The effects of intergrin-linked kinase on angiogenesis in hypertrophic scar / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar.</p><p><b>METHODS</b>The human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups.</p><p><b>RESULTS</b>(1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05).</p><p><b>CONCLUSIONS</b>The ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.</p>

Treatment of extensive acute radiation burn and its complications / 中华烧伤杂志

This article reports the treatment of a patient suffered from acute radiation burn covering 41% TBSA, with deep partial-thickness and full-thickness injury, produced by exposure to a large-scale industrial electron accelerator. An open wound began to appear and enlarged gradually 10 weeks after the exposure. Serious wound infection with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, pneumonia, respiratory failure, systemic inflammatory response syndrome, nephropathy and hypoproteinemia developed successively since 3 weeks after the wound formation. Skin grafts failed to survive, resulting in enlargement of the wound. After being treated with proper measures, including parenteral nutrition, respiratory support with a ventilator, appropriate antibiotics, steroid administration for nephropathy, deep debridement for wounds followed by skin grafting, the patient was cured and discharged after undergoing 15 operations in 500 days. The clinical condition of an extensive acute radiation burn is complicated. We should pay close attention to the changes in functions of organs, and strengthen the therapeutic strategies to support the function of organs to reduce the incidence of systemic complications. The control of the infection and the timely and effective repair of the wound are still the key points of the treatment of an extensive local radiation injury.

Expression of integrin-linked kinase in fibroblasts of scar induced by cobalt chloride and its effect on cell proliferation / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation.</p><p><b>METHODS</b>The human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro. Cells from the 5th to the 6th passages were used in the experiment. Six bottles of Fbs were obtained from each of the seven patients, and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0, 50, 100, 150, 200, and 250 µmol/L for 24 h. The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR. Fbs were stimulated by CoCl2 in the most suitable concentration (100 µmol/L) and the protein expression of ILK was determined 0, 1, 2, 4, 12, and 24 h after the stimulation. Then the Fbs were divided into control group (cultured with nutrient solution), negative control group (transfected with con-siRNA), and ILK siRNA group (transfected with ILK siRNA). They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection, with 4 wells for each concentration in each group. The cell proliferation was detected by XTT assay. Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement, and LSD method was used in multiple comparisons.</p><p><b>RESULTS</b>The expression level of ILK mRNA was highest in Fbs cultured with 100 µmol/L CoCl2 for 24 h, with significant difference compared with those of Fbs cultured with other concentrations of CoCl2 (F = 50.958, P < 0.001). The expression of ILK protein in Fbs cultured with 100 µmol/L CoCl2 for 1 h (0.243 ± 0.009) was lower than that cultured for 0 h (0.387 ± 0.017), and it started to increase from 2 h (0.361 ± 0.010), and exaggerated at 4 h (0.584 ± 0.028), 12 h (0.730 ± 0.029), and 24 h (0.785 ± 0.031). The expression levels of ILK protein at 1, 4, 12, 24 h were statistically different from that at 0 h (P values all below 0.05). XTT showed that cell proliferation level was highest in control group when cultured with 100 µmol/L CoCl2 (F = 488.026, P < 0.001), which decreased from 150 µmol/L. The cell proliferation level in control group cultured with 250 µmol/L CoCl2 was significantly lower than that with 0 µmol/L (P values all below 0.05). There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F = 2.542, P = 0.056). The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F = 2519.542, P < 0.001).</p><p><b>CONCLUSIONS</b>ILK may be a key protein in response of hypoxia in Fbs. The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs, while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.</p>

The effect of integrin-linked kinase on VEGF expression in fibroblasts from human hypertrophic scar / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the expression of integrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar.</p><p><b>METHODS</b>Fibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: (1) Cells were cultured only in DMEM containing 10% FCS in the control group; (2) Cells were transfected with empty plasmid in the empty plasmid group; (3) Cells were transfected with plasmid expressing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VEGF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR (RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA.</p><p><b>RESULTS</b>Before ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 +/- 0.060) than that in control group (0.022 +/- 0.001) and empty plasmid group (0.028 +/- 0.005, P < 0.05). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0.819 +/- 0.019) than that in control group (0.607 +/- 0.033) and empty plasmid group (0. 591 +/- 0.024, P<0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P < 0.05).</p><p><b>CONCLUSIONS</b>ILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.</p>

