Objective To investigate the influence of gender differences on NF-kB activation in livers in septic rats. Methods Total 20 female and 20 male Wistar rats were randomly divided into four groups.Tissue samples of the livers were collected to measure NF-kB activation by EMSA.The level of plasma ALT,TNF-?and estrogen were measured also. Results NF-kB activation in normal male and female rats has no significant difference (P＞0.05).After stimulated by LPS,the level of NF-kB activation and ALT,TNF-?in plasma were markedly upregulated,and the index of female group lower than that in male group (P＜0.01).The level of NF-kB activation in livers and ALT,TNF-?in plasma both in male and female have significantly negative correlation with the level of estrogen in plasma (P＜0.05 ).Conclusion There are significantly gender differences on NF-kB activation in livers in septic rats.Estrogen may decrease the injury of livers in septic rats.

OBJECTIVETo study the effects of glucocorticoid on skeletal muscle protein metabolism in burn sepsis and its possible mechanism.

METHODSThe rats were randomly divided into four groups with 15 rats in each group. Group B, 30% TBSA full-thickness burn was produced on the back and endotoxin (6 mg/kg bw) was given intraperitoneally after the injury to simulate burn sepsis. Groups C and D, glucocorticoid receptor antagonist RU38486 (10 mg/kg bw) was given by gavage 2 hours before or 2 hours after burn with endotoxin, respectively. Group A, the rats received only normal saline in same volume as endotoxin. Plasma levels of cortisol were determined with standard procedure. Extensor digitorium longus muscles (EDL) were procured from both legs 12 hours after the injury. After weighing, the proteolytic rate was determined in vitro in an incubation system with oxygen rich environment by high performance liquid chromatography. The gene expressions of ubiquitin, E(2)-14kDa and C2 in the muscles were determined by Northern blot analysis.

RESULTSThe weight of EDL was significantly lower in group B than in group A (t = 9.03, P < 0.01). Although the weight of EDL muscles was also lower in groups C and D than in group A, it was significantly higher than in group B (t = 2.26, 6.42, P < 0.05 or P < 0.01). The concentrations of plasma cortisol were markedly higher in groups B, C and D than in group A (t = 9.03 - 22.94, P < 0.01). A 58.8% (210/357) of the total and 335.5% (4.16/1.24) of myofibrillar proteolytic rate in group B was higher than in group A (t = 36.99 and t = 46.19, P < 0.01), respectively. The total and myofibrillar proteolytic rate in group D was 28.3% (161/567) and 49.6% (2.68/5.40) and in group C 18.9% (108/567) and 23.2% (1.25/5.40), which were lower than those in group B (t = 5.34 approximately 34.68, P < 0.01), respectively. Although the expressions of ubiquitin mRNA (2.4 kb), E(2)-14 kDa mRNA (1.2 kb) and C2 mRNA in groups C and D were significantly higher than in group A, all the values were lower than those in group B (t = 3.22, 11.32, P < 0.01), especially in group C.

CONCLUSIONSThe proteolytic rate of skeletal muscle, especially the myofibrillar proteolytic rate, was enhanced during burn with sepsis. Hypersecretion of glucocorticoid could upgrade the gene expression of ubiquitin system, resulting in hyperdegradation of skeletal muscle protein during burn with sepsis. Glucocorticoid receptor antagonist RU38486 could decrease the hyperdegradation of skeletal muscle during burn with sepsis.

Animals ; Burns ; metabolism ; Gene Expression Regulation ; Glucocorticoids ; physiology ; Hydrocortisone ; blood ; Male ; Mifepristone ; pharmacology ; Muscle Proteins ; metabolism ; Muscle, Skeletal ; metabolism ; Rats ; Rats, Wistar ; Sepsis ; metabolism ; Ubiquitin ; metabolism

OBJECTIVETo investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs.

METHODShECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture day 1, 3, and 5. (4) Cells were collected and divided into non-transfection group and transfection group, with 12 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in transfection group were transfected with Ad-hEGF gene in the optimum MOI (50). The expression levels of cytokeratin 14 (CK14) and CK19 of cells were measured by immunofluorescence staining at TH 24. (5) Cells were collected, grouped, and treated as in (4), with 6 wells in each group. At post scratch hour (PSH) 0 (immediately after scratch), 12, 24, and 48, the migration distance of cells was observed and measured with inverted phase contrast microscope. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, and LSD test.

