AIM:To investigate LPS(lipopolysaccharide)stimulated cytokine secretion from normal rat kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor - α (TNF- α) and endothelin- 1 (ET- 1 ) secreted by LPS stimulated kupffer cells were detected. RESULTS: LPS had an stimulative effect on kupffer cell activity. LPS in definite concentrations promoted kupffer cell secretion. CONCLUSION: LPS promotes kupffer cell secretion, which may be associated with liver injury induced by LPS.

AIM:To investigate LPS(lipopolysaccharide)stimulated cytokine secretion from normal rat kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured.Tumor necrosis factor -? (TNF-?) and endothelin-1(ET-1) secreted by LPS stimulated kupffer cells were detected. RESULTS: LPS had an stimulative effect on kupffer cell activity. LPS in definite concentrations promoted kupffer cell secretion.CONCLUSION: LPS promotes kupffer cell secretion, which may be associated with liver injury induced by LPS.

Objective To investigate the effects and mechanisms of extraction centipede extraction (CE) on grafting hepatocellular carcinoma (HCC) in nude mice.Methods We set up model of Bel-7404 cells heterotopic grafting HCC in nude mice,and observed the tumor growth and metastasis after intragastric administration of CE (treatment group).Immunohistochemical methods were used to detect X-chromosome linked inhibitor of apoptosis protein(XIAP),bax gene,vascular endothelial growth factor(VEGF) and angiopoietin-2(Ang-2) in tumor tissue.Results There was distinctive inhibitive effect of CE on in Bel-7404 cells heterotopic grafting HCC in nude mice.The staining cells population,staining intensity and the optical density(OD)of XIAP,VEGF and Ang-2 in the control group were higher than those in treatment group,but those of BAX were lower than that in treatment group,and there was significant difference between the 2 groups(P

Objective To investigate the effect of hepatic microcirculatory disturbance induced by intestinal endotoxemia in liver injury. Methods The model of rats with intestinal endotoxemia induced by thioacetamide(TAA) was established. 25 Male Wistar rats were divided into 4 groups as normal control group (N), Heparin control group(H), TAA treated group(T), and TAA + heparin treated group(T +H). The changes of plasma biochemistry and hepatic histopathology were measured. Results The plasma endotoxin level, alanine transaminase (ALT) activity and malondialdehyde (MDA) content in TAA treated rats were markedly higher than that in the control ones (P < 0.05), while plasma endotoxin level was lower ( P > 0.05) and ALT as well as MDA were decreased significantly ( P < 0.05) in TAA + heparin group than in TAA group. Conclusion Intestinal endotoxemia could induce disturbance of hepatic microcirculation, which results in ischemia and hypoxia of liver cell. Heparin could not only correct disturbance of hepatic microcirculation induced by intestinal endotoxemia, but also reduce liver injury induced by ischemia and hypoxia.

