To investigate the optimum transfection condition in transfecting human keratinocytes with plasmid-liposome complexes in vitro,the cultured human keratinocytes at 60% ～ 100% confluences were exposed to the eukaryotic expression plasmid,pCMV*SPORT-β-gal,coated with LipofectAMINE in different DNA/liposome mixing concentration ratios.After cultured for another 48 hours following the ends of 6 ～ 24 hours exposures to the DNA-liposome complexes,the transfected human keratinocytes were visualized by β-galactosidase staining.Then,the transfection efficiency was determined by calculating the rate of β-galactosidase staining positive cells.β-galactosidase expression was showed clearly in human keratinocytes transfected with the DNA-liposome complexes.The highest efficiency was achieved with cultured cells at 80% and 90% confluences,demonstrating by the transfection rates of (31.35±1.35)% and (32.32±2.47)% respectively.Meanwhile,the essential transfection conditions for these efficiencies were in coating pCMV*SPORT-β-gal DNA of 1.5μg/100μL with LipofectAMINE of 12.5μL/100μL,and exposing the cells to the DNA-liposome complexes for 8 hours.These results indicate that LipofectAMINE could effectively transfer eukaryotic expression plasmid into human keratinocytes in vitro,for which the optimization of transfection conditions involve in cells growth state,DNA/liposome mixing concentration ratio,and exposure time of cells to DNA-liposome complexes.

Objective To study how to reduce the complication rate of laparoscopic cholecystectomy (LC) for acute cholecystitis. Methods A retrospective analysis was made on clinical data of 158 cases of acute cholecystitis treated by LC from September 2001 to December 2003. Results Operations were accomplished laparoscopically in 151 cases, while conversions to open surgery were required in 7 cases (1 case of Mirizzi’s syndrome, 2 cases of gallbladder carcinoma, 1 case of cholecystoduodenal fistula, 2 cases of "ice-like" adhesions in the Calot triangle, and 1 case of common bile duct stones). Open re-exploration was performed in 1 case because of biliary leakage. Intraoperative cholangiography (IOC) was successfully conducted in 10 cases, 3 of which were found common bile duct stones. Out of the 3 cases, 2 cases underwent an intraoperative endoscopic sphincterotomy and 1 received a conversion to open choledochotomy with T-tube drainage. Conclusions When utilizing LC for acute cholecystitis, the rate of conversion and the incidence of complication may be reduced as long as the patients were properly selected, the principles of safety were abided by, and the intraoperative cholangiography was performed routinely.

Objective To explore the similarities and differences in amplitude of low frequency fluctuation ( ALLF) between patients with first-episode drug-naive schizophrenia and offspring of schizophrenia patients.Methods ALFF values were estimated by measuring the Blood Oxygen Level-Dependent ( BOLD) signal using resting state function-al magnetic resonance imaging ( rs-fMRI) .The fMRI date were acquired from 23 patients with first-episode drug-naive schizophrenia (SZ), 25 offsprings of schizophrenia patients (OS) and 29 age -and gender -matched health controls ( HC) .The ALFF value of each subject was calculated by MATLAB-based DPARSF software.Results Compared with HC, the ALFF values of SZ and OS were significantly different in the left posterior part of the inferior temporal gyrus, left parahippocampal gyrus, left hippocampus, right postcentral gyrus and bilateral precuneus.The ALFF values were not signif-icantly different between these two groups in the aforementioned regions.Compared with OS and HC, the ALFF values of SZ were significantly different in the left anterior part of the inferior temporal gyrus, left temporal pole and bilateral calcarine cortex.But there was no significant difference between OS and HC.Conclusions The brain function is abnormal in pa-tients with early schizophrenia and offspring of schizophrenia patients.The significant difference of ALFF in the left posterior part of the inferior temporal gyrus, left parahippocampal gyrus, left hippocampus, right postcentral gyrus and bilateral pre-cuneus may suggest the heredodiathesis-related brain functional alterations.Significant difference of ALFF in the left ante-rior part of the inferior temporal gyrus and the left temporal pole bilateral calcarine cortex may suggest the disease-related brain alterations.