Expression of integrin-linked kinase in human hypertrophic scar and its relationship with angiogenesis / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the expression of integrin-linked kinase (ILK) in scar in different growth stages, as well as its relationship with angiogenesis.</p><p><b>METHODS</b>(1) Fifteen burn patients with scar formation time shorter than 6 months, ranging from 6 to 12 months, and longer than 12 months were hospitalized from December 2009 to December 2010. They were divided into A, B, and C groups according to the scar formation time, with 5 patients in each group. Scar specimens were harvested for observation of ILK expression with immunohistochemistry method, and ILK mRNA expression with real time fluorescence quantitative RT-PCR. (2) Microvascular endothelial cells (MEC) were isolated from scar tissue in A group and cultured in vitro, and then they were purified by immunomagnetic beads and identified with coagulation factor VIII marked by immunofluorescence (fibroblasts from human normal skin were used as control). The cultured cells in logarithmic growth phase were divided into control group (cultured with M131 medium containing microvascular growth supplement), transfection 1 group (transfected with empty plasmid), and transfection 2 group (transfected with ILK cDNA plasmid) according to the random number table. After 24 hours, the expressions of ILK mRNA, Flt-1 mRNA, and KDR mRNA were determined with real time fluorescence quantitative RT-PCR. Data were processed with one-way analysis of variance.</p><p><b>RESULTS</b>Immunohistochemical observation showed that ILK in A group mainly expressed in the basal layer cells of epidermis, cytoplasm of fibroblasts, and MEC in scar, while ILK in B group only distributed in the basal layer cells of epidermis, but ILK expression in C group was not obvious. The expression of ILK mRNA in A group (0.34 ± 0.16) was significantly higher than those in B and C groups (0.17 ± 0.06, 0.07 ± 0.13, F = 37.007, P = 0.000). MEC grew up showing cobble stone formation after purification. The expression of coagulation factor VIII was positive in cytoplasm of purified MEC, while that was negative in fibroblast of human normal skin. The expressions of ILK mRNA (57.807 ± 5.556), KDR mRNA (0.836 ± 0.014), and Flt-1 mRNA (0.162 ± 0.005) in transfection 2 group were higher than those in control and transfection 1 groups (0.018 ± 0.003, 0.028 ± 0.020, 0.023 ± 0.004 and 0.042 ± 0.005, 0.039 ± 0.007, 0.046 ± 0.003; F(ILK) = 87.110, F(KDR) = 11.241, F(Flt) = 18.199, with P values all below 0.01).</p><p><b>CONCLUSIONS</b>ILK mainly expressed in scar tissue with formation time shorter than 6 months, and it may affect vascularization of scar by regulating gene expressions of KDR and Flt-1 in MEC, which plays an important role in early scar formation.</p>

An experimental study of using chitinous membrane as the culture scaffold for epidermal stem cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of constructing a skin tissue engineering covering on chitinous membrane using rat epidermal stem cells (ESCs).</p><p><b>METHODS</b>Rat ESCs were isolated and cultured by cold digestive method and collagen type IV adherent method. Cell colonies were observed with inverted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal microscope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of surface markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expressions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chitinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the vehicle was observed.</p><p><b>RESULTS</b>Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive; CD71 and CD34 were negative; CK19, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P > 0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chitinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope.</p><p><b>CONCLUSIONS</b>Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.</p>

Influence of skin-derived progenitor cell combining with hyaluronic acid on the wound healing of diabetic rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the effect of skin-derived progenitor cell (SKP) combined with hyaluronic acid( HA) on the wound healing in diabetic rats.</p><p><b>METHODS</b>SKP of Spraque-Dawley (SD) neonate rats were isolated and cultured and mixed with HA. The differentiation characteristics of SKP in the culture were observed. Sixty SD rats were injected intraperitoneally with 65 mg/kg streptozotocin( STZ) to induce diabetes. Two symmetrical full-thickness cutaneous wounds( 1.0 cm in diameter) were made on the back of each SD rat and randomly divided into A (n = 20, with treatment of 100 mircol SKP-HA) , B (n = 20, with treatment of 100 mirol HA) , and C ( n = 20, with treatment of DMEM/F12 culture medium) groups. Tissue samples from wound in each group were harvested on 1, 2, 3, 4 weeks after the treatment. Wound healing rate, changes in histomorphology, the content of hydroxyproline ( HYP) , and immigration of labelled SKP were determined and analyzed.</p><p><b>RESULTS</b>SKP grew well when cultured with HA. The characteristics of SKP to differentiate into lipocyte, neuron, and neurogliocyte remained in the culture. Compared with that in C group, epithelization in the wounds of A and B groups appeared earlier. The wound healing rate in A group [ (72.1 +/- 2. 8)% ] and B group [ (53.7 +/- 2. 9)% ] were obviously higher at 2 post-treatment weeks(PTW) than that in group C [(42. 5 +/- 1.5)% ( P <0.05) , and that in A group was obviously higher compared with B and C groups at 3 PTW ( P < 0. 05 or 0. 01). The wound healing rates in A and B groups were (100. 00 +/- 0.00) % at 4 PIW, which were obviously higher than that of group C( P <0.01) . There was no obvious difference in the HYP content among the 3 groups at 1 PIW ( P > 0. 05) , but it was obviously higher in A and B groups than that in C group at 2,3,4 PTW( P <0.01) , and that in A group was significantly higher than that in B group at 3 and 4 PTW( P <0. 01). SKP survived well on the wound, and migrated towards the dermis as time elapses.</p><p><b>CONCLUSION</b>SKP-HA composition can promote wound healing in diabetic rats.</p>

