Objective To evaluate the effect of low-molecular weight heparin (LMWH) on biological behavior of a human cutaneous squamous cell carcinoma cell line A431.Methods To optimize the concentration of LMWH,A431 cells were treated with different concentrations (12.5,25,50,100 and 200 IU/ml) of LMWH for 24,48 and 72 hours followed by CCK-8 assay for the detection of cell viability.Then,A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24,48 and 72 hours.Subsequently,flow cytometry was performed to assess cell cycle,real time quantitative PCR (RT-qPCR) and Western blot to quantify the expression of vascular endothelial growth factor (VEGF) mRNA and protein respectively,double-antibody sandwich enzyme linked immunosorbent assay (ELISA) to determine the expression level of VEGF protein in the supernatant of A431 cells,wound-healing assay,Transwell assay,and adhesion assay to observe the migration and adhesivity of A431 cells.Analysis of variance and t test were carried out for statistical analysis.Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay,and used in the following experiment.The LMWH of 200 IU/ml resulted in a decrease in cell viability,cell cycle arrest,an increase in cell percentage in G1 phase,and a reduction in cell percentage in S phase.The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml,significantly lower than that in untreated A431 cells (48.62 ± 4.50,t =6.14,P ＜ 0.05; 46.86 ± 3.51,t =9.78,P ＜ 0.05).A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA (10.16 ±0.07 vs.18.77 ± 0.11,4.11 ± 0.01 vs.17.39 ±0.05,t=114.38,451.10,both P＜ 0.05),VEGF protein (0.16 ± 0.01 vs.0.20 ± 0.01,0.12 ± 0.01 vs.0.21 ± 0.01,t =4.90,11.02,both P ＜ 0.05),and in the supernatant level of VEGF protein ((67.17 ± 3.34) ng/L vs. ( 122.63 ± 23.17) ng/L, (28.14 ± 3.14) ng/L vs.(86.76 ± 1.18) ng/L,t =4.10,30.27,both P＜ 0.05).The percentage of adherent cells was 29.7％ ± 1.92％ and 17.5％± 0.79％ in LMWH-treated A431 cells at 48 and 72 hours,respectively,significantly lower than that in untreated A431 cells (36.9％ ± 0.35％,34.6％ ± 0.96％,respectively,both P＜ 0.05).The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24,48 and 72 hours.Conclusion LMWH may suppress the proliferation,migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.

Objective To observe the effect of short hairpin RNA (shRNA)-mediated vascular endothelial growth factor (VEGF) gene silencing on the growth of human skin squamous cell carcinoma(SCC) xenografts in nude mice.Methods Two eukaryotic expression plasmids targeting VEGF gene,including psilencer-VEGF1-shRNA (VEGF-s1) and psilencer-VEGF2-shRNA (VEGF-s2),as well as one negative control plasmid containing random target sequence (psilencer-Target-off-shRNA,T-off),were chemically synthesized,and transfected into a human skin SCC cell line A431 to develop stably transfected cell lines.Real time quantitative PCR (RT-qPCR) and double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were carried out to measure the expression of VEGF mRNA and protein respectively in A431 cells.Twelve nude mice were divided into 4 goups to be subcutaneously inoculated in the axillary region with untransfected A431 cells as well as A431 cells transfected with VEGF-s1,VEGF-s2 and T-off,respectively.The tumor growth was observed in nude mice every 5 days.Twenty days after the inoculation,the mice were sacrificed,and transplanted tumors were obtained from the mice and subjected to an immunohistochemical study for the measurement of VEGF,proliferating cell nuclear antigen (PCNA) and CD34 expression.Data were statistically analyzed by using the Stata 7.0 software,and t test was conducted to compare the differences between groups.Results The mRNA and protein expression levels of VEGF were significantly lower in A431 cells transfected with VEGF-s1 and VEGF-s2 than in untransfected A431 cells (27.85 ± 3.95 and 24.69 ± 2.83 vs.54.06 ± 6.38,t =6.05,7.29,both P＜ 0.01; 32.67 ± 2.52 and 29.27 ± 1.10 vs.52.85 ± 2.23,t =8.04 and 11.53,both P＜0.01 ).Twenty davs after the inoculation,the volume and weight of xenografted tumors in mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells were significantly lower than those in mice with untransfected A431 cells ( ( 192.50 ± 10.90) mm3 and (203.67 ± 3.21 )mm3 vs.(272.00 ± 21.07) mm3,t =5.80 and 5.55,both P＜ 0.01; (0.05 ± 0.03) g and (0.13 ± 0.04) g vs.(0.25 ± 0.02) g,t =9.60 and 4.64,both P＜ 0.01 ).Decreased expression rate of VEGF,PCNA and number of CD34-positive vessels were observed in the xenografted tumor tissue from mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells compared with that from mice with untransfected A431 cells (52.00％ ± 2.00％ and 56.67％ ± 3.06％ vs.70.00％ ± 2.00％,both P ＜ 0.01;37.01％ ± 2.41％ and 33.94％ ± 3.25％ vs.72.11％ ± 3.02％,both P＜ 0.01; 2.05 ± 0.07 and 1.72 ± 0.10 vs.4.01± 1.27,both P ＜ 0.01).No significant differences were observed in the above parameters between cells transfected with VEGF-s1- and VEGF-s2-transfected A431 cells,between untransfected and T-off-transfected A431 cells,between tumor xenografts derived from VEGF-sl- and VEGF-s2-transfected A431 cells,or between tumor xenografts derived from untransfected and T-off-transfected A431 cells (all P ＞ 0.05).Conclusions The shRNA targeting VEGF gene can significantly inhibit the expression of VEGF in A431 cells and A431-derived tumor xenografts in nude mice,in turn suppress the growth and attenuate the malignant phenotype of tumor.

