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Objective@#To investigate the protective effects and mechanism of keratinocyte growth factor (KGF) combined with hypoxia inducible factor-1α (HIF-1α) on intestinal crypt epithelial cells (IEC-6) of rats with hypoxia stress.@*Methods@#(1) The routinely cultured IEC-6 of rats were collected and divided into normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group, according to the random number table, and then the previous mediums were respectively replaced with dulbecco′s modified eagle medium (DMEM), medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α. And the cells were cultured in cell incubator with oxygen volume fraction of 21% for 24 hours. (2) Another batch of routinely cultured IEC-6 were collected and divided into normoxia control group, hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group, according to the random number table. The previous mediums were replaced with DMEM, DMEM, medium with 0.5 ng/mL KGF, medium with 10.0 ng/mL HIF-1α, and medium with 0.5 ng/mL KGF and 30.0 ng/mL HIF-1α respectively. And then, the cells in normoxia control group were cultured routinely for 24 hours, and cells in the other 4 groups were cultured in cells incubator of 3 gases, with oxygen volume fraction of 5% for 24 hours. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and morphological changes of cells were observed with optical microscope. Cells cultured in normoxic and hypoxic incubators were collected, with 3 samples in each group, and survival rates of cells were detected by cell count kit 8. Cells in normoxia control group and cells cultured in hypoxic incubator were collected, with 3 samples in each group. The cell cycle changes and apoptosis rates were detected by flow cytometer, the content of adenosine triphosphate (ATP) was detected by ultraviolet spectrophotometer, and protein expression of p53 was detected by Western blotting. Data were processed with one-way analysis of variance and least significant difference test.@*Results@#(1) After being cultured for 24 h, cells cultured in normoxic incubator grew well with oval or round shapes and clear cytoplasm, and cells cultured in hypoxic incubator showed irregular shapes such as fusiform or starlike shape, with black particle in cytoplasm. (2) After being cultured for 24 h, cell survival rates of normoxia blank group, normoxia KGF group, normoxia HIF-1α group, and normoxia combine group were (107.4±8.7)%, (109.8±2.9)%, (115.8±7.4)%, and (112.8±10.6)% respectively. There was no significantly statistical difference in general comparison of cell survival rates among the above groups (F=0.685, P=0.586). After being cultured for 24 h, cell survival rates of hypoxia control group, hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were (35.1±4.6)%, (52.9±6.8)%, (56.2±3.1)%, and (71.2±9.6)% respectively, which were significantly lower than (106.3±12.3)% of normoxia control group (P<0.001). Survival rates of cells in hypoxia KGF group, hypoxia HIF-1α group, and hypoxia combine group were significantly higher than the rate of cells in hypoxia control group (P=0.023, 0.009, <0.001). Survival rate of cells in hypoxia combine group was significantly higher than the rates of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.017, 0.045). (3) After being cultured for 24 h, percentage of cells in G1 phase in hypoxia control group was significantly higher than that of cells in normoxia control group (P=0.030), percentages of cells in S phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously lower than the percentage of cells in normoxia control group (P=0.020, 0.031, 0.026), and percentages of cells in different phases in other groups were close to those of cells in normoxia control group (P=0.516, 0.107, 0.052, 0.985, 0.637, 0.465, 0.314, 0.591). After being cultured for 24 h, percentages of cells in G1 phase in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than the percentage of cells in hypoxia combine group (P=0.001, 0.030, 0.014), and percentages of cells in S phase in the above 3 groups were obviously lower than the percentage of cells in hypoxia combine group (P=0.001, 0.012, 0.010). (4) After being cultured for 24 h, compared with that of cells in normoxia control group, apoptosis rate of cells in hypoxia control group obviously increased (P=0.018), and apoptosis rate of cells in hypoxia combine group obviously decreased (P=0.008). After being cultured for 24 h, compared with that of cells in hypoxia control group, apoptosis rates of cells in hypoxia KGF group and hypoxia combine group obviously decreased (P=0.004, 0.001). Apoptosis rate of cells in hypoxia combine group was obviously lower than those of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.032, 0.002). (5) After being cultured for 24 h, compared with that of cells in normoxia control group, the content of ATP of cells in hypoxia combine group changed unobviously (P=0.209), and content of ATP of cells in the other groups obviously decreased (P= <0.001, 0.001, 0.002). Content of ATP of cells in hypoxia HIF-1α group and hypoxia combine group was obviously higher than that of cells in hypoxia control group (P=0.044, 0.001). Content of ATP of cells in hypoxia combine group was obviously higher than that of cells in hypoxia KGF group and hypoxia HIF-1α group (P=0.011, 0.020). (6) After being cultured for 24 h, protein expressions of p53 of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group were obviously higher than that of cells in normoxia control group (P<0.001), and protein expression of p53 of cells in hypoxia combine group was obviously lower than those of cells in hypoxia control group, hypoxia KGF group, and hypoxia HIF-1α group (P=0.001, 0.001, 0.002).@*Conclusions@#KGF combined with HIF-1α have significant protective effects on IEC-6 of rats with hypoxia stress, and can improve its survival in hypoxic environment by inhibiting cell cycle arrest, reducing the level of apoptosis, and increasing level of energy metabolism.