Objective To investigate the difference in expression profiles of micro-RNA (miR-NA) between human epidermal stem cells and epidermal keratinocytes.Methods (1) Human primary epidermal stem cells and keratinocytes were obtained with enzyme digestion method and type Ⅳ collagen coated chosen method.Growth of cells cultured in vitro was observed by inverted microscope.Monoclonal antibody of integrinβ1,keratin 1 (CK1),CK10,and CK19 were detected by immunocytochemical staining.(2)Total RNA was respectively isolated from epidermal stem cells and epidermal keratinocytes by Trizol-based single-step procedure,detected by formaldehyde denaturing gel electrophoresis,purified by mirVanaTM miRNA isolation kit,and then labeled and hybridized by miRNA labeling and hybridization kit.Images of hybridization were analyzed using the Feature Extraction (Version 10.7).Data normalization and difference analysis were performed with GeneSpring (GX 10.0).Moreover,miRNA microarray results were confirmed by RT-PCR.(3) Target genes of differently expressed miRNA were predicted.Results Epidermal stem cells exhibited rapid adherence to type Ⅳ collagen and formed distinct clones after 3 days of culture; expressions of integrinβ1 and CK19 were positive.Keratinocytes could not adhere rapidly to type Ⅳ collagen and formed few clones after 3 days of culture ; expressions of CK1 and CK10 were positive.(2) Of the epidermal stem cells,31 miRNAs were up-regulated and 153 down-regulated.Besides,significantly up-regulated miRNAs were hsa-miRNA-125b-3p,hsa-miRNA-197-5p,and hsa-miRNA-376a-3p,while significantly down-regulated miRNAs were hsa-miRNA-203,hsa-miRNA-29b-3p,and hsa-miRNA-34a-3p.Findings of RT-PCR on hsa-miRNA-197-5p and hsa-miR-29b-3p revealed high concordance with the microarray results.(3) Some miRNAs target genes indicated that miRNA related to cell proliferation,differentiation,apoptosis,aging and other biological characteristics.Conclusion Distinct differences in miRNA expression profiles are detected between human epidermal stem cells and keratinocytes and this may correlate closely with their different biological characteristics such as proliferation and differentiation ability.

In the human body, the structure of the organ and tissue is its functional foundation. With the human body skin losing, the skin also loses its function. When the skin defect area is quite large, it is the question that the source of the autologous skin is deficient. But with the engineering research progressing, the application of artificial skin to repair the skin defect is possible. In order to make the tissue-engineered skin have "normal" skin function, the structure research also should make them approach as far as possible to be similar. This article reviews the constructs, properties and applications of the epidermal substitutes, the dermal substitutes and the skin substitutes. With continued development of multi-discipline and continued progress of basic and clinical research, the constituted tissue-engineered skin substitutes will more approach the normal skin in the structure and properties.

BACKGROUND:Vaginal smooth muscle cells are mainly cultured by enzyme digestion method and explant method.The former method requires a short culture time with a high output,but this method demands a large number of tissues with a high opportunity of pollution,and the optimal digestion time is difficult to control.The latter method is simple and effective,but the culture needs a long time.OBJECTIVE:To observe the culture time of rabbit vaginal smooth muscle cells using explant + enzyme digestion methods,and to compare with the explant method.DESIGN,TIME AND SETTING:The in vitro observation experiment was performed at the Burn Institute and Animal Laboratory of First Affiliated Hospital,Nanchang University from February 2005 to February 2006.MATERIALS:Female New Zealand rabbits aged 4-5 months,with the body mass of 2.0-2.5 kg were selected for this study.METHODS:Vaginal smooth muscle cells using explant method:1 mm?1 mm?1 mm explants were uniformly incubated in a culture medium,and then placed in a incubator at 37 ℃ for 3.0-4.0 hours.After tissue confluence,tissues were immersed in DMEM containing 0.1 volume fraction,stored at 37 ℃ for 3.0-4.0 days.Following cells grew from the tissue islets,the medium was changed about once every three days.Vaginal smooth muscle cells using explant + enzyme digestion methods:The tissue pieces were digested by 0.1% collagenase type Ⅳ at 37 ℃ for 0.5-1 hour,and then enzymatic digestion was interrupted when the edge of tissue pieces became coarse.These pieces were cultivated in culture dishes.The remaining steps were the same as the explant method.Serial subculture:The 1st subculture was made when 50%-60% cells were confluent.After the 2nd passage of cells was obtained,subculture was made when 80% cells were confluent.MAIN OUTCOME MEASURES:Time of primary culture;Surface structure and ultrastructure of the 3rd passage of vaginal smooth muscle cells.RESULTS:The eruption time and the confluent time of vaginal smooth muscle cells could be shortened by the explant + collagen digestion methods about 1-2 days and 3-4 days respectively compared with the explant method only.Vaginal smooth muscle cells,which were obtained by the two methods mentioned above,could propagate for 5-6 passages.The attached cells were polygonal or spindle and after confluence could be seen,the marked smooth muscle cells "Peak-Valley" structure in some domains under the inverted microscope.The third passage of smooth muscle cell nuclei were serration;the cytoplasm contained the marked smooth muscle cell structural myofilaments and dense bodies and plenty of golgi bodies;the rough endoplasmic reticulum broadened;the free ribosome were abundant under the transmission electron microscope.This indicated that vaginal smooth muscle cells with synthesis phenotype could be greatly collected.CONCLUSION:Explant + enzyme digestion methods need a short cycle of primary culture.Vaginal explants should be kept moisture during drawing materials,isolation and the whole.The time of enzymatic digestion should not be too long,to the point that palpi pilosi is seen surrounding the explants.Primary culture should remain static state and use of plastic culture dishes.