Effects of 5F from Pteris semipinnate L on growth of human pathological scars in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of 5F from Pteris semipinnate L on the growth of human pathological scar in nude mice.</p><p><b>METHODS</b>5F from Pteris semipinnate L was administered at different doses in nude mouse models bearing human pathological scars. The morphology, histology, tumor growth factor-beta1 and type I collagen content of the scar tissues were examined after the administration.</p><p><b>RESULTS</b>Administration of 5F significantly reduced the volume of the implanted pathological scars in the nude mouse models, and histologically, the scar tissue exhibited a transition to the normal scar architecture with decreased TGF-beta1 and type I collagen content.</p><p><b>CONCLUSION</b>5F could effectively inhibit the growth of pathological scars in nude mice.</p>

The antagonistic effect of recombinant human decorin on TGF-beta1 stimulation of fibroblasts in collagen lattices / 中华烧伤杂志

<p><b>OBJECTIVE</b>To mimic contact pattern between decorin and TGF-beta1, in vivo, and investigate the antagonistic effect of recombinant human decorin on TGF-beta1 stimulation of hypertrophic scar fibroblasts in collagen lattices.</p><p><b>METHODS</b>Fibroblasts populated collagen lattices (FPCL) model was adopted in the study, and they were divided into control group, decorin group [2mg/L recombinant human decorin (rh-decorin) was administered to FPCL], TGF-beta1 group (5 microg/LTGF-beta1 was administered to the culture medium), and TGF-beta1 + decorin group (2mg/L rh-decorin was administered to FPCL, then culture medium containing 5 microg/L TGF-beta1 was added into FPCL). Changes in PAI-1 and alpha-SMA protein expression in scar fibroblasts in collagen lattices were detected with Western blotting at 12 post-administration hour (PAH), 24 PAH, 48 PAH, and 72 PAH, and expressions of PAI-1 and alpha-SMA mRNA were concomitantly examined by RT-PCR.</p><p><b>RESULTS</b>The contraction of FPCL at each time-point in control group was obviously attenuated compared with that in decorin group, but it was significantly intensified compared with that in TGF-beta1 group. The expression of PAI-1 and alpha-SMA mRNA and protein in TGF-beta1 group (3482 +/- 211, 4320 +/- 272, 0.89 +/- 0.15, 0.56 +/- 0. 11) were markedly increased than those in control group (1764 +/- 147, 1699 +/- 146, 0.29 +/- 0.06, 0.21 +/- 0.06, P < 0.01), while no obvious difference of them was found between control and other two groups.</p><p><b>CONCLUSION</b>Stimulation of scar fibroblasts by TGF-beta1, can be suppressed when rh-decorin is blended into collagen lattices, indicating that decorin is effective in neutralizing TGF-beta1 in vitro. The pathogenesis of hypertrophic scar might be related to up-regulation of TGF-beta1 with the lack of decorin after cutaneous injury.</p>

The complications of radiotherapy for breast cancer and the treatment for radiation ulcer / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the effective treatment for chronic ulcer following radiotherapy for breast cancer and reveal the universality and severity of radiation-induced brachial plexus neuropathy.</p><p><b>METHODS</b>The TRAM flap, the local expanded flap or the delayed skin flap were applied to repair the ulcer wounds in 16 patients. Electromyogram examinations were used to evaluate the radiation lesions of the brachial plexus.</p><p><b>RESULTS</b>All the flaps survived successfully with satisfactory results except one, which sustained partial necrosis due to infection. Ten patients underwent regular electromyogram examinations, seven of them were found to have radiation-induced brachial plexus neuropathy.</p><p><b>CONCLUSIONS</b>Radiation ulcer following radiotherapy for breast cancer is often concomitant with brachial plexus neuropathy. These injuries presented a chronically progressive and irreversible course. Application of the flaps that have adequate blood supply can reconstruct the wounds effectively.</p>