Objective To investigate the methods and techniques of simplified binding pancreaticojejunostomy for patients with periampullary malignant tumor after radical pancreatoduodenectomy (RPD). Methods From March 2005 to May 2010, 323 patients with periampullary malignant tumor received RPD at the Tongji Hospital of Huazhong University of Science and Technology, and their clinical data were retrospectively analyzed.Simplified binding pancreaticojejunostomy was applied after RPD: the distal end of pancreas was freed for 3-4 cm;a No. 6 or No. 8 silicone urinary catheter was inserted into the pancreatic duct for 4-5 cm, and the remaining urinary catheter (6-8 cm) out of the pancreatic duct was sutured to the pancreatic stump with absorbable sutures.The cutting end of the jejunum (2-3 cm) was everted, and the everted mucosa of the jejunum ( 1 cm) was injured by electrocautery, then the everted jejunum was reverted to its normal position. The cutting end of the mesentery of jejunum and its opposite side, as well as the mid-point of these two parts were sutured symmetrically with the lower and upper edges of the pancreas, and with the capsule of pancreas between them. The everted jejunum was wrapped over the pancreatic stump and sutured it to the pancreas for fixation. The cutting end of the jejunum was bound to the pancreatic stump with 1-0 absorbable suture after confirming the jejunum was completely invaginated into the pancreas. The alimentary tract was reconstructed by using Child's method. Results Simplified binding pancreaticojejunostomy was successfully completed in all patients, Pancreatic fistula was detected in one patient who was complicated with anastomotic bleeding on the third day after secondary laparotomy. The patient was discharged with catheter and spontaneously recovered one month later. Pancreatic fistula was also detected in two patients with distal bile duct carcinoma and two patients with carcinoma in the uncinate process of pancreas at postoperative day 3, 6, 8 and 11, and they were cured by expectant treatment. The incidence of pancreatic fistula was 1.5% (5/323). Conclusion Simplified binding pancreaticojejunostomy is simple, safe and feasible, and it can significantly reduce the incidence of pancreatic fistula.

Objective To explore the repair possibilities of endothelial progenitor cells (EPCs)in peripheral blood in patients with different extents of obstructive sleep apnea (OSA) through measuring the levels of pro-angiogenic factors and different subgroups EPCs in peripheral blood in patients with OSA. Methods Ninety adult patients with OSA, 30 healthy controls with matched age and gender were enrolled for this study. The subjects performed Polysomnography, were divided in-to four group based on Apnea Hypopnea Index (AHI). The serum levels of HIF-1α, SDF-1αand VEGF were assessed by ELISA. Mononuclear cells were isolated from peripheral blood with density gradient centrifugation, and flow cytometry was used to detect levels of CD133+KDR+EPC, CD133+CD34+EPC, CD34+KDR+EPC and ALDHloCD34+KDR+EPC based on AL-DH activity, and CD133, CD34, PE-KDR related cell surface markers. Results The levels of CD133+KDR+EPC, CD133+CD34+EPC, CD34+KDR+EPC were higher in OSA groups than those of control group, both of which were higher in severe OSA group than those of in mild and moderate OSA groups. The levels of ALDHloCD34+KDR+EPC were higher in mild and moderate OSA groups than that of the control groups, and the levels of ALDHloCD34+KDR+EPC were significantly lower in se-vere OSA group than those of control, mild and moderate OSA groups. Serum levels of HIF-1α. VEGF were significantly high-er in OSA groups compared to those in control groups, both of which were higher in severe OSA group than those of mild and moderate OSA groups. Serum levels of SDF-1αwere significantly lower in severe OSA groups than those of mild, moderate OSA and control groups (P<0.05). Conclusion The mobilization and recruitment of different subtypes of EPCs are obvious-ly increased in patients with OSA, but ALDHloCD34+KDR+EPC with vascular repair capacity keeps to invariability, even de-creases in patients with severe OSA, which results in endothelial damage, and increases the risk of cardiovascular disease.

