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Objective To investigate the dynamic changes of osteopontin (OPN) in the guinea pig model of cholesterol gallstone disease.Methods Forty-four guinea pigs were randomly assigned to cholesterol gallstone group and control group.The animals were sequentially killed on Day 7,14,28,42,56 and 70 after operation.The expressions of OPN mRNA in gallbladder and liver tissues were detected by real-time PCR.The changes of OPN,mucin,α1-acid glycoprotein (AAP) and apolipoprotein A1 (APOA1) in bile and blood plasma were detected by ELISA kits.Results The expression of OPN mRNA in gallbladder and liver tissues increased gradually and reached the peak in the 6th week,and then decreased.The increased expression of OPN in bile in the gallstone group started from the 1st week and reached the peak in the 6th week (P ＜ 0.05),which gradually decreased to the baseline in the 10th week (P ＞ 0.05).The expressions of OPN in bile and blood demonstrated similar trends,while the peak time in blood samples was much earlier (4th week).The changes of APOA1 in bile and blood were similar to OPN,although there was no advanced peak value in blood.The levels of mucin and AAP in bile and blood increased after operation,and were kept at high level throughout the study.Conclusions OPN is involved in the whole process of cholesterol gallstone formation,which may be associated with other nucleation-active factors.
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Objective To detect the nucleation-promoting potency of retinol binding protein (RBP) in the simulated bile system,and observe the process of cholesterol nucleation and crystallization.Methods Small and synthetic bile systems were synthesized.RBP (RBP group),α1-acid glycoprotein (AAG,AAG group) and albumin ( Alb,Alb group) were added into the Small bile respectively.The derived indices of nucleation time,crystal growth rate and final concentration of crystal were studied by Busch method.Alb ( control group) and RBP (experimental group) were added into the Small and synthetic bile systems respectively.The nucleation time was detected by polarizing microscopy according to the Holan method and the nucleation-promoting potency was calculated according to the Holzbach method. The morphological dynamics of cholesterol nucleation and crystallization were observed under a light microscope.All data were analyzed by using the analysis of variance or the t test.Results The derived indices of nucleation time,crystal growth rate and final concentration of crystal were 0.66,1.29 and 1.01 in the AAG group,and 0.73,1.02,0.95 in the RBP group,respectively.In the Small bile system,the average nucleation time of cholesterol monohydrate crrystals were ( 12.2 ± 1.2 )days in the control group and (8.2 ± 1.5)days in the experimental group,respectively,with a significant difference between the 2 groups (t =2.97,P ＜ 0.05 ).The nucleation activity of RBP was 0.67 in the Small bile system.In the synthetic bile system,the average nucleation time of cholesterol monohydrate crystals were (9.5 ± 1.1 ) days in the control group and (7.5 ± 1.1 )days in the experimental group,respectively,with a significant difference between the 2 groups ( t =2.35,P ＜ 0.05 ).The nucleation activity of RBP was 0.79 in the synthetic bile system.The process of cholesterol nucleation and crystallization was divided into 4 stages,and the split phenomenon appeared in the bile system at day 21.Conclusion RBP is a pro-nucleation factor in simulated bile system.
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Objective To investigate the role of osteopontin (OPN) in the pathogenesis ot cholesterol gallstone formation in bile.Methods The nucleation time of OPN in model bile and human gallbladder bile was studied by the nucleation time assay,the effect of OPN on cholesterol crystal growth in model bile was examined by the cholesterol crystal growth assay.The effect of OPN on vesicle was detected by the transmission electron microscopy in model bile and gallbladder bile; then the content of OPN and calcium were detected via the commercial kits in human bile.Results Osteopontin prolonged nucleation time in a dose dependent manner in model bile and human bile,and this effect was correlated with calcium.Compared with control group,the nucleation times were prolonged by 1.50and 1.93 times in lithogenic bile at the concentration of osteopontin 50 μg/ml and 100 μg/ml (P＜0.01),respectively. Nucleation time were prolonged by 1.17 and 1.33 times in normal bile (P＜0.01) and by 1.29 and 1.48 times in model bile (P＜0.01),respectively.The rate of cholesterol crystals growth was not influenced by calcium ions,but inhibited by osteopontin in a dose dependent manner in the model bile.Furthermore,the formation,aggregation and fusion of vesicles were delayed by osteopontin in bile samples as indicated by the transmission electron microscopy.The concentration of osteopontin [(0.53± 0.08) mg/ml vs. (0.65 ± 0.14) mg/ml,P＜0.05] and the calcium ions [ (0.71 ± 0.17) mmol/L vs. ( 0.84 ± 0.08 ) mmol/L,P ＜ 0.05 ] were lower in lithogenic bile than in control.Conclusions Osteopontin can inhibit the cholesterol gallstone formation in model and human gallbladder bile as the anti nucleating factor.
