The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Polymorphism of ornithine decarboxylase antizyme inhibitor 1 gene is associated with liver cirrhosis in Chinese hepatitis B patients / 中华肝脏病杂志

A cirrhosis risk score (CRS) comprised of single nucleotide polymorphisms (SNPs) in seven genes that predicts the risk of cirrhosis in Caucasian hepatitis C has been reported. The present study was to evaluate the association of 11 separate but related SNPs and the CRS with cirrhosis risk in Chinese hepatitis B patients. A total of 563 Chinese subjects with persistent HBV infection (349 with evident liver cirrhosis and 214 without cirrhosis clinically or pathologically) were studied. The candidate SNPs were detected with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method. The allele frequency and genotype distribution of each polymorphism as well as the CRS value within the cirrhosis and non-cirrhosis subjects were compared. The rs2679757 polymorphism of the antizyme inhibitor 1 (AZIN1) gene was associated with the risk of cirrhosis (x2 = 6.79, P = 0.03, odds ratio for GG+AG versus AA = 1.63, 95% confidence interval = 1.13-2.35). A gene variant (rs886277) in the transient receptor potential cation channel subfamily M, member 5 gene (TRPM5) was associated with liver cirrhosis, but did not reach statistical significance (x2 = 5.77, P = 0.06). Two SNPs (rs4986791, rs62522600) are not polymorphic in Chinese. Genotype frequencies of other SNPs were not different between the cirrhosis and non-cirrhosis groups. The overall CRS values were not different between the cirrhotic and non-cirrhotic groups (median value 0.57 versus 0.62, Z = -1.05, P = 0.29). SNP rs2679757 in the AZIN1 gene is associated with the risk of HBV-related liver cirrhosis in Chinese. The CRS for Caucasian population has limited applicability for predicting liver cirrhosis in Chinese hepatitis B patients. SNPs associated with cirrhosis prognosis in hepatitis B patients and liver diseases with other etiologies warrant further clinical validation.

The role of HO-CO system in the hemodynamic disturbance of cirrhotic rats / 中华肝脏病杂志

To investigate the role of heme oxygenase(HO), a catalyzing enzyme of heme to produce CO, in modulation of systemic circulation in CCl4-induced cirrhotic rats. Saline(vehicle) and ZnPP were s.c. injected into the posterior necks of rats respectively and the rats were then anesthetized by pentobarbital sodium in four hours. Mean arterial pressure (MAP, kPa), heart rate (HR, b/min) and portal pressure (PP, cm/H2O) were measured by indwelling catheter. Plasma CO was determined by Chalmers method. Heme oxygenase acivity was determined by the rate of bilirubin formation. The cirrhotic rats showed significant hyperdynamic circulation indicated by decreased mean arterial pressure [MAP, (15.6+/-1.7) vs (18.9+/-0.9) kPa, t = 4.52, P less than 0.01] and increased portal pressure [PP, (16.7+/-0.8) vs (8.8+/-0.3) cm H2O, t = 23.10, P less than 0.01] as compared to normal control rats(NS). ZnPP could cause a significant increase in MAP [(17.3+/-1.5) vs (15.6+/-1.7) kPa, t = 2.18, P less than 0.05] and significant decrease in PP [(13.2+/-0.7) vs (16.7+/-0.8) cm H2O, t = 8.53, P less than 0.01] in cirrhotic rats. The cirrhotic group presented a significant increase in plasma CO [(18.0+/-1.9) vs (10.4+/-1.3)mumol/L, t = 8.42, P less than 0.01] and HO activity in the spleens [(11.1+/-0.9) vs (6.5+/-0.9) nmol bilirubin/mg protein/h, t = 9.28, P less than 0.01] and intestines [(2.5+/-0.1) vs. (1.3+/-0.2) nmol bilirubin/mg protein/h, t = 15.1, P less than 0.01]. ZnPP could cause significant decreases in plasma CO and HO activity in liver, spleen and intestine of both control and cirrhotic rats. HO-CO system activation may be an important reason for the hemodynamic disturbance of liver cirrhosis.