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Objective@#To investigate the effect of recombinant human keratinocyte growth factor 2 (rhKGF-2) on lung tissue of rabbits with severe smoke inhalation injury.@*Methods@#A total of 120 New Zealand rabbits were divided into 5 groups by random number table after being inflicted with severe smoke inhalation injury, with 24 rats in each group. Rabbits in the simple injury group inhaled air, while rabbits in the injury+phosphate buffer solution (PBS) group inhaled 5 mL PBS once daily for 7 d. Rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group received aerosol inhalation of 1 mg/kg, 2 mg/kg, and 5 mg/kg rhKGF-2 (all dissolved in 5 mL PBS) once daily for 7 d, respectively. On treatment day 1, 3, 5, and 7, blood samples were taken from the ear central artery of 6 rabbits in each group. After the blood was taken, the rabbits were sacrificed, and the tracheal carina tissue and lung were collected. Blood pH value, arterial oxygen partial pressure (PaO2), arterial blood carbon dioxide pressure (PaCO2), and bicarbonate ion were detected by handheld blood analyzer. The expressions of pulmonary surfactant-associated protein A (SP-A) and vascular endothelial growth factor (VEGF) in lung tissue were detected by Western blotting. Pathomorphology of lung tissue and trachea was observed by hematoxylin-eosin staining. Data were processed with analysis of variance of two-way factorial design and Tukey test.@*Results@#(1) Compared with those in simple injury group, the blood pH values of rabbits in the latter groups on treatment day 1-7 had no obvious change (P>0.05). The PaO2 of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were (75.0±2.4) and (71.0±4.5) mmHg (1 mmHg=0.133 kPa), respectively, which were significantly higher than (62.0±6.8) and (63.0±3.0) mmHg in simple injury group (q=4.265, 8.202, P<0.05 or P<0.01). The PaO2 of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was (82.0±4.9) mmHg, which was significantly higher than that in simple injury group (q=6.234, P<0.01). Compared with that in simple injury group, the PaCO2 of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 was significantly decreased (q=4.876, P<0.01) and significantly increased on treatment day 5 (q=5.562, P<0.01); the PaCO2 of rabbits in injury+5 mg/kg rhKGF-2 group was significantly increased on treatment day 5 and 7 (q=5.013, 4.601, P<0.05 or P<0.01). Compared with that in simple injury group, the serum bicarbonate ion of rabbits in injury+1 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=5.142, P<0.01); the serum bicarbonate ion of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 5 and 7 were significantly increased (q=4.830, 6.934, P<0.01); the serum bicarbonate ion of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 5 were significantly increased (q=3.973, P<0.05). (2) The expressions of SP-A in lung tissue of rabbits in simple injury group and injury+PBS group in each treatment time point were close (P>0.05). The expressions of SP-A in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group on treatment day 3 were 0.091±0.007 and 0.101±0.009, respectively, significantly higher than 0.069±0.009 in simple injury group (q=10.800, 13.580, P<0.01). The expressions of SP-A in lung tissue of rabbits in injury+1 mg/kg rhKGF-2 group, injury+2 mg/kg rhKGF-2 group, and injury+5 mg/kg rhKGF-2 group on treatment day 5 and 7 were 0.127±0.008, 0.132±0.006, 0.194±0.006, 0.152±0.017, 0.166±0.004, 0.240±0.008, significantly higher than 0.092±0.003 and 0.108±0.005 in simple injury group (q=6.789, 12.340, 17.900, 9.875, 31.480, 40.740, P<0.01). (3) On treatment day 1 and 5, there was no significant difference in the expression of VEGF in lung tissue of rabbits among the 5 groups (P>0.05). Compared with those in simple injury group, the expressions of VEGF in lung tissue of rabbits in injury+2 mg/kg rhKGF-2 group on treatment day 3 and 7 were significantly increased (q=4.243, 8.000, P<0.05 or P<0.01), and the expression of VEGF in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group on treatment day 7 was significantly increased (q=20.720, P<0.01). (4) On treatment day 1, the injury of rabbits in each group was similar, with a large number of neutrophils infiltrated and abscess formed in the alveolar and interstitial tissue, thickened alveolar septum, some collapsed alveolar and atelectasis; large area of tracheal mucosa was degenerated and necrotic, with a large amount of inflammatory exudates blocking in the cavity. On treatment day 3, the inflammation of lung tissue and trachea in each group were improved, but the inflammation in simple injury group and injury+PBS group was also serious. On treatment day 5, the inflammation in lung tissue and trachea of rabbits in injury+2 mg/kg rhKGF-2 group and injury+5 mg/kg rhKGF-2 group were improved much obviously than those in the other groups. On treatment day 7, the inflammation in lung tissue of rabbits in injury+5 mg/kg rhKGF-2 group alleviated obviously than those in the other groups, most alveoli had no obvious exudative fluid, the alveolar cavity was intact and clear, the local alveolar dilated like a cyst, and the alveolar septum thinning; the improvement of inflammation of trachea was more obvious than the other groups, the tracheal mucosa tended to be more complete, and few neutrophils were infiltrated in the endotracheal cavity.@*Conclusions@#Atomization inhalation of rhKGF-2 can improve the PaO2 level of rabbits with severe smoke inhalation injury, reduce airway inflammation, increase the expression of SP-A and VEGF in lung tissue, thus promoting the repair of lung tissue.