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Objective To observe the effect of combined treatment with rhTRAIL(recombinant human TNF-related apoptosis inducing ligand) and selective Cox-2 inhibitor Celecoxib on gallbladder carcinoma in vitro and to explore the possible mechanism of the effect. Methods Western blot analysis was used to detect the expression of c-FLIP and death receptors after treatment by Celecoxib. Apoptosis of gallbladder cell line SGC-996 after the combined treatment with Celecoxib and rhTRAIL was detected in three ways: (1) phase microscopy of the cells, (2) detection of effector caspase-3 and caspase-7 activity, and (3) determination of the proportion of apoptotic cells labeled by Annexin V-PI flow cytometric analysis using CELLQUEST software. Results Celecoxib down-regulated the expression of c-FLIPs and up-regulated the expression of DR5 in a dose- and time-dependent mode on cell line SGC-996. Apoptotic levels in the combined treatment group in cell line SGC-996 were significantly higher than those in the single drug treatment group and control group. Conclusion Celecoxib markedly sensitized rhTRAIL-induced apoptosis through the down-regulation of c-FLIPs and up-regulation of DR5 in gallbladder carcinoma cell line SGC-996.
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Objective To investigate the relationship between thyroid nodules with calcification and thyroid carcinoma and its significance in the screening of thyroid carcinoma in high risk group.Methods The clinical data of 1771 patient undergoing surgery for thyroid nodules from March, 2006 to March, 2009 in Huashan Hospital, Fudan University were retrospectively analyzed. Results Among 1771 patients, 500 were finally identified as having malignant tumors. Incidence of calcification in thyroid carcinoma was 68. 4%, and that in benign thyroid nodules was 27.0% ( χ2 = 259. 5, P ＜ 0. 05 ). The specificity of microcalcification for the diagnosis of carcinoma was 89. 4%, and its positive predictive value was 66. 3% ( χ2 = 368.6, P ＜ 0. 01 ). The incidence of thyroid carcinoma in patients ＜ 45 years was 39.2%, while that in patients ≥ 45 years was 22.9% ( χ2 = 51.12, P ＜ 0. 05 ). The incidence of carcinoma in patients of single thyroid nodule was 31.7% and that in those with multiple nodules was 26. 4% (χ2 =4. 766,P ＜ 0. 05). Metastasis was pathologically diagnosed in 26. 8% of lymph nodes found by preoperative ultrasonography. Conclusions The specificity of thyroid nodule calcification, especially microcalcification is high for the diagnosis of thyroid carcinoma. High-risk index for carcinoma includes thyroid nodules with microcalcification, ＜ 45 years old and single thyroid nodule.
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Objective To investigate the role of osteopontin (OPN) in cholesterol gallstone formation.MethodsGallbladder bile was obtained from patients with cholelithiasis (n=36,the experimental group) and from donors of liver transplantation (n=19,the control group).OPN,calcium ion and lipid were analysed quantitively.The nucleating role of OPN in bile was evaluated using nucleating time (NT) approach.ResultsOPN inhibited cholesterol nucleation in a dose dependent manner.OPN (50 μg/ml and 100 μg/ml) prolonged NT by 48.90％ (91.51％) and 17.07％ (32.93％) in lithogenic and control bile,respectively.OPN (100 μg/ml) also inhibited the nucleating effect induced by calcium ion.Furthermore,a combination of OPN (50 μg/ml) and calcium prolonged NT by 75.78％ and 33.96％ in lithogenic and control bile,respectively.A combination of OPN (100 μg/ml) and calcium prolonged NT by 125.9％ and 62.26％ in the 2 groups.The contents of osteopontin and calcium were significantly lower in lithogenic bile than control bile (P＜0.05).On the other hand,the cholesterol saturation index and the contents of cholesterol,phospholipid and bile acid were significantly higher (P＜0.05).ConclusionsOPN inhibited cholesterol gallstone formation.It may be involved in the pathogenesis of cholelithiasis.