Effect of collagens and growth factors (TGFß1, PDGF-BB) on cytoskeletal reorganization and cell migration in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the effects of collagens and growth factors TGFß1 and PDGF-BB on the cytoskeletal components and migration in cultured rat hepatic stellate cells (HSCs).</p><p><b>METHODS</b>Primary rat hepatic stellate cells were isolated and cultured. A Transwell Chamber system was used to observe the changes of serum starved HSCs haptotactic migration (direct stimulation) and chemotactic migration (indirect stimulation) after collagens and growth factors treatment. Changes in actin cytoskeletal organization were visualized by fluorescence staining using FITC-labeled phalloidin and the fluorescence images were recorded using confocal microscopy.</p><p><b>RESULTS</b>TGFß1 enhanced significantly the motility of primary HSCs at 5 ng/ml: for haptotactic migration cells: 131.37+/-3.15 vs 102.93+/-1.01, F=40.84, P<0.05; for chemotactic migration cells: 210.17+/-1.78 vs 102.93+/-1.01, F=64.53, P<0.05. PDGF-BB enhanced significantly the motility of primary HSCs at 10 ng/ml (haptotactic migration cells: 203.67+/-7.54 vs 102.93+/-1.01, F=40.90, P<0.05; chemotactic migration cells: 319.56+/-11.71 vs 102.93+/-1.01, F=54.57, P<0.05); Both chemotactic and haptotactic stimuli with 100 microg/ml collagen type I significantly increased HSCs migration: for haptotactic migration cells: 127.20+/-6.47 VS 102.93+/-1.01, F=41.01, P is less than 0.05; for chemotactic migration cells: 201.52+/-11.28 vs 102.93+/-1.01, F=36.49, P<0.05; however, collagen type IV had no effect on HSCs migration. Serum-starved, untreated cells had a rounded-up morphology. PDGF-BB and TGFß1 induced a rapid morphological change concomitant with a robust reorganization of actin cytoskeleton in HSCs.</p><p><b>CONCLUSIONS</b>Collagen type I, PDGF-BB and TGFß1 could induce cell migration in HSCs, but collagen type IV has no such effect. PDGF-BB and TGFß1 could induce the actin cytoskeleton reorganization in HSCs.</p>

Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although the migration of hepatic stellate cells (HSCs) is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases (especially RhoA) in platelet-derived growth factor (PDGF)-BB-induced migration of HSCs.</p><p><b>METHODS</b>The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases (RhoA, Rac1 and Cdc42) within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active (CA, Q63L) or dominant negative (DN, T19N) RhoA mutants. Data were analyzed using SPSS 16.0 software. Student's t test was used to analyze differences between two groups and one-way analysis of variance (ANOVA) was used among multiple groups.</p><p><b>RESULTS</b>Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic (direct) and chemotactic (indirect) stimulation by PDGF-BB. PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Rac1 and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls.</p><p><b>CONCLUSIONS</b>PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC migration.</p>

Binding characteristics between RGD-containing cyclic peptide and rat hepatic stellate cells: an in vitro study / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the binding characteristics between an artificial Arg-Gly-Asp (RGD)-containing cyclic peptide [cyclo(CGRGDSPK)] and rat hepatic stellate cells (HSC).</p><p><b>METHODS</b>An artificial RGD-containing cyclic peptide was labeled with fluorescein isothiocyanate (FITC). HSCs were isolated by collagenase in situ liver recirculating and purified by density gradient centrifugation from normal rats. The cells were cultured for 5 days of primary culture (quiescent phenotype) or for 7 days of secondary culture (activated phenotype). To access the binding and uptake, HSCs were incubated with FITC-cRGD of different concentrations at 4 degree C or 37 degree C, and then the binding and uptake were investigated by flow cytometry. The location of FITC-cRGD in HSC was investigated by fluorescent microscopy. Kd and maximal binding sites per cell were calculated by radioligand binding assay (RBA) of receptors using 3H-cRGD. In the interim, FITC-cAGA was used as a peptide control devoid of any binding site.</p><p><b>RESULTS</b>The binding between FITC-cRGD and HSC was saturable, time- and dose-dependent and could compete with overdosed unlabeled cRGD. The fluorescence was mainly distributed in cytoplasma, especially near the nuclei. Kd was 7.05 x 10(-9) mol/L and Bmax per cell was nearly 6.79 x 10(5).</p><p><b>CONCLUSIONS</b>The results demonstrate that cRGD are specifically taken up by HSC through a receptor-mediated pathway. The information is useful for understanding the ligand-receptor interaction of HSC. FITC labeled cyclic RGD-peptides meet the standards of special ligands and FITC does not change the binding activation of cyclic RGD-peptides.</p>