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Objective To determine the protective role of keratinocyte growth factor 2 (KGF-2) on dextran sodium sulfate (DSS)-induced murine colitis and investigate the mechanism of its effect on intestinal mucosal barrier.Methods A total of 36 male C57BL/c mice were divided into 4 groups:normal control group,DSS model group,model + KGF-2 (5 mg/kg) group and model + KGF-2 (10 mg/kg)group.Groups except the control were added 3.5 ％ DSS in drinking water.Disease activity index (DAD,weight change,colon length loss and histological score were detected to evaluate the protective effect of KGF-2 on colitis.Serum fluorescein isothiocyanate-dextran (FITC-D) permeability were assayed by multiscan spectrum.In order to explore the protective role of KGF-2 on murine intestinal mucosal barrier,ZO-1 and occludin protein concentration were detected by immunohistochemical staining and Western blot.Meanwhile,cytokines including TNF-α,IL-6,IL-10,TGF-1β,IFN-γ and IL-1β in colonictissue were detected by ELISA.Results Compared with DSS-induced colitis model group,10 mg/kg KGF-2 significantly reduced DAI (P =0.021 1),weight loss (P =0.017 6),colon length loss (4.956 ±0.2583 vs.6.289±0.215 7,P =0.001 1),histologicalscore (12.17±1.222 vs.7.000±0.6325,P =0.001 1),and FITC-D permeability (168.5 ± 27.01 vs.14.62 ± 1.812,P =0.004 7) and reversed the downregulation of tight junction (TJ) proteins (ZO-1:0.158 6 ± 0.010 51 vs.0.387 9 ± 0.028 64,P＜0.000 1;occluding:0.300 5 ± 0.026 56 vs.0.445 0 ± 0.056 62,P =0.043 4).Significant decrease of TNF-α (68.93 ± 3.379 vs.40.41 ± 1.576,P＜0.000 1) and increase of IL-6 (3 755 ± 309.8 vs.5 640 ± 418.0,P =0.004 7) and IL-10 (304.0 ± 21.47 vs.521.2 ± 49.40,P =0.002 4) levels were noted in the 10 mg/kg KGF-2 treated mice.The effect of the KGF-2 was dose-dependent.Conclusions KGF-2 could ameliorate DSS-induced colitis and it may be associated with the decrease of the damage of mucosal barrier structure and funcntion by preventing ZO-1 and occludin from downregulating.The protective effect of KGF-2 on intestinal barrier function may also be exerted by inhibition of TNF-α and stimulation of IL-6 and IL-10 secretion.
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Objective To develop chitosan composite keratinocyte growth factor-2 mutant(KGF-2M)temperature-sen-sitive dressing and evaluate its physicochemical properties and dynamic release rule were used.Methods Chitosan, chi-tosan quaternary ammonium salt,β-glycerophosphate and other adjuvant materials to configure different formulations which were compounded with KGF-2M in order to develop temperature-sensitive dressing.Gelling time, temperature,the release rate of KGF-2M and other indicators were measured to analyze the physical and chemical properties of the temperature -sen-sitive dressing.Results Chitosan-KGF-2M composite dressing with temperature-sensitive properties was obtained by opti-mizing the formulation components of chitosan and related adjuvant materials.When the liquid dressing was above 35℃,it could be converted from liquid to solid gelatin within 10 minutes.The compound KGF-2M released from the gel was more than 98%at 4 h,and its bioactivity remained stable.Conclusion The thermo-sensitive gel has the characteristics of good conformability,moisturizing(moisture),isolation,wound healing,and a controlled release effect,which has great potential in wartime for wound repair.
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Objective To observe the effects of keratinocyte growth factor-2 (KGF-2) on the expressions of chemokine FKN and tight junction protein claudin-5 in lung tissue of rats with acute lung injury (ALI). Methods Healthy male Sprague-Dawley (SD) rats were randomly divided into normal saline (NS) control group, ALI model group and KGF-2 pretreatment group, with 10 rats in each group. The rat ALI model was reproduced by injection of 0.01 mL/kg oleic acid into the tail vein, and the rats in NS control group were injected with the same amount of NS. The rats in KGF-2 pretreatment group were instilled with 5 mg/kg KGF-2 in the airway at 72 hours before the model reproduction, and the rats in the NS control group and the ALI model group were instilled with the same amount of NS. The abdominal aortic blood of rats was collected at 8 hours after model reproduction, and then the rats were sacrificed, bronchoalveolar in left lung was lavage, and the bronchoalveolar lavage fluid (BALF) was collected for determination of protein levels in plasma and BALF, and the lung permeability index (LPI) was calculated. The lung tissues were harvested, after hematoxylin-eosin (HE) staining, the histopathological changes were observed under light microscope, and the ALI pathology score (LIS) was calculated. The lung wet/dry weight (W/D) ratio was determined. Immunohistochemistry and Western Blot were used to qualitatively and quantitatively analyze the expressions of FKN and claudin-5 in the lung tissue. The correlation between two variables was analyzed by linear or curve fitting correlation analysis. Results In the ALI model group, the lung tissue was severely damaged, and obvious pathological changes were observed, including thickened alveolar space and inflammatory cell infiltration, and LIS score, lung W/D ratio and LPI were significantly higher than those of the NS control group (LIS: 3.56±0.28 vs. 0.62±0.36, lung W/D ratio: 6.37±0.29 vs. 4.39±0.33, LPI: 3.46±0.69 vs. 0.98±0.17, all P < 0.01). Compared with the NS control group, the positive expression of FKN in the lung tissue of the ALI model group was significantly increased, and the expression level was significantly increased (FKN/GAPDH: 0.97±0.18 vs. 0.62±0.04, P < 0.01); the positive expression of claudin-5 was significantly decreased, and the expression level was significantly decreased (claudin-5/GAPDH: 0.56±0.11 vs. 1.06±0.13, P < 0.01). There was a significant negative correlation between FKN and claudin-5 protein expression (r = -0.817, P = 0.025). After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced, and the pathological changes were significantly improved, and the LIS score, lung W/D ratio and LPI were significantly lower than those of the ALI model group (LIS: 2.41±0.17 vs. 3.56±0.28, lung W/D ratio: 5.45±0.55 vs. 6.37±0.29, LPI: 2.42±0.19 vs. 3.46±0.69, all P < 0.01). Compared with the ALI model group, the positive expression of FKN in the lung tissue of KGF-2 pretreatment group was significantly decreased, and the expression level was significantly decreased (FKN/GAPDH: 0.79±0.03 vs. 0.97±0.18, P < 0.01); the positive expression of claudin-5 was significantly increased, and the expression level was significantly increased (claudin-5/GAPDH: 0.80±0.05 vs. 0.56±0.11, P < 0.01). There was still a significant negative correlation between FKN and claudin-5 protein expression (r = -0.847, P = 0.012). Conclusion KGF-2 may restore the expression of tight junction protein claudin-5 by down-regulating the expression of chemokine FKN, thereby reducing the damage of blood barrier in ALI.
