Objective To study the ability of silica nanoparticles as gene carriers for adult human epidermal keratinocytes gene transfection.Methods The silica nanoparticles-DNA conjugated with the enhanced green fluorescence protein plasmid DNA(pEGFP-N_1) was transfected into adult human epidermal keratinocytes.The expression of green fluorescence protein was investigated in transfected keratinocytes by eletromicroscope examine and the efficiency of gene transfection was revealed.Results The silica nanoparticles-DNA complexes can be effectively transfected into adult human epidermal keratinocytes and the efficiency of gene transfection was about 20%～30%.Conclusion The silica nanoparticles can be used as DNA carriers for gene transfection,and can efficiency transfect the pEGFP-N_1 into adult human epidermal keratinocytes.

Objective To study the expression characteristics of cytokeratin 19(CK19) in different live tissues from victims with third-degree burns to explore the possible mechanisms of tissue reparation and regeneration in orthophoria.Methods 10 young victims with third-degree burns were included in this study.Alive tissues at different regeneration stages were studied by immunohistochemistry with streptavidin-biotin-peroxidase method using specific anti-CK19 monoclonal antibody. Only cytoplasm expression was considered as specific.Normal skin tissue and chronic skin ulcer were used as controls.The study was focused on the distribution and morphological features of CK19-positive cells.Results ⑴In normal skin ,CK19-positive cells were seen in basialis layer of epiderm and cutaneous appendages.⑵CK19-positive cells were not found in subcutaneous tissue at 3～6 days after the injury,but they were seen in granulation tissues.⑶A lot of CK19-positive cells were detected in early regeneration tissues.⑷In late regeneration tissues,CK19-positive cells were detected in basialis layer of epiderm and cutaneous appendages.⑸Advanced regenerationed epidermal tissue was similar to that of normal.⑹No CK19-positive cells were observed in all of 6 cases with chronic skin ulcer.Conclusion It is possible that victims with third-degree burns could be healed by tissue regeneration orthophoria.

Objective To probe the effects of recombinant adenovirus containing Akt on carbon tetrachioride-induced rat liver cirrhosis and portal hypertension. Methods Cirrhosis was induced in rats by a complex method of carbon tetrachloride. Recombinant adenovirus Ad-myr-HA-Akt was produced by homologous recombination in 293 cells. Rats received Ad-myr-HA-Akt via the tail vein at the second and the sixth week respectively. The pathological changes in liver tissues were observed after Van Gieson (VG) staining. Fas antigen in rat livers were determined by immunohistochemical method. The levels of alanine minotransferase( ALT), aspartate aminotransferase ( AST), albumin( ALB ) and hydroxyproline (Hyp) were measured. Fas antigen in rat livers were determined with immunohistochemical method. Expression of Akt, p-Akt, Fas and DR5 were evaluated by Western blotting. Frozen sections of the liver, heart,lung,kidney, brain,spleen and testis were made to examine the expression of enhance green flourescent protein (EGFP) by fluorescence microscopy in EGFP group. After 8-week CCl4 treatment, portal hypertensive rats in the saline group and Ad-Akt group received saline and Ad-myr-HA-Akt via the tail vein respectively. Portal vein pressure, mean arterial pressure and heart rate were measured in all rats on Day 3. Results In comparison with other cirrhosis rats, the pathological changes in the Akt group was markedly attenuated, and the levels of ALT, AST and Hyp were significantly lowered. Western blotting showed that the protein expression of p-Akt in the Akt group was higher significantly as compared with those in the negtive control group, saline group and EGFP group. Western blot also showed that the protein expression of Fas and DR5 in the Akt group was lower significantly. EGFP expression was mainly demonstrated by fluorescence microscopy on the frozen section of liver, very little fluorescene were detected in lung and kidney and there was no detectable EGFP in the other organs. Conclusions Ad-myr-HA-Ak inhibits CCl4-induced liver cirrhosis and is a potential pharmacological target for gene therapy in liver cirrhosis.

Objective To investigate the clinical efficacy and mechanism of vacuum-assisted closure (VAC) in the preoperative and postoperative treatment of bedsore united with skin flap.Methods Twenty two cases with bedsore were randomly divided into experimental and control groups.In the control group,the surgery of flap was performed after the treatment of continuous negative pressure about 7-10 days and the VAC was not applied after operation.While in the experimental group,VAC was not used before operation.It was applied on flaps as soon as sutured the border of flap and decubitus ulcers and removed after 7-10 days.By comparing the general appearance of two groups,microvessel count and the detection rate of bacterial culture and other indicators,the clinical effects of two treatments were investigated and the preliminary mechanism was analyzed.Results After preoperative VAC treatment,11 cases of control group showed a little granulation tissue growth,less subcutaneous hematoma and wound effusion,increased microvessel count and negative bacterial culture.However,there were 4 cases of death cavity residual,subcutaneous hematoma and wound effusion,positive bacterial culture and another 4 cases of delayed healing with skin flap repairing bedsore.The application of VAC in experimental group showed close contact of flap with the basement,less effusion,increased microvessel count and negative bacterial culture.One case of skin flap had a small area of separation,after the dressing of skin and the flap survived.The other wounds healed by first intention.Conclusions The use of VAC to repair bedsore can reduce the number of operation,and it is beneficial to the flap survival.