Effects of glycyrrhizin on TGFbeta1 stimulated hepatic stellate cell signaling transduction / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the role of glycyrrhizin on TGFbeta1 stimulated signaling transduction in rat hepatic stellate cells (HSCs).</p><p><b>METHODS</b>The mice HSCs were isolated and cultured with or without glycyrrhizin (1 micromol/L-1000 micromol/L) in vitro after TGFbeta1 stimulation. The mRNA level of Smad2, 3, 7 were measured with RT-PCR; protein expression level of Smad2, 3, 7 and collagen I, III were analyzed with Western blot.</p><p><b>RESULTS</b>TGFbeta1 increased the mRNA level and protein expression of Smad2, 3, 7 in HSC; it also increased protein expression of collagen I and III. 1 micromol/L-1000 micromol/L glycyrrhizin decreased the mRNA level and protein expression of Smad2, 3, 7; it also inhibited protein expression of collagen I and III gradually.</p><p><b>CONCLUSION</b>Interventing the TGFbeta signaling pathway and decreasing the synthesis of collagen, might be involved in the anti-fibrosis mechanism of glycyrrhizin.</p>

Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.</p><p><b>METHODS</b>Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type I collagen. In addition to hepatic hydroxyproline content, hepatic collagen types I and III were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.</p><p><b>RESULTS</b>Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P < 0.01), hepatic hydroxyproline content and the accumulation of collagen types I and III were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P < 0.01) as compared with the model group.</p><p><b>CONCLUSION</b>The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.</p>

Diisopropylamine dichloroacetate in the treatment of nonalcoholic fatty liver disease: a multicenter random double-blind controlled trial / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the efficacy and safety of diisopropylamine dichloroacetate in the treatment of nonalcoholic fatty liver diseases (NAFLD).</p><p><b>METHODS</b>A randomized, double-blind, dose-paralleled control trial was carried out with NAFLD patients. The patients were randomly assigned to 2 groups treated with either a high dosage (120 mg/d) or a low dosage (60 mg/d) of diisopropylamine dichloroacetate for 8 weeks and the efficacy and safety of the drug were examined.</p><p><b>RESULTS</b>127 cases were recruited for the trial, 63 in the high dosage group, and 64 in the low dosage group. No case dropped out in the trial but four cases were eliminated (4/127, 3.1%). The final number in this trial was 123, with 61 in the high dosage group and 62 in the low dosage group. After 8 weeks of treatment, the overall improvement of clinical symptoms in the high dosage and in the low dosage group was 87.8% and 79.6%, respectively. ALT normalization was found in 55.7% and 69.4% of the cases in the two groups, serum lipids were lowered in 67.2% and 67.7% and ultrasound grading of the liver alteration severity was lowered in 51.7% and 43.5% in the two groups. The differences found between the two groups were of no statistical significance. One case from each group was found having an adverse drug reaction of dryness of the mouth (1.6%). No severe adverse drug reactions were found.</p><p><b>CONCLUSION</b>Diisopropylamine dichloroacetate could be used as a safe and effective drug in the treatment of nonalcoholic fatty liver diseases.</p>

A randomized controlled Multi-center clinical trial on mosapride in the treatment of functional dyspepsia / 中华流行病学杂志

<p><b>OBJECTIVE</b>To evaluate the effectiveness and safety of mosapride on treatment of functional dyspepsia.</p><p><b>METHODS</b>Randomized controlled clinical trial was conducted and patients suffered from functional dyspepsia were included. 5 mg mosapride was given three times daily for 4 weeks in the treatment group. 10 mg domperidone was given three times daily for 4 weeks as control. Changes on symptom score, gastric empty or new occurring events were included as outcomes.</p><p><b>RESULTS</b>231 patients suffered from functional dyspepsia were selected by inclusion and exclusion criteria from August 15 to Oct 22, 1999. Of these, 108 (46.8%) were males, versus 123 (53.2%) females and 118 (51.2%) in the treatment group and 113 (48.9%) as controls. 222 (96.1%) patients were followed up. Results showed that the total efficacy rates in early satiety and abdominal distension were 84.5% and 90.1% in mosapride after the 2 weeks of treatment. Mosapride seemed to be more effective in improving symptoms of belching and heartburn than that in controls (P < 0.05). In 4 weeks, the total efficacy in improving symptoms