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Objective To explore the effect of keratinocyte growth factor(KGF)on the lung tissue and expres-sion of transforming growth factor - β1(TGF - β1 )in newborn rats with hyperoxia. Methods The 108 newborn SD rats were randomly divided into air group,hyperoxia group and KGF intervention group,and each group had 36 rats. The rats in every group were randomly divided into the 3,7,14 days subgroups,and each group had 12 rats. The rats in the hy-peroxia group and KGF intervention group were continually exposed to more than 950 mL/ L of oxygen box until the end of the experiment. KGF intervention group simultaneously undertook oxygen inhalation,hypodermic injection of 1 mg/ d recombinant human KGF(rhKGF)on the back on the first 3 days and 0. 5 mg/ d 3 days later till the end of the experi-ment. Air group and hyperoxia group were offered equivalent 9 g/ L saline. The rats in the air group took air. The sub -groups of the 3,7 and 14 days were cut for lung tissue in the corresponding time,observing lung tissue by light micro-scope for pathological changes and TGF - β1 protein expressed in the lungs was determined by immunohistochemistry. Results Air group sprout pulmonary alveolus on the 7th day,and the alveolaration finished on the 14th day,while hy-peroxia group had alveolar growth retardation and pulmonary fibrosis,but pulmonary fibrosis was not obvious in KGF in-tervention group. There was a significant difference on 7th day and 14th day between hyperoxia group and air group in TGF - β1(9. 43 ± 0. 64 vs 8. 62 ± 0. 52,P ﹤ 0. 05;9. 97 ± 0. 49 vs 8. 66 ± 0. 48,P ﹤ 0. 01). There was no significant difference between KGF intervention group and air group in TGF - β1(8. 67 ± 0. 55 vs 8. 56 ± 0. 43,8. 77 ± 0. 52 vs 8. 62 ± 0. 52,8. 81 ± 0. 47 vs 8. 66 ± 0. 48,all P ﹥ 0. 05). There's a significant difference on 7th day and 14th day be-tween hyperoxia group and KGF intervention group in TGF - β1(all P ﹤ 0. 05). Conclusions KGF can inhibit the pro-tein expression of TGF - β1 ,and this may be one of the possible mechanism underlying the protective effect of KGF a-gainst lung injury.
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Objective To explore the survival/proliferation,apoptotic and death effects of keratinocyte growth factor (KGF) in alveolar epithelial type Ⅱ cells (AT Ⅱ Cs) exposed to hyperoxia.Methods Primary culture of AT Ⅱ Cs from the Sprague-Dawley rat fetuses was studied under room air condition (210 mL/L O2) and hyperoxic condition (950 mL/L O2) for 0.5-12.0 h.Various concentrations of KGF (15 μg/L,25 μg/L,50 μg/L,75 μg/L,100 μg/L)were added into the cell cultures.Cells were randomly divided into room-air group,room-air-KGF group,hyperoxic-exposure group and hyperoxic-exposure-KGF group.The levels of intracellular reactive oxygen species (ROS),cleaved cysteinyl aspartate specific proteinase-3 (Caspase-3),cell death and proliferation of AT Ⅱ Cs were measured by flow cytometer,Western Blot,release of lactate dehydrogenase assays (LDH assays) and 3-(4,5-Dimethyhhiazol-2-yl)-2,5-diphenyhetrazolium bromide assays (MTT assays),respectively.Results Under room air condition,KGF could significantly increase AT Ⅱ Cs proliferation with 15-100 μg/L in a dose-dependent manner and significantly decrease LDH production at concentrations of 25-100 μg/L.Exposure to hyperoxia resulted in a significant increase in intracellular ROS production in AT Ⅱ Cs in a time-dependent manner compared with that of the room air group.Cell viability decreased and LDH release increased significantly in a time-dependent manner when AT Ⅱ Cs were exposed to 950 mL/L O2 for more than 4 h.After exposure to hyperoxia for 0.5 h and 1 h,KGF could significantly increase AT Ⅱ Cs proliferation in 15-75 μ g/L and significantly decrease LDH production at concentrations of 25-75 μg/L.After exposure to hyperoxia up to 4 h,higher viability was observed in 15 μg/L and 25 μg/L KGF group,and lower death rate presented in 25-100 μg/L KGF group.Further,prolnged hyperoxic exposure for 8 h,high viabilitv was shown only in 50 μg/L KGF group,and less death rate was observed only in 75 μg/L KGF group.In addition,no significant difference in viability and mortality was found between hyperoxic group and hyperoxic-KGF group after hyperoxic exposure for 12 h.Expression of cleaved Caspase-3 was significant higher after 4 h and 8 h hyperoxic exposure than that in room-air group ;at the same time,by adding 25 μg/L and 75.μg/L KGF led to decreased expression of Caspase-3 was detected,compared to hyperoxic group.Conclusions KGF may promote survival/proliferation,inhibited apoptosis and death of rat fetal AT Ⅱ Cs in room air condition or under temporary exposure to hyperoxia in vitro.However,prolonged exposure to hyperoxia may decrease the sensitivity of AEC Ⅱ Cs to KGF and limit its protective effects on lung injury.