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<p><b>OBJECTIVE</b>To construct the plasmid expression vector pSIH1-H1-copGFP for RNA interference against vascular endothelial growth factor C (VEGF-C) and to evaluate its effect on the expression of VEGF-C mRNA in gastric cancer cells after transfection.</p><p><b>METHODS</b>Three siRNAs of genome sequence of VEGF-C gene were retrieved from GenBank and one negative chain was used as control. Four siRNAs were cloned into plasmid pSIH1-H1-copGFP,which were then transfected into gastric cancer cells (SGC7901). The expression of VEGF-C mRNA was analyzed by RT-PCR.</p><p><b>RESULTS</b>The recombinant plasmid of pSIH1-H1-copGFP specific for VEGF-C was confirmed by gene sequencing analysis. The target sequence obtained was completely consistent with the design. Transfection efficiency of the three siRNAs ranged from 60% to 70%. After transfection, the expression of VEGF-C mRNA in SGC7901 cells was significantly inhibited. Inhibition rates of VEGF-C mRNA expression were 35.4%, 33.8% and 81.5% in the three siRNA plasmid vectors, respectively.</p><p><b>CONCLUSION</b>The siRNA expression plasmid vector against VEGF-C mRNA is successfully constructed, and RNAi may be a useful technique to inhibit the lymphangiogenesis of gastric cancer.</p>
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Humans , Cell Line, Tumor , Genetic Vectors , Plasmids , RNA Interference , RNA, Small Interfering , Stomach Neoplasms , Genetics , Transfection , Vascular Endothelial Growth Factor C , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of Lidan Granule (, LDG) on bile lithogenic tendency and biliary 33.5 kd vesicular protein (VP) and to explore its mechanism.</p><p><b>METHODS</b>Sixty patients with choledocholithiasis combined with cholecystolithiasis were randomly assigned to the LDG treated group, the sodium cholate treated group for positive control, and the untreated control group, 20 patients in each group. The 4 bile lithogenic trend indexes, including lithogenic index (LI), unconjugated bilirubin percent (UCB%), unconjugated bilirubin saturation index (BSI) and Z-value, were determined before and after treatment. The content of VP in bile was determined as well.</p><p><b>RESULTS</b>Before treatment, the LI, UCB%, BSI and Z-value in the LDG treated group were 1.298+/- 0.265, 34.72+/-2.96, 0.353+/-0.093 and 0.556+/-0.499, respectively, which was decreased after the 2-week treatment to 0.926+/-0.208, 8.93+/-1.19, 0.154+/-0.056 and 0.257+/-0.211, respectively (all P<0.05). Meantime, the content of VP was also lowered from 0.050+/-0.005 g/L to 0.032+/-0.005 g/L. However, no significant change in any of the above-mentioned indexes was found in the other two groups.</p><p><b>CONCLUSION</b>LDG could effectively suppress bile lithogenic trend and reduce 33.5 kd VP in bile.</p>
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Aged , Female , Humans , Male , Middle Aged , Bile , Metabolism , Cholecystolithiasis , Drug Therapy , Choledocholithiasis , Drug Therapy , Drugs, Chinese Herbal , Therapeutic UsesABSTRACT
Objective To investigate the relevant risk factors for fungal infection following operation of the gastrointestinal neo- plasm and offer supporting data for the prevention of fungal infection.Methods Medical records from 116 patients who under- went the operation of gastrointestinal neoplasm in the special group of this hospital from January 2006 to June 2006 were retro- spectively reviewed on the relevant risk factors by univariate and multivariate Logistic regression analysis.Results Of the 116 patients reviewed, 18 had fungal infection.Forty-six samples were positive for fungal pathogen.The most frequently isolated fungal strain was Candida albicans (15/20) and the most common infection site was gastrointestinal tract (14/18).Fungal in- fection after the operation of gastrointestinal neoplasm was significantly relevant with the duration of antibiotic use, duration of post-operative fasting, low serum albumin, high blood glucose and complication of bacterial infection.The duration of antibiotic use was a significantly independent risk factor.Conclusions Reasonable antibiotic use, nutritional support, early enteral nutri- tion and control of blood glucose should be taken into account after the operation of gastrointestinal neoplasm in order to prevent fungal infections.