Objective To determine the relationship of cytochrome P-450 epoxygenase metabolites of arachidonic acids with impaired endothelium-dependent vasorelaxation in the healthy elderly. Methods A case-control study was employed.The study enrolled 60 healthy young adults (group A) and 60 healthy senior citizens (group B) of Han population in Shanghai. Serum contents of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) (a stable metabolite of 14,15-EET), 6-Kote-PGF1a (a stable metabolite of prostaglandin) and TXB2 (a stable metabolite of thromboxane A2) were measured using ELISA kits. The endothelium-dependent vasorelaxation (EDV) and endothelium-independent vasorelaxation (NEDV) in brachial arteries were determined by color Doppler ultrasound. Results Compared with group A, group B had significant higher levels of hemoglobin A1c, triglyceride and cholesterol levels (P＜0.01), and significant lower levels of 14,15-DHET and 6-Kote-PGF1a (P＜0.001), leading to increased values of TXB2/6-Kote-PGF1a and TXB2/14,15-DHET. There was bigger basal interior diameter of brachial arterials with reduced EDV and NEDV response (P＜0.01 and P＜0.05, vs. group A respectively) in group B. Moreover, the age was negatively correlated with 14,15-DHET and TXB2/14,15-DHET. Conclusions Our results indicate that the impaired EDV and NEDV in aging are associated with reduced production of arachidonic acid metabolites through cytochrome P-450 epoxygenase pathway and cyooxygenase pathway of endothelium in the healthy elderly.

OBJECTIVETo observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

METHODSFive SD rats were collected to prepare ADSCs with over-expression of GDNF. One hundred and fifty SD rats were divided into normal control group (N), GDNF-ADSCs group (GA), ADSCs group (A), GDNF group (G), and physiological saline group (P) according to the random number table, with 30 rats in each group. Rats in group N were routinely fed without treatment, and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg. Rats in groups GA, A, G, and P were respectively injected with 100 µL suspension of ADSCs with over-expression of GDNF (1 x 10(7) cells per mL), 100 [µL ADSCs suspension (1 x 10(7) cells per mL), 100 µL GDNF solution (100 mg/L) , and 100 µL physiological saline to the surface of the injured nerves immediately after injury. Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8, and morphology of the sciatic nerves was observed in PIW 8. In PIW 4, the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting. Data were processed with one-way analysis of variance, analysis of variance of repeated measurement, and SNK test.

RESULTSCompared with that of group N, the hind limb stride values in groups GA, A, G, and P were significantly lower at each time point (with P values below 0.05). Compared with those of group P, the hind limb stride values in group GA from PIW 3 to 8, in group A in PIW 3, 5, and 7, and in group G in PIW 3, 5, 7, and 8 were significantly longer (with P values below 0.05). The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97), (13.25 ± 1.40), (12.86 ± 1.42), (14.06 ± 1.50), and (15.09 ± 1.17) cm, which were significantly longer than those in group A [(8.90 ± 0.82), (9.03 ± 0.57), (9.27 ± 0.36), (9.86 ± 0.36), and (9.52 ± 0.58) cm] and group G [(8.87 ± 0.69), (8.51 ± 1.18), (9.34 ± 0.87), (9.76 ± 0.67), and (9.50 ± 1.22) cm], with P values below 0.05. Compared with that of group N, the number of myelinated nerve fibers of sciatic nerves was obviously decreased in group P but obviously increased in groups GA, A, and G; the diameter of axons was obviously shorter, and the myelin thickness was obviously increased in groups GA, A, G, and P in PIW 8 (with P values below 0.05). The number of myelinated nerve fibers in group GA was 31.2 ± 0.8, which was significantly higher than that in group A (23.7 ± 2.7), group G (22.3 ± 2.7), or group P (9.3 ± 2.8), with P values below 0.05. The diameter values of axons among groups P, A, G, and GA were similar (with P values above 0.05). The myelin thickness of rats in group GA was (3.41 ± 0.34) µm, which was significantly thicker than that in group A [(2.64 ± 0.37) µm] or group G [(2.41 ± 0.34) µm], with P values below 0.05. In PIW 4, the protein expression of GDNF of sciatic nerves was significantly higher in groups P, A, G, and GA than in group N (with P values below 0.05), and the protein expression of GDNF in group GA was significantly higher than that in group P, A, or G (with P values below 0.05).