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BACKGROUND:The repair and management of ful-thickness skin defects resulting from burns and chronic wounds remain a significant unmet clinical chal enge. Using epidermal stem cel s and keratinocyte growth factor for ful-thickness wound repair is a promising approach. Low-frequency electromagnetic fields which are a non-invasive physical stimulation therapy have been recognized as a good method to enhance wound healing. OBJECTIVE:To develop a new strategy to accelerate wound healing by transplanting transfected epidermal stem cel s and keratinocyte growth factor and treating with low-frequency electromagnetic fields in a mouse model. METHODS:Epidermal stem cel s from Sprague-Dawley neonatal rats were isolated and cultured in vitro, then the cel s were labeled with 5-bromo-2-deoxyuridine and transfected by Ad-KGF, a recombinant adenovirus carrying the keratinocyte growth factor. Mice were given to create ful thickness skin wound on the dorsum and randomly assigned to four groups:control group, transplantation of epidermal stem cel s group, transplantation of keratinocyte growth factor gene modified epidermal stem cel s group, and transplantation of keratinocyte growth factor gene modified epidermal stem cel s plus low-frequency electromagnetic field exposure group. RESULTS AND CONCLUSION:The best healing pattern was observed in the keratinocyte growth factor gene modified epidermal stem cel s plus low-frequency electromagnetic field exposure group (P<0.05) at days 9 and 16. 5-Bromo-2-deoxyuridine labeled cel s existed in the wound in the treated groups at day 9. A significantly increased expression of endogenous keratinocyte growth factor was detected in the transplantation of Keratinocyte Growth Factor gene modified epidermal stem cel s group, and transplantation of keratinocyte growth factor gene modified epidermal stem cel s plus low-frequency electromagnetic field exposure group at day 16. A wel-advanced epithelialization was observed in transplantation of keratinocyte growth factor gene modified epidermal stem cel s plus low-frequency electromagnetic field exposure group at days 16 and 30. These results suggest that low-frequency electromagnetic fields enhanced wound healing fol owing the transplantation of keratinocyte growth factor gene modified epidermal stem cel s.
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Objective To investigate the expression of lung keratinocyte growth factor receptor (KGFR) in rats with acute spinal cord injury (ASCI) in different time points and its role in lung edema.Methods Thirty-two adult Wistar rats weighing 240 g to 260 g were assigned to experimental group (n =16) and control group (n =16) according to the random number table.Each group consisted of time points of 24 hours,3 days,1 week and 2 weeks after the modeling (4 rats per time point).A rat model of ASCI in experimental group was induced at C7 segment by dropping a weight of 10 g from the height of 2.5 cm (Allen' s method).In control group,laminas were removed only,leaving spinal cord at C7 intact.Rats were sacrificed at each time point for measurement of lung wet/dry weight ratio,Western blot analysis of expression of lung KGFR protein and RT-qPCR detection of lung KGFR mRNA expression.Results After ASCI in rats,the expressions of lung KGFR protein and mRNA began to drop at 24 hours (0.23 ±0.06,0,012 1 ±0.002 3),reached the trough at 3 days (0.17 ±0.04,0.008 5 ±0.001 7)and picked up at 1 week.Expression of lung KGFR mRNA in experiment group showed statistically significant difference from that in control group at 24 hours and 3 days (P ＜ 0.05),whereas in each time point the difference of KGFR protein expression between experiment and control groups was statistically significant(P ＜0.05).Variation trend of KGFR expression was in parallel with the severity degree of pulmonary edema.Conclusion Lung KGFR presents significant down-regulation in ASCI rats and this may be associated with the development of pulmonary edema after ASCI.