RESULTS(1) At TH 24 and 48, morphology of cells in each transfection group and non-transfection group were similar. Compared with that in non-transfection group, the cell debris increased significantly in 200 fold transfection group at TH 72. At TH 24, 48, and 72, the expression of green fluorescent protein was not seen in cells of non-transfection group, whereas it increased in cells of transfection group over transfection time. (2) The transfection rate of Ad-hEGF gene of cells in each transfection group increased gradually over transfection time. At TH 72, the transfection rates of Ad-hEGF gene of cells in 50-200 fold transfection groups were all above 90%, while the transfection rates of Ad-hEGF gene of cells in non-transfection group, 5, and 20 fold transfection groups were (0.51±0.20)%, (62.44±6.23)%, and (75.00±5.43)% respectively, which were obviously lower than the rate in 50 fold transfection group [(93.12±2.55)%, with P values below 0.01]. The mass concentration of EGF in culture supernatant of cells in each transfection group increased gradually over transfection time. At TH 72, the mass concentration of EGF in culture supernatant of cells in 50 fold transfection group was obviously higher than that in each of the other groups (with P values below 0.01). The proliferation activity of cells in each group at TH 24 and 48 was similar (with P values above 0.05). At TH 72, the proliferation activity of cells in 200 fold transfection group was obviously lower than that in other groups (with P values below 0.05). (3) On culture day 1, the biological activity of EGF secreted by cells in two groups was similar (P>0.05). On culture day 3 and 5, the biological activity of EGF secreted by cells in transfection group were obviously higher than that in non-transfection group (with P values below 0.01). (4) At TH 24, the expression levels of CK14 and CK19 of cells in transfection group were higher than those in non-transfection group. (5) The width of scratch in two groups was nearly the same at PSH 0. At PSH 12-48, the migration distance of cells in transfection group was obviously longer than that in non-transfection group (with P values below 0.01).

CONCLUSIONSThe suitable range of MOI of hECs transfected with Ad-hEGF gene is 50-150, and 50 is the optimum. hECs transfected with Ad-hEGF gene with MOI 50 can effectively express the EGF gene and keep its good abilities of proliferation, differentiation, and migration, as well.

Adenoviridae ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; EGF Family of Proteins ; genetics ; metabolism ; Epidermis ; cytology ; Genetic Vectors ; Humans ; Keratins ; metabolism ; Male ; Transfection

OBJECTIVESTo observe the effect of recombinant human growth hormone on metabolism in severely burned patients.

METHODSFrom January 1999 to January 2001, 50 patients, aged 12 to 50 years, with over 30% total body surface area (TBSA) and 10% full-thickness burns, were randomized in a double-blind study. In the control group normal saline was used as a placebo (control group), while 0.3 IU/kg(-1) /d(-1) recombinant human growth hormone was given from postoperative day 1 to day 10 in the rhGH group. The excised burn wounds were closed with microautograft and allograft skin. Blood samples were collected at 6:00 am for assaying of growth hormone, blood glucose, blood insulin, anti-insulin antibody, glucagon, cortisol, serum amino acid profile, transferring, proalbumin, total protein, dielectric, and resting energy expenditure (REE) was also measured.

RESULTSThe concentration of blood GH in both groups was lower (t = 2.806, P < 0.05) than that of physiological values before surgery. However, the concentration of GH on POD 3 in the rhGH group was significantly higher than that of normal values, but a higher level was observed on POD 7 in the rhGH group than that of the control group (t = 3.142, P < 0.05). Although the concentration of anti-insulin antibody was slightly increased, there was no significant difference between the two groups. The concentration of glucagons tended to decrease with an increase in the concentration of blood glucose, and it was marked in the rhGH group. There was no significant difference between the two groups. The concentration of cortisol was higher than normal values, but no significant difference was observed between the two groups. With the administration of rhGH, the plasma concentration of amino acids was lower than that of the control group (t = 2.714, P < 0.05), and the urinary output of 3-MH in the rhGH group was lower than that of the control group (t = 2.207, P < 0.05).

CONCLUSIONSAdministration of rhGH in patients with major burn after surgery could improve their metabolic status, namely, increased lipolysis energy, accelerated protein synthesis, accelerated gluconeogenesis, reduced muscle proteolytic rate, and reduced REE expenditure. There is no effect on stress hormone. rhGH exerts a beneficial effect on metabolism in severely burned patients, but hyperglycemia is apt to occur, and water, Na(+), Cl(-) retention are suggested.

Adolescent ; Adult ; Amino Acids ; blood ; Burns ; blood ; drug therapy ; surgery ; Child ; Female ; Growth Hormone ; therapeutic use ; Human Growth Hormone ; genetics ; therapeutic use ; Humans ; Insulin ; blood ; Male ; Middle Aged ; Recombinant Proteins ; therapeutic use ; Sodium Chloride ; blood