ObjectiveTo discuss the role of transient receptor potential vanilloid 1 (TRPV 1) in the regulation effect of electroacupuncture on gastric motility.MethodTRPV1 gene knock-out mice (KO mice) and wild-type C57BL/6 mice (WT mice) were selected to receive acupuncture at Zusanli (ST36),Quchi (LI11), Zhongwan (CV12), and Weishu (BL21), and the intragastric pressure was observed before and after acupuncture.ResultElectroacupuncture at Zusanli caused both excitation and inhibition in WT mice, predominated by mild excitation, while electroacupuncture at Weishu, Quchi and Zhongwan all caused inhibition effect; in the KO mice, electroacupuncture at Zusanli, Quchi, Zhongwan, and Weishu all inhibited gastric motility.Conclusion TRPV1 bears certain regulating effect on gastric motility, andacupuncture can inhibit the gastric motility in TRPV gene KO mice.

Objective To explore the mechanism of vessel endothelial dysfunction in rats under intermittent hypoxia (IH). Methods The respiratory simulation system was used to simulate IH. Sixty C57BL/6J rats (male) were randomized into control group and IH group. The rats of IH group were exposed to IH 8 hours per day for 6 weeks. The serum levels of hypoxia inducible factor (HIF)-1a and stromal cell derived factor (SDF)-1a were assessed by ELISA. The serum levels of reactive oxygen species (ROS) were detected in two groups. The serum expression of miR-199a-5p was detected by real-time fluorescent quantitative PCR in two groups. The dual luciferase report system and point mutation test were used to verify target gene for HIF-1a. Results The serum levels of HIF-1a and SDF-1a were significantly higher in IH group than those of control group (μg/L:1.60±0.02 vs. 1.19±0.02, 1 823.00±8.97 vs. 1 444.00±17.90, P<0.01). The serum level of ROS was significantly higher in IH group than that of control group (U/mL:487.66±35.73 vs. 211.57±23.82, P<0.01). The serum level of miR-199a-5p expression was significantly lower in IH group compared to that of control group (1.31±0.07 vs. 3.47± 0.17, P<0.01). The result of dual luciferase reporter gene detection confirmed that target gene of miR-199a-5p was HIF-1a. Conclusion The serum level of miR-199a-5p is decreased first due to IH, and then its target gene (HIF-1a) is increased. HIF-1a can induce the increased level of SDF-1a, and its receptor (CXCR-4 ) is also increased. Finally, HIF-1a can increase the serum level of ROS, resulting in the endothelial dysfunction.

Objective To prospectively assess the nutritional status in the patients with alimentary tract malignancy,and to elucidate the factors related to malnutrition.Methods The nutritional status of 328 patients with newly diagnosed alimentary tract malignancy was assessed using subjective global assessment(SGA)and serum levels of prealbumin and albumin.And the factors influencing the nutritional status of the patients with alimentary tract malignancy in different locations were analyzed.Results The prevalence of malnutrition was 64.43% in all,75.81% in colon cancer,63.24% in esophageal cancer,62.40% in gastric cancer and 60.27% in rectal cancer.The changes of nutritional status mainly manifested weight loss with the incidence of 67.39%,serum prealbumin level under 200 g/L with the incidence of 24.1% and serum albumin level less than 35 g/L with the incidence of 31.70%.And there was significant difference in weight loss and serum levels of prealbumin and albumin among the patients with different nutritional status(P=0.000).The factors that influence the nutritional status of the patients with alimentary tract malignancy include the location and TNM staging of tumors,and the age,appetite and digestive symptoms of the patients.Conclusion The patients with alimentary tract malignancy are susceptible to malnutrition due to the multiple factors such as the tumor location and metabolic impacts of tumor on host.Nutritional screening,assessment and early intervention should be emphasized in the inpatients with alimentary tract malignancy.