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<p><b>OBJECTIVE</b>To explore the relationship of bacteria identified in cholesterol gallstones and gallstone formation.</p><p><b>METHODS</b>Observe the bacteria activity in model bile and the influence of bacteria on the cholesterol nucleation time (NT).</p><p><b>RESULTS</b>(1) Model bile were suitable for the growth of E. coli, Pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecalis, clostridium difficile and Clostridium. Propionibacterium acne grew weakly and the growth of Bacteroides fragilis was restrained in model bile. (2) Only pseudomonas aeruginosa and enTerococcus faecalis could ly shorten the cholesterol nucleation time. (3) With pseudomonas aeruginosa or enTerococcus faecalis added in model bile, the formation of cholesterol crystals presented a progressive course of evolution.</p><p><b>CONCLUSIONS</b>Pseudomonas aeruginosa and enterococcus faecalis, not propionibacterium acne, have pro-nucleating ability in model bile.</p>
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Bile , Metabolism , Microbiology , Cholelithiasis , Microbiology , Cholesterol , Metabolism , Crystallization , Enterococcus faecalis , Models, Biological , Propionibacterium acnes , Pseudomonas aeruginosaABSTRACT
Objective To investigate the effects of electrical stimulation on the expression of vascular endothelial growth factor(VEGF)mRNA and receptor FLK 1/KDR in a ischemic model of rat hindlimb. Methods The model of hindlimb ischemia on the right side was established by ligation of the superficial femoral artery in 10 rats. The rats were then randomized into an experimental group and a control group. The rats in the experimental group were intervened with electrical stimulation ( 25 Hz, 0.1 V) on the tibialis anterior (TA) of the right side, while those in the control group were not. RT PCR and immunohistological methods were used to detect the expressions of VEGF mRNA and protein in TA muscles. FLK 1/KDR was detected by means of Western blot and immunofluorescence. Results After 7 days of continuous stimulation, there was a significant increase in blood flow within the muscle. VEGF mRNA and VEGF protein had 4 fold and 2 fold increases, respectively, in the stimulated TA muscles as compared to the control(2.58 vs 0.93, 0.48 vs 0.24, P
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<p><b>OBJECTIVE</b>To identify genetic abnormalities in primary pancreatic carcinoma in humans.</p><p><b>METHODS</b>Comparative genomic hybridization (CGH) was used to investigate genomic imbalances in 27 cases of pancreatic carcinomas. Multiple deletions and gains were observed in all tumor specimens.</p><p><b>RESULTS</b>Losses affecting chromosomes 9p, 17p, 4q and 6p and gains involving 8q, 7q, 3q and 1q were commonly observed.</p><p><b>CONCLUSIONS</b>There are multiple regions of chromosomes with changes copy number in pancreatic carcinoma. The altered chromosomal regions may contain several candidate genes which are involved in the development and progress of pancreatic carcinogenesis.</p>
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Adult , Aged , Female , Humans , Male , Middle Aged , Chromosome Aberrations , Chromosome Disorders , Nucleic Acid Hybridization , Pancreatic Neoplasms , GeneticsABSTRACT
OBJECTIVE: To establish a rapid and precise detective method of 33.5 kd vesicular protein and to screen an effective treatment of cholelithiasis. METHODS: Specific antibody of the biliary vesicular protein was obtained by immunizing rabbits and enzyme-linked immunosorbent assay (ELISA) kit was developed. The concentrations of 33.5 kd vesicular protein in serum and bile of gallstone patients and control were examined respectively. The effects of Cholagogue Dry Syrup and Eulektrol Capsule on decreasing 33.5 kd vesicular protein were also studied by ELISA kit. RESULTS: One-step ELISA equation was Y=0.035 X (r=0.99). The vesicular protein concentrations in serum and bile of cholesterol gallstone group [(179.8+/-97.9) mg/L and (213.4+/-70.1) mg/L respectively] were significantly (P<0.05) higher than in the pigment stone group and control. Data showed that, with 2-week administration, Cholagogue Dry Syrup significantly decreased both biliary and serum 33.5 kd vesicular protein of cholesterol gallstone patients, while Eulekrol Capsule and control groups didn't have the same results. CONCLUSION: The concentrations of 33.5 kd protein are different in cholesterol gallstone patients and healthy groups which might be related to cholesterol nucleation process. Cholagogue Dry Syrup is of cholagogic and litholytic effect by decreasing biliary lithogenesis.