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Objective To investigate the expression and significance of keratinocyte growth factor-1(KGF-1) in the kidneys of septic rats.Methods Eighty male Wistar rats were randomly divided into the sepsis group and sham operation group.Either group was divided into 3-hour,6-hour,12-hour,24-hour and 36-hour subgroups,with 8 rats in each.Sepsis models were established by cecum ligation perforation (CLP).The kidney and blood samples were respectively taken for pathological study.Serum creatinine (Cr) and blood urea nitrogen (BUN) level determination and leukocyte counting were performed.KGF-1 protein was detected by immunohistochemical method and the expression of KGF-1 genes by real time PCR.Results The renal tubules in sepsis group were injured most at 24h after operation,which were infiltrated by considerable neutrophilic granulocytes,leukomonocytes and mononuclear cells,with vacuolar degeneration,focal hemorrhage and necrosis under the light microscopy.Serum Cr and BUN increased from 6h after operation,and reached the peak at 24h after operation in sepsis group,which were significantly different in those of sham operation group (P< 0.01).Compared with sham operation group,the expression of KGF-1 protein and genes of sepsis group increased significantly and reached the peak at 6h after operation.KGF-1mRNA was positively correlated with BUN and Cr (r=0.504,P<0.01;r=0.382,P<0.05).Conclusion KGF-1 was also expressed in normal kidneys,mainly in tubule epithelial cells of renal medulla and also in that of renal cortex.The expression of KGF-1 in sepsis group was obviously higher than that of sham operation group,and was positively correlated with the degree of kidney damage after infection.KGF-1 was speculated to be one of the protective factors affecting the repair of impaired kidneys caused by infection.
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Objective To investigate the role of hepatocyte growth factor(HGF),keratinocyte growth factor(KGF) and interleukin-1β(IL-1β) in tracheal aspirates (TA) in the genesis and development of acute respiratory distress syndrome (ARDS).Methods The levels of HGF,KGF and IL-1β in TA of 25 children with ARDS (ARDS group) and 23 children with non-ARDS (control group) were assayed by ELISA.Lung injury score was applied to all patients.Results The levels of HGF,KGF and IL-1β in TA were significantly higher in ARDS group than in control group(P<0.0 1).As compared with survivors,the levels of HGF,KGF and IL-1β in TA were markedly higher in dead patients(P<0.01).LIS had a positive correlation with the levels of HGF,KGF and IL-1β(P<0.01).Conclusion HGF,KGF and IL-1β participate in the development of ARDS.The degree of lung injury and prognosis of ARDS may be early estimated by the levels of HGF,KGF and IL-1β in TA.
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Objective To explore the effects of keratinocyte growth factor receptor (KGFR) transgene on sodium channel in alveolar type Ⅱ cells with LPS-induced acute lung injury, to provide the evidence for gene treatment in acute lung injury. Method Totally 40 male Sprague-Dawtey rats were randomly divided into four groups, including normal control (n=8), injured control (n=10), normal transgene (n=10) and injured transgene (n=12). The models of acute lung injury were produced using LPS, and the successful criteria was the obvious enlargement in the lung tissue. The rats in normal transgene group and injured transgene group were injected with 1 mL of KGFR adenovirus vector through rats' tail vein. At 72 hours later, the rats in injured control group and injured transgene group were injected with LPS in dose of 5 mg/kg (BW). While rats in normal control group and normal transgene group were injected with equivalent saline simultaneously. Another 48 hours later, rats in the four groups were killed. The lung tissue were collected for analysis. The expression of sodium channel in rats' alveolar type Ⅱ cells were detected by immunohistochemistry and immunoeectron microscope. Difference among the experimental groups were estimated by ANOVA analysis (LSD-t-test). There was statistical signifi-cance when P<0.05. Results The levels of sodium channel expression in rats' alveolar type Ⅱ cells were differ-era, with normal control group (47.7±3.33), normal transgene group (46.9±5.21), injured tramgene group (29.19±4.11) and injured control group (5.1±2.3). The level of sodium channel expression in injured trans-gene group was lower than that in normal transgene group (t=9.134, P<0.001) and normal control group (t=10.601,P<0.001), but signifieantly higher than that in injured control group (t=16.466, P<0.001). Conclusions The transgene vector can effectively promote the expression of sodium channel in alveolar type Ⅱ cells in rats with LPS-indueed acute lung injury, and can alleviate sodium and water reteraion in alveolar.
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PURPOSE: Growth factors such as keratinocyte growth factor (KGF) and epidermal growth factor (EGF) have been shown to stimulate alveolar proliferation and pulmonary surfactant production in neonatal animals, raising the question of their antenatal uses. We studied the effects of antenatal administration of recombinant human KGF (rhKGF), recombinant human EGF (rhEGF), or dexamethasone (Dexa) in mouse pups on mRNA synthesis of surfactant proteins A, B, and C. METHODS: Time-dated pregnant mice were divided into 5 groups. At gestational day 16, the pregnant mice received intraperitoneal injection of saline, rhKGF, rhKGF+Dexa, Dexa alone, or rhEGF. Fetuses were delivered by cesarean section 24 h later. Lung tissues were obtained for isolation of RNA and realtime RT-PCR for SP-A, -B, and -C. RESULTS: Relative SP-A mRNA levels of any of the treatment groups were not significantly different from the control group. Either KGF or Dexa group did not show higher levels of SP-B mRNA than control group. Relative mean values of SP-B mRNA of KGF+Dexa and EGF groups were higher than the control group, but not statistically significant. Even though there was a trend of increasing levels of SP-C mRNA in all the treatment groups, the differences were not statistically significant. CONCLUSION: Antenatal intraperitoneal administration of KGF, EGF, or dexamethasone to pregnant mice did not increase the mRNA expressions of surfactant proteins in preterm mouse pups. However, the effects of different doses, timing, and routes of administration are important factors that may influence the outcomes and should further be investigated in the future.