CONCLUSIONSADSCs over-expressing GDNF protein can obviously promote the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.

Adipose Tissue ; Animals ; Electrophysiology ; Glial Cell Line-Derived Neurotrophic Factor ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; metabolism ; Nerve Crush ; Nerve Regeneration ; physiology ; Rats ; Rats, Sprague-Dawley ; Sciatic Nerve ; pathology ; physiology

Objective To establish and verify the personalized reference interval of blood cells.Methods The results of blood cells from 2 089 health subjects in 2014,2015 and 2016 were collected.The ratio of the later results to the previous results was defined as the fluctuation (λ).The ratio (λ1) of the results of 2015 to the results of 2014 was calculated and λ1 was analyzed statistically to establish the fluctuation reference interval (CIλ).The ratio (λ2) of the results of 2016 to the results of 2015 was calculated.λ2 was used to verify λ2.The personalized reference interval (CIp) was established by multiplying each result of 2015 and CIλ.CIp was verified by results of 2016.The ratio of the upper and lower limits of CIp was calculated.The ratio of the upper and lower limits of the reference interval (WS/T 405) was calculated.Results The values of CIλ were as follows:WBC (0.66 to 1.53),L(0.67 to 1.51),M (0.50 to 2.00),N(0.56 to 1.78),E(0.4 to 2.51),PLT(0.76 to 1.32),RBC(0.92 to 1.12),Hb(0.92 to 1.11),Hct(0.91 to 1.12),MCV(0.95 to 1.07),MCH(0.95 to 1.05)and MCHC(0.94 to 1.06).The validation tests of CIλ and CIp showed that both CIλ and CIp were suitable for this laboratory.Compared with the reference interval of professional criteria,the ratio of the upper and lower limits of the CIp was smaller than that of traditional criteria.Conclusion CIp for this laboratory was established and verified.Compared with traditional criteria,CIp should be more personalized and highly sensitive.

Objective　To investigate the effects of DiBaiRen decoction on the proliferation of human keratinocytes and its in vitro anti-oxidation role .　Methods　Human keratinocytes were cultured in vitro in the media containing gradient diluted DiBaiRen decoction. The cell proliferation rates at different culture times were recorded. Cellular oxidation injury inflicted by superoxide anion and hydroxyl anion was produced by adding hypoxanthine-xanthine oxidase and hydrogen peroxide to the culture media. MTT colorimetry was employed to determine cellular activity.The antagonistic effects of DiBaiRen decoction on cellular oxidation injury were analyzed.　Results　In the case that DiBaiRen decoction was added to the culture in concentrations of 30ml/L to 100ml/L, there exhibited obvious accelerated proliferation of the keratinocytes,with evident shortening of the cell fusion time. In addition, the oxidation injury of cultured keratinocytes caused by hypoxanthine-xanthine oxidase and hydrogen peroxide was ameliorated or eliminated by the addition of DiBaiRen decoction,and the effects were dose-dependent.　Conclusion　DiBaiRen decoction might possess anti-oxidation effects and could promote the in vitro proliferation of cultured human keratinocytes,and it might be beneficial in wound protection and epithelization.