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<p><b>OBJECTIVE</b>To evaluate the pro-nucleating activity in 33.5 x 10(3) vesicular protein.</p><p><b>METHODS</b>The model biles were established according Kibe and Zhu. The pro-nucleating activity of 33.5 x 10(3) vesicular protein were examined by polarized light microscopy. The protein and its enzymatic deglycosylation and proteolysis fractions nucleation promoting activity were detected by cholesterol crystal growth assay.</p><p><b>RESULTS</b>33.5 x 10(3) vesicular protein displayed apparent potency of pro-nucleation with activity of 0.310, and derived crystal growth curve indices It, Ig, Ic were presented as 0.57, 1.52, 1.63 respectively, but after treated by N-glycanase enzyme and pronase, no promoting activity were found.</p><p><b>CONCLUSION</b>The 33.5 x 10(3) vesicular protein may be involved in the nucleation process of gallstone formation, which is regulated by its peptide and sugar chain.</p>
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Humans , Bile , Metabolism , Cholelithiasis , Cholesterol , Metabolism , Macromolecular Substances , Microscopy , Models, Biological , Protein Transport , Proteins , MetabolismABSTRACT
Objective To study the effect of CO 2 pneumoperitoneum and enteric disturbance on serum ?-endorphin (?-EP) in SD rats, in order to investigate their influence on peri-operative stress responses in SD rats. Methods A total of 120 SPF-grade male SD rats were randomly divided into four groups with 30 rats in each group. The four groups received CO 2 pneumoperitoneum (Group A), a 5 cm abdominal incision (Group B), a 5 cm abdominal incision with gastroenteric disturbance (Group C), and intraperitoneal anesthesia (Group D or Control Group), respectively. Concentrations of serum ?-EP of these groups were measured 10 min, 20 min, and 40 min after the beginning of surgery, respectively. Results Concentrations of serum ?-EP in the Group A 10 min, 20 min, and 40 min after the beginning of surgery were 274 7?66 6 pg/dl, 157 3?63 8 pg/dl, and 163 9?74 5 pg/dl, respectively, which were all extremely significantly higher than those in the Control Group ( P
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Objective To clarify the significance of bacteria DNA in cholesterol gallstone. Methods Semi-quantitative PCR and comparative 16S rRNA analysis were used to detect bac teria DNA in gallbladder mucosa, bile and cholesterol gallstones. The influence of peri-operative use of antibiotics on the results of detection was a lso observed. Results (1) The positive rate of bacteria DNA in core and periphery part of gallstones, bile and gallbladder mucosa was 79%, 82%, 77% and 64% respectively, with no posi tive correlation existed among them. (2) The bacteria concentrations in core and periphery part of gallstones, bile and gallbladder mucosa were 3.19?2.09, 3.26 ?2.05, 3.23?2.14 and 3.28?2.70 cfu (log value) respectively. The bacteria con centration in core part of gallstone correlated with those of periphery ones ( r=0.822, P
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Objective To explore the structure and type of sugar chain in 33 500 pro-nucleating protein, and its role in gallstone formation. Methods The 33 500 vesicular protein was examined by dot-immunobinding assay of lectin coupled to a peroxidase (HRP-DSA,HRP-ConA,HRP-WGA). The morphology of biliary vesicles was observed under transmission electron microscopy in process of massive vesicular aggregation, culmination and crystal formation. The protein and its enzymatic deglycosylation fractions nucleation promoting activity were detected by cholesterol crystal growth assay. Results 33 500 vesicular protein with multiantennary and complicated glycan displayed apparent potency of nucleation promotion, which clearly reflected by HRP-DSA immunobinding, and derived crystal growth curve indices. It, Ig, Ic were presented as 0.57, 1.52, 1.63 respectively, but after treated by N-glycanase enzyme, no promoting activity was found. Conclusions Our data suggest the sugar chain play an important role in pro-nucleating process, and may be involved in the gallstone formation.