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Animales , Femenino , Humanos , Ratones , Embarazo , Animales Recién Nacidos , Cesárea , Dexametasona , Factor de Crecimiento Epidérmico , Feto , Factor 7 de Crecimiento de Fibroblastos , Inyecciones Intraperitoneales , Péptidos y Proteínas de Señalización Intercelular , Queratinocitos , Pulmón , Surfactantes Pulmonares , ARN , ARN MensajeroRESUMEN
Objective To investigate the effect of recombinant human keratinocyte growth factor variant(K102) on mechanical corneal injury in rabbits.Methods Mechanical corneal injury was made in 108 rabbits surgically.K102 eye drop was given respectively at dose of 0.5,0.25,0.125 mg/ml,taking Beifushu eye drops and Dexamethasone sodium phosphate eye drops as the positive control,and K102 eye drop solvent as the blank control;4 times per day.Corneal haze was determined with slit lamp microscope and the histopathological changes were observed under light and electron microscope.Results Three months after operation,the corneal haze was less severe and the average number of corneal cells in shallow 50 ?m corneal basement was less than that in Beifushu group and blank group after 0.5 mg/ml K102 treatment.The results of light and electron microscopy showed the epithelial hypertrophy,disorders of cells and collagen arrangement after 0.5 mg/ml K102 treatment were much lighter than that in Beifushu group and blank group.Conclusion Local application of K102 eye drops reduces the number of stromal keratocyte,then effectively inhibits corneal haze at early stage after mechanical corneal injury in rabbits.
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BACKGROUND AND OBJECTIVES: Epidermal-mesenchymal interactions control epidermal growth and differentiation and thus regulate tissue homeostasis in the epidermis. So the function of fibroblasts is, in addition to producing extracellular matrix as a structural framework, to act as a cellular communication bridge between epidermis and dermis by synthesizing various mediators, such as growth factors and cytokines. Although the epithelial-mesenchymal cell interaction is still not known clearly, cytokines like interleukine-1beta from keratinocyte promote Keratinocyte growth factor (KGF) which is a member of the fibroblast growth factor (FGF-7) group. IL-1beta was shown to be an important modulator of KGF expression by fibroblast cells. Like hepatocyte growth factor, KFG is best characterized as paracrine mediators of stromal-epithelial interactions produced by fibroblast cells to regulate the functions of epithelial cells and the KGF receptors (KGFR) which is a transmembrane tyrosine kinase that is a splice variant of the FGFR-2/bek gene. To study the regulation of epidermal cell proliferation and differentiation by fibroblasts via paracrine effects of oropharyngeal mucosa, an in-vitro model system has been developed to mimic epidermal-dermal interactions. Material and Method: A co-culture of fibroblasts and keratinocytes in three-dimensional collagen gels was treated with IL-1beta after carrying out the tissue culture from oropharyngeal mucosa. Immuno-histochemistry for localization of KFG and KFGR was done in these artificial tissue and in the mucosa. RESULTS: KGF and KGFR proteins were strongly expressed in cytoplasm of intermediate and superficial layers of the epithelium of the oropharyngeal mucosa. In four out of the five cases, three-dimensional oral mucosa cultures were successfully reconstructed on fibroblast-populated collagen lattice. KGF expression was found focally in the keratinocyte of epithelial layer and diffusely in fibroblast-populated collagen lattice. KGFR was only expressed focally in Keratinocyte of epithelial layer. CONCLUSION: Epidermal-mesenchymal interactions in oropharyngeal mucosa via IL-1beta, KGF and KGFR were observed in a three-dimesional culture system, showing that this system could be used as a study model of epidermal-mesenchymal interactions in oropharyngeal mucosa with some limitations.
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Comunicación Celular , Proliferación Celular , Técnicas de Cocultivo , Colágeno , Citocinas , Citoplasma , Dermis , Epidermis , Células Epiteliales , Epitelio , Matriz Extracelular , Factor 7 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos , Fibroblastos , Geles , Factor de Crecimiento de Hepatocito , Homeostasis , Péptidos y Proteínas de Señalización Intercelular , Interleucina-1beta , Queratinocitos , Mucosa Bucal , Membrana Mucosa , Proteínas Tirosina QuinasasRESUMEN
BACKGROUND: Dendritic epidermal T cell(DETC) resident in the mouse epidermis is gammadelta-T lymphocytes. Most of them express Vgamma5/Vdelta1 T cell receptor. Compared with alphabeta-T lymphocytes, physiologic and/or pathologic role of DETC are not fully understood. Keratinocytes under the conditions such as irradiation, viral and bacterial infection, and exposure to carcinogen may express keratinocyte self-antigen. DETC may have the ability to recognize stressed and malignant keratinocytes. Moreover, specific chemokines released from DETC may contribute to inflammatory response, which maintain skin integrity and homeostasis. OBJECTIVE: The purpose of this study was to investigate the effects of transfection of keratinocyte, growth factor(KGF) gene derived from DETC on the keratinocyte proliferation and differentiation in cell culture. METHODS: RT-PCR using KGF primer to confirm the KGF mRNA in DETC and Keratinocyte MTT assay to evaluate the effect of keratinocyte proliferation and immunohistochemical staining using anti keratin(K)14 mAb, anti K1,10 mAb and anti involucrin mAb as a marker to elucidate the effect of keratinocyte differentiation were done. RESULTS: 1.The KGF specific PCR product was demonstrated in DETC, normal positive control of cultured fibroblast and KGF gene transfected cultured keratinocytes, but it was not observed in normal cultured keratinocytes by RT-PCR using KGF-specific primer. 2.Numbers of keratinocytes in normal control group were (2.5+/-0.5)X10(4), (8.8+/-1.5)X10(4), (8+/-0.5)X10(4), (10.5+/-0.7)X10(4) after 1st, 2nd, 3rd and 4th days, respectively. Numbers of keratinocytes with KGF gene transfected group were (1.5+/-1.0)X10(4), (9.5+/-0.2)X10(4), (9.8+/-0.5)X10(4), (13+/-2.0) X10(4) after 1st, 2nd, 3rd and 4th days, respectively. In comparison to the control, the numbers of keratinocytes were increased in the KGF gene transfected group from the 3rd and 4th day(p<0.05). On the MTT assay, compared with the control, the numbers of keratinocytes were also increased in the KGF gene transfected group at 3rd day(p<0.05). 3.In the immunohistochemical staining with mAb to K1,10 and involucrin, to evaluate the degree of differentiatin of kerationcyte reveals that KGF gene transfected cells with Ca++(0.5mM) were stained weaker than the degree of differentiation of Ca++(0.5mM) treated group. Immunohistochemical staining with mAb to K14 reveals that KGF gene transfected cells with Ca++(0.5mM) were stained stronger than the degree of differentiation of Ca++(0.5mM) treated group. CONCLUSION: The above results strongly suggest that KGF, which produced by activated DETC, have a role in stimulating the proliferation and suppressing the differentiation of keratinocytes in the skin. In conclusion, DETC may involved in the control of proliferating activity of neighboring kerationcytes through KGF communication, which play an important role in the maintenance of epithelial integrity.