Objective To construct the eukaryotic expression plasmid containing human epidermal growth factor(hEGF) gene with signal peptide(SP).Methods After two pairs of primers were designed and synthesized,the cDNA fragment of hEGF and SP genes were amplified from total RNAs. The amplified cDNA fragments were cloned into pGEM-T vector.The expression plasmids were verified by double endonuclease digestion and DNA sequence analysis. Results With RT-PCR using two pairs of primers,two bands(about 90bp and 180bp) were obtained and confirmed as signal peptide and EGF cDNA fragment with electrophoresis analysis and DNA sequencing after cloned into pGEM-T vector.The SP and EGF cDNA fragments were inserted into plasmid pcDNA3.1(+).The bands of 240bp and 5.4kb were obtained and identified as the full length of SP-EGF cDNA fragment by DNA sequence analysis.Conclusion The eukaryotic expression plasmids containing hEGF gene is successfully constructed.

Objective To confirm whether or not let-7b and miR-199a were significantly associated with malignant melanoma growth and proliferation. Methods An over -expression plasmid and an inhibitor, which targeted on let-7b and miR-199a, was constructed. B16F10 cells were divided into seven groups: control group, let-7b plasmid group, miR-199a plasmid group, empty plasmid group, let-7b inhibitor group, miR-199a inhibitor group, inhibitor control group. Foreign gene was transfected into B16F10 cells, let-7b and miR-199a expression were validated from RNA level, protein level and cell level. Results The relative let-7b or miR-199a gene expression of the let-7b plasmid group (3.8776±0.1372)and miR-199a plasmid group (2.8660±0.2821)were significantly higher than control group (P＜0.05), the relative let-7b or miR-199a gene expression of the let-7b inhibitor group (0.2057±0.0263) and miR-199a inhibitor group(0.2656±0.0253) were significantly lower than control group(P＜0.05). The cyclinD1 expression of the let-7b plasmid group(2.023±0.315) and let-7b inhibitor group (1.857±0.377) were significantly higher than control group (0.997±0.041) (P＜0.05), whereas, the Met expression of themiR-199a plasmid group (5.19±0.309) and miR-199a inhibitor group (4.87±0.044) were significantly higher than control group (2.2±0.198) (P＜0.05). The let-7b plasmid group and miR-199a plasmid group B16F10 cell growth rate were slower than control group, especially on the third day after transfection, the growth rate gradually dropped to the lowest value (P＜0.05). In addition, the apoptosis rates of the let-7b plasmid group and miR-199a plasmid group reach to (11.8±1.19)% and (11.3±1.59)%,which were significantly higher than control group (P＜0.05). Conclusions let-7b and miR-199a may be a negative regulator on the B16F10 cell growth and proliferation.

Objective To study the influence of let-7b on cell proliferation and aerobic glycolysis of human melanoma cell A375.Methods Transfect A375 cell line with hsa-let-7b oligonucleotide or antisense.Glucose and lactate in medium were determined by spectrophotometry at 24 h and 48 h time point after transfection.The cell proliferation was determined by methylthiazol tetrazolium (MTT) assay.Results Over expression of let-7b in melanoma cell reduced cell proliferation notably,compared to the other groups by MTT(P ＜0.05).However,the glucose consumption and lactate production differences were not observed during 24 h or 48 h ( P ＞ 0.05 ),the blank control group transformed about 57％ and 43％ glucose to lactate during 24 h and 48 h.Conclusions Melanoma cell line A375 has notably aerobic glycolysis hallmark,let-7b could inhibit proliferation of melanoma cell line A375,but it may has no influence on glucose metabolism.

OBJECTIVE: To observe the expression of high mobility group box chromosomal protein 1(HMGB1) in RAW264.7 macrophages after interfering with burning serum and qinghuobaidu-yin (QHBDY), and to find out the endogenous protection mechanism of QHBDY resisting inflammation reaction. METHODS: RT-PCR was used to detect the expression of HMGB1 in RAW264.7 macrophages after interfering RAW264.7 macrophages with normal SD rat serum, burning SD rat serum, and QHBDY feeding SD rat serum. RESULTS: Small quantity of HMGB1 mRNA was expressed in RAW264.7. The expression of HMGB1 mRNA fluctuated around the standard level after interfering with normal serum of SD rats. The expression of HMGB1 mRNA rose at 3 h, and then decreased to the standard level; at 18 h, it rose rapidly; at 36 h, it reached the peak; and at 48 h, it remained at the high level after interfering with burning serum. The expression of HMGB1 mRNA increased at 3 h, and then decreased to the standard level. At 24 h, it started to rise after interfering with herb serum, and was lower than that of; the burning serum group (P<0.05). CONCLUSION Burning serum can increase the expression of HMGB1 mRNA in RAW264.7. QHBDY can decrease the high expression of HMGB1 mRNA in RAW264.7 caused by burning serum.
Animals ; Burns ; complications ; immunology ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; HMGB1 Protein ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; Male ; Mice ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Systemic Inflammatory Response Syndrome ; etiology ; metabolism ; prevention & control

MicroRNAs have been identified as a new class of regulatory molecules that affect many biological functions by interferring the target gene expressions. Latest studies demonstrate that microRNAs can influence many pivotal bio-processes and deeply involve in the metabolism of glucose, lipid and amino acid and biological oxidation. For glucose metabolism, microRNAs are related to insulin secretion, insulin sensitivity, glucose uptake, glycolysis, oxidation and mitochondrial function. For lipid matebolism, microRNAs can regulate the target genes related to lipid biosynthesis, catabolism and transportation. MicroRNAs can influence glutamine catabolism.
Animals ; Glucose ; metabolism ; Glutamine ; metabolism ; Humans ; Insulin ; metabolism ; Insulin Secretion ; Lipid Metabolism ; physiology ; Metabolism ; physiology ; MicroRNAs ; physiology