Objective To investigate the changes of human epidermal stem cells after transfected with human telomerase reverse transcriptase(hTERT)gene.Methods The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into in vitro cultured human fetal epidermal stem cells by liposome-mediated transfection.Then,the positive cells were selected with G418.The mRNA and protein expressions of hTERT were detected by reserve transcriptase-polymerase chain reaction(RT-PCR)and Western blot.The telomerase activity and the proliferation and cycle of human epidermal stem cells were detected by telomeric repeat amplification protocol(TRAP)-ELISA and flow cytometry respectively.Results RT-PCR and Western blot techniques detected weak mRNA and protein expressions of hTERT gene in untransfected and vacant vector transfected cells but high level of mRNA and protein expressions of hTERT gene in pIRES2-EGFP-hTERT transfected cells.Compared with untransfected and vacant vector transfected cells,the pIRES2-EGFP-hTERT transfected cells had higher telomerase activity,with lower proportion of cells at G_0/G_1 phase,higher proportion of cells at S and G_2/M phases and enhanced proliferation ability.Conclusion Transfection with hTERT gene can markedly enhance mRNA and protein expressions,telomerase activity and proliferation ability of hTERT gene of human epidermal stem cells euhured in vitro.

Objective To investigate the cell cycle and mechanisms of HCC Bel-7404 cell line by treatment of extract of centipede ( ECP). Method Bel-7404 cell line was cultured in vitro. ECP was applied to the interference of the growth of Bel-7404 with different drug concentration. Cell cycles and apoptosis ratios of ECP group were detected by flow cytometry (FCM). To investigate the expression of apop-tosis related genes XIAP and Bax, we also detect HCC Bel-7404 cell line after 48 hours treated with ECP by immunohistochemical method. Result The results of FCM displayed that ECP in treatment group mainly retard cell cycle phase in G0/G1 after 48 hours. The ratios of G0/ G1 .proliferation index (PI) and apoptosis ratios were significantly different between treatment group and control group when the concentrations were different. It was also significantly different between treatment group and control group when the same concentration of ECP was used. It showed that XIAP gene expression decreased gradually while Bax gene expression increased with the increase of ECP concentration, and these were statistical significance in contrast to control group ( P <0.05). Conclusion ECP mainly retarded cell cycle phase of Bel-7404 in G0/G1, suppressed the proliferation, and could induce it to apoptosis. The inhibitory effect was time - concentration dependent. The mechanisms were due to promote Bax gene and inhibit XIAP gene expression in HCC Bel-7404 cell line.

Objective In order to know the effective of local oxygen therapy to promoting renovation of residual surface of bums wound.Methods Divided 60 patients with burns into the experimental group and the control group,there were 30 cases in the each group.Routine nursing cares were used in the control group,while the local oxygen therapy was used in the experimental group.Compare the condition of secretion of wound between the two groups.Results The wound healing time of experimental grou was(5.87±3.29)d,which significant shorten than that of in the control group,the kinds of bacteria in the control group was more than in the experimental group.Conclusions The local oxygen therapy can effective promote the healing of residual surface of bums wound,which can reduce the infection rate of bacteria.