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Objective To evaluate the diagnostic accuracy of ELISA kit for cholesterol gallstone. Methods The ELISA kit of 33?500 vesicular protein was established by sandwich method, and the concentrations of the protein in gallbladder bile were examined among cholesterol, pigmental gallstone patients and control groups. Results The gallbladder 33?500 vesicular protein (213?70) ?g/ml is much higher in cholesterol gallstone patients than in pigmental gallstone patients (72?55) ?g/ml and control groups (65?52) ?g/ml (F=60.9, P
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Objective To explore the protective effect of treatment of somatostatin combined with growth hormone on brain injury in early severe acute pancreatitis(SAP)rats, and to investigate the relationship between brain injury and ratio of endothelin 1(ET 1) /nitric oxide(NO). Methods SAP model was established by retrograde pancreatic bile duct injection of 3.5% sodium taurocholate at a dose of 2.5 ml/kg in rats. Eighty SAP rats were equally divided into SAP group, somatostatin (S) group (introvenous injection of somatostatin at a dose of 42 ?g/kg once a day for 2 days), somatotropin (G) group (subcutaneous injection of somatotropin at a dose of 0.5 ?g/kg once a day for 2 days) and S+G group. Twenty normal rats were used as controls. The changes of encephaledema and blood brain barrier permeability were measured by dry wet method and Evan′s blue staining, respectively. Apoptosis of brain cells was detected by TUNEL method, and the ratio of serum ET 1/NO was also determined. Results The ratio of serum ET 1/NO was remarkably increased in SAP rats, which was correlated with the intensity of brain edema, permeability of blood brain barrier and apoptosis of brain cells. The treatment of somatostatin combined with growth hormone reduced the ratio of ET 1/NO and thus decreased the intensity of encephaledema, the permeability of blood brain barrier and apoptosis of brain cells. The changes of the brain slow wave were found simultaneously by electroencephalography in SAP rats after the therapy. Conclusions The treatment of somatostatin combined with growth hormone in SAP rats can inhibit serum ET 1/NO, prevent the development of brain edema, decrease the permeability of blood brain barrier, and alleviate the brain cells apoptosis. All these result in the relief of the brain injury.
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Objective To study the expression changes of matrix metalloproteinases (MMPs), protein kinase C (PKC) and nuclear factor-?B (NF-?B ) on the injured rabbit vascular smooth muscular cells (VSMC) transfected with tissue inhibitor of metalloproteinase-2 (TIMP-2) vector. Methods Lipofectin method was used to transfect TIMP-2 vector into VSMC, Western blot analysis to detect TIMP-2 peptides and zymography assay to determine MMPs. The activities of MMPs and NF-?B were detected by electrophoretic mobility shift assay (EMSA). The mRNA and protein expressions of PKC? were determined by RT-PCR and immunocytochemistry. Results The injured VSMC showed increased enzyme activity of MMP-2. There was very lower level expressions of PKC? and NF-?B in the normal VSMC but high in the injured VSMC. However, in injured VSMC transfected with TIMP-2 vector, the activity of MMP2/9 was suppressed and the expressions of PKC? and NF-?B decreased (P