Biopsy ; Histological Techniques ; instrumentation ; methods ; Humans ; Indicators and Reagents ; Paraffin Embedding ; Ultrasonics

Objective To examine the advanced oxidation protein products (AOPP) in patients with acute coronary syndrome(ACS) and discuss the relationship between oxidative stress with the development of atherosclero-sis(AS). Methods Plasma were collected in 59 acute myocardial infarction (AMI) patients including 35 patients underwent selective PCI,24 patients underwent emergency PCI,43 unstable angina pectoris(UA) patients and 10 non-coronary artery disease (non-CAD) patients. All cases underwent coronary angiography (CAG). Plasma was collected immediately,post-24 hours and post-48 hours after admission. AOPP was determined by measurements of absorbance (A) at 340 nm under acidic conditions via spectrophotometry. Results AOPP was (236.42±30.41) ( n = 35 ), ( 207.84±29.50 ) mmol/L ( n = 35 ), ( 227.79 ± 35.18 ) mmol/L ( n = 31 ) respectively immediately, post-24 hours and post-48 hours after admission in AMI ( selective PCI ), ( 239.95 ±39.94 ) mmol/L ( n = 43 ), (175.92 ±29.46) mmol/L(n =38) ,and (156.54 ±28.29) mmol/L(n =35) in UA group and (57.41 ± 13.60) mmol/L( n = 9 ), (56.11 + 11.90) mmol/L ( n = 10 ) and ( 61.75 ± 12.28 ) mmol/L ( n = 8 ) in non-CAD group. Compared with normal group ( without CAD ) , significantly higher plasma AOPP was detected in AMI ( selective PCI) and UA patients ( P < 0.05 ). AOPP level was significantly increased in AMI selective PCI patients as compared with that of emergency PCI group immediately and post-24 hours after admission( P <0.01 ) ,and post-48 hours after admission( P < 0.05 ), but there was no statistical significance between emergency PCI and UA group( P > 0.05 ). Conclusions Oxidative stress is an important step in the development of atherosclerosis, and the higher levels of AOPP in ACS patients show that AOPP may be as good markers in these patients.

OBJECTIVETo reveal the quality variation of polysaccharides, triterpenoids and proteins in spores and fruiting bodies of Ganoderma lucidum from producing areas, different varieties, harvesting parts and periods, and wall-breaking treatments.

METHODSpores and fruiting bodies from varieties of Longzhi No. 1 and Hunong No. 1 were collected as test samples, together with wall-broken spores sold in domestic main producing areas. The anthrone-sulfuric acid colorimetric method was used to determine the content of total polysaccharides. The vanillin-glacial acetic acid-perchloric acid colorimetric method was used to determine the content of total triterpenoids. The Lowry method was used to determine the content of total proteins.

RESULTThe content ranges of total polysaccharides, total triterpenoids, and total proteins from 6 domestic main producing areas were 0.40% - 2.25%, 1.36%-3.15% and 0.74% -1.91% respectively. The content ranges of total polysaccharides, triterpenoids, and proteins in the fruiting bodies from 2 varieties cultured in Zhejiang were 0.25% -1.42%, 0.44% -1.42% and 1.82% -3.67% respectively. In addition, the ranges of samples from wall-unbroken spores were 0.41% - 0.91%, 0.09% - 0.12%, 0.78% - 0.90% respectively and wall-broken spores are 1.03% - 2.25%, 1.89% - 3.15%, 0.96% - 1.04% respectively.

CONCLUSIONThere are significant differences in the contents of main chemical ingredients of wall-broken G. lucidum spores saled in the markets. The samples from Zhejiang contain high content of total polysaccharides and triterpenoids, and samples from Fujian contains more proteins. Between the 2 major varieties cultured in Zhejiang, Longzhi No. 1 contains higher content of triterpenoids, but Hunong No. 1 has more polysaccharides. Contents of triterpenoids and polysaccharides from wall-broken spores are much higher than those of fruiting bodies. The stipes from fruiting bodies contains more polysaccharides than those of the pileus, while the triterpenoids contents are higher in the pileus than stipes. The pileus and stipes collected in the second year contain higher content of polysaccharides than the first year's samples, but the contents of triterpenoids are lower. Wall-breaking treatment would significantly improve the extraction and dissolution rate of total triterpenoids and polysaccharides.

Fungal Proteins ; analysis ; Polysaccharides ; analysis ; Reishi ; chemistry ; Spores, Fungal ; chemistry ; Triterpenes ; analysis