Objective To evaluate the therapeutic effects of Thesium chinensis Turcz extracts on IgA nephropathy (IgAN) rats .Methods The hot water extracted solution of Thesium chinensis Turcz flow through the AB‐8 macroreticular res‐in column repeatedly ,then being eluted with different contents of ethanol to get four part of extract .The IgAN model of SD rats were established by oral intake of bovine serum albumin(BSA ,400 mg/kg) together with injection of lipopolysaccharide(LPS) and CCl4 .Beginning with 8th week ,the positive group(LGT ,10 mg/kg) ,water extract group(TT ,1 .0 g/kg) ,water soluble group(TTW ,0 .4 g/kg) ,high polarity group(TT20 ,0 .4 g/kg) and medium polarity group(TT50 ,0 .4 g/kg) rats were intragas‐tric administrated drug daily ,the normal group control and model control group rats were given with saline(n=8) .During the next 5 weeks treatment process ,the 24 h urine volume ,24 h urine protein concentration ,and quantity of erythrocyte in urine were observed .The levels of TP ,ALP ,ALT ,AST ,Scr and BUN in serum were determined .The expression of IgA deposition was measured by immunofluorescence staining ,and the pathological changes of kidney tissue were assayed after the therapy process .Results After 5 weeks therapy ,the 24 h urine protein concentration ,the quantity of erythrocyte in urine ,and the level of Scr and BUN of TT and TT50 groups were significantly lower than model control .The fluorescence intensities in the me‐sangial area were weaker than model control .Also ,the degrees of renal capsule expansion and mesangial matrix proliferations , and the kidney tubules damage of TT and TT50 groups were weaker than those of model control .Conclusion The water extract and its medium polarity fraction of Thesium chinensis Turcz show good therapeutic effects on IgAN rats .

OBJECTIVETo lower down the antigenicity of heterogenous swine acellular dermal tissue, and to explore the feasibility of clinical using it as a composite graft for human patients.

METHODSSplit-thickness skin was harvested from healthy swines and then processed by two methods. The swine acellular dermal matrix (sADM) was prepared by removing cells from the skin with trypsin and Triton X-100. Then the cross-linked sADM (sADM(1)) and non-cross-linked sADM (sADM(0)) were embedded subcutaneously in rabbits and also transplanted onto the burn wounds of patients. The histological changes and also transplantation results were observed.

RESULTS(1) In animals with sADM(0) embedded subcutaneously, the grafted tissue was invaded immediately by host cells with obvious inflammatory reaction and tissue degradation. But there was less inflammatory reaction, and with no obvious skin degradation and contraction with sADM(1). (2) In ten burn patients with III degree burn wounds and one patient with wound in chest after scar removal, sADM and ultra-thin skin (UTS) composite graft were grafted on the wounds with autologous thin skin (ATS) and autologous razor-thin or UTS as the control. Nineteen pieces of composite skin of sADM with UTS were grafted on the wounds with survival rate of 78.9%, exhibiting no evident difference with that of ATS. When sADM(0) and UTS were grafed, there exhibited remarkable early inflammatory reaction and wound contraction with similar external appearance with that of UTS. Whereas when sADM(1) and UTS were grafted, there appeared less early inflammatory reaction and wound contraction, resulting in an even appearance and soft to touch similar to that with ATS. But ulceration occurred, with exposure of sADM(1), exposure and severe macrophage reaction to foreign body in 6 wounds of 3 cases 12.8 +/- 6.9 weeks after sADM(1) and UTS grafting.

CONCLUSIONGrafting of sADM as a dermal substitute of composite skin could alleviate early post-grafting immune reaction and improve UTS grafting results. But the delayed graft rejection couldn't be avoided.

Animals ; Burns ; surgery ; Dermatologic Surgical Procedures ; Dermis ; immunology ; transplantation ; Humans ; Rabbits ; Skin ; immunology ; injuries ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Time Factors ; Transplantation, Heterologous ; Wound Healing

OBJECTIVETo investigate the role of substance P in the formation of hypertrophic scar.

METHODSDermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt. The effect of substance P on proliferation of fibroblasts was measured by MTT assay. Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis.

RESULTSSubstance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media. By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P. The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt.

CONCLUSIONThe results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.

Cell Division ; Cells, Cultured ; Dermis ; cytology ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression ; drug effects ; Neurokinin-1 Receptor Antagonists ; Quinuclidines ; pharmacology ; RNA, Messenger ; Substance P ; metabolism ; pharmacology ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1 ; Up-Regulation