%).</p><p><b>CONCLUSIONS</b>Substrate stiffness may have a great effect on proliferation and migration of fibroblast during the process of wound healing and scar formation, which can be related to regulation of integrin β(1) expression.</p>

Effect of calcium on the activity and expression of integrin beta1 promoter in HaCaT cells and cell migration / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of calcium on the activity and protein expression of integrin beta1 promoter in human immortal keratinocyte colony HaCaT cell and cell migration.</p><p><b>METHODS</b>(1) HaCaT cells were cultured in vitro (12-slot plate) and divided into 5 groups according to the random number table, with 18 slots in each group: reporter plasmid pGL3 promoter (positive control group, PC), pGL3 empty vector (negative control group, NC), pGL3-1756 bp (total length promoter group, TL), pGL3-1442 bp (distal promoter group, D), and pGL3-261 bp (proximal promoter group, P) was respectively used to transfect HaCaT cells in non-serum RPMI 1640 culture medium with 0.00, 0.03, 0.09, 0.30, 0.80, or 1.20 mmol/L calcium (3 slots in each group with each concentration). Luciferase activity was detected with dual-luciferase reporter assay system 24 hours after transfection. (2) HaCaT cells steadily transfected with small interfering RNA-integrin beta1 vector (steadily transfected in brief) constructed in our laboratory were normally cultured and divided into 6 parts according to the random number table. And then they were treated with former 6 different concentrations of calcium, with 3 samples for each concentration. Expression level of integrin beta1 protein was determined with Western blot. (3) Normal and steadily transfected HaCaT cells were cultured in 6-slot plate, 18 slots for each kind of cells. They were cultured with former 6 kinds of calcium culture media (divided according to the random number table, with 3 slots of cells for each concentration) for 12 hours after scratch test. Cell migration rate was observed and determined. (4) Data were processed with one-way analysis of variance and independent samples t test.</p><p><b>RESULTS</b>(1) The luciferase activity of cells in TL group increased from 0.16+/-0.09 to 0.39+/-0.09 and 0.35+/-0.05 (with t value respectively 3.143, 3.140, P values all below 0.05) as calcium concentration increasing from 0.00 mmol/L to 0.09 and 0.30 mmol/L, and it decreased as calcium concentration increased to 0.80 and 1.20 mmol/L. The change pattern of luciferase activity of cells along with calcium concentration in D group was similar to that in TL group, but its activity (0.56+/-0.32, 0.64+/-0.06) at the concentration of 0.09, 0.30 mmol/L was respectively higher than that in TL group (with t value respectively 0.887, 6.122, P values all below 0.05). There was no obvious influence of calcium in either concentration on the luciferase activity of cells in P group. (2) The expression amount of integrin beta1 of steadily transfected HaCaT cells cultured with 0.03, 0.09, 0.30, 0.80, 1.20 mmol/L calcium (0.58+/-0.09, 1.40+/-0.29, 1.41+/-0.09, 0.99+/-0.10, 1.16+/-0.15) were all increased as compared with that cultured with 0.00 mmol/L calcium (0.53+/-0.10, with t value respectively 0.687, 4.880, 11.210, 5.578, 6.199, P values all below 0.05). (3) Migration speed of normal HaCaT cells cultured with 0.09, 0.30 mmol/L calcium increased obviously as compared with that cultured with 0.00 mmol/L calcium, and it slowed down when cultured with 0.80, 1.20 mmol/L calcium. There was no obvious difference of migration rate among steadily transfected HaCaT cells treated with different concentration of calcium.</p><p><b>CONCLUSIONS</b>Distal promoter region of integrin beta1 plays a vital role in regulating integrin beta1 transcription in human epidermal cells. And calcium regulates activity, protein expression of integrin beta1 promoter and cell migration.</p>