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Animales , Ratones , Infecciones Bacterianas , Técnicas de Cultivo de Célula , Quimiocinas , Epidermis , Factor 7 de Crecimiento de Fibroblastos , Fibroblastos , Homeostasis , Queratinocitos , Linfocitos , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T , ARN Mensajero , Piel , TransfecciónRESUMEN
Purpose:To investigate expression of keratinocyte growth factor(KGF) messenger ribonuclunc acid in human NSCLC and to study the role of KGF in the development of NSCLC. Methods:The expression of KGF mRNA in 50 cases with NSCLC was detected by in situ hybridization (ISH). Results:On ISH slides, positive KGF mRNA was mainly shown as fusiform stain in plasma of fibroblast and blood vessel smooth muscle cell in mesenchymal of NSCLC, also, some parenchyma cell plasma was stained. The positive rate of KGF mRNA in tumor(86%) is statistically higher than that in normal tissue(24%)(P
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Objective To investigate the effects of keratinocyte growth factor(KGF) and keratinocyte growth factor receptor(KGFR) on the malignant transformation of gestational trophoblastic disease(GTD).Methods Immunolocalization of KGF/KGFR was performed on sections prepared with the samples from 26 hydatidiform mole,18 invasive mole and 12 choriocarcinoma.The in situ hybridization was used to detect the mRNA of KGF/KGFR in the tissues of hydatidiform mole and GTD.Analysis was performed according to intensity of staining and number of positive cells.Results It was revealed that specific staining for mRNA and protein of KGF/KGFR existed in hydatidiform mole and gestational trophoblastic tumor(GTT).The mRNA and protein of KGF/KGFR were allocated in cytoplasm of syncytiotrophoblasts and cytotrophoblasts of malignant hydatidiform mole,and the KGF/KGFR protein was also expressed in benign tissue,while the expression of KGFR in malignant hydatidiform mole was significantly higher than that in benign tissue(?2=12.775,P
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OBJECTIVE: To investigate the effect of recombined human keratinocyte growth factor variant (K102) on corneal alkali burn in rabbit. METHODS: 60 New Zealand rabbits were randomized to 5 groups. Corneal alkali burn models were made and in the left eyes of the rabbits with 1 mol?L-1 NaOH solution. Then the trial groups (groups A, B and C) were treated with 0.5, 0.25, 0.125 mg?mL-1 K102 eye drops, respectively; group D (positive control group) with bFGF eye drops, and group E (negative control group) with solvent of K102 eye drops. The rate of corneal reepithelialization and the area of neovascularization (CNV) were observed under microscope. RESULTS: Within 24 h, the corneal epithelium growth rate in the trial groups (A, B and C) were 1.52, 1.57 and 1.46 mm2?h-1, respectively, which were significantly different as compared with negative control group (0.98 mm2?h-1) (P
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BACKGROUND: Human keratinocyte growth factor(KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblast. In skin, KGF has been shown to stimulate keratinocyte proliferation. OBJECTIVE: In the present study, we sought to determine whether KGF is expressed in human squamous and basal cell carcinoma. METHODS: Using a reverse transcriptase polymerase chain reaction(RT-PCR), we examined the expression of KGF mRNA in normal and cancerous human skin tissues. RESULTS: 1. The 583 bp KGF specific PCR product was observed in normal human cultured fibroblast, but no PCR product was seen in normal human cultured keratinocyte, A431, PAM212, HaCaT, and XB2 cell lines by RT-PCR of KGF mRNA using KGF-specific primer as visualized on an ethidium bromide stained agarose gel. 2. The 583 bp KGF specific PCR product was observed in the normal human dermis, squamous and basal cell carcinoma samples. 3. All three squamous and basal cell carcinoma samples revealed significant overexpression of KGF mRNA transcript in comparison with normal skin tissue. CONCLUSION: These results suggest that KGF mRNA level may be elevated in squamous and basal cell carcinoma and elevated KGF may enhance cellular proliferation of these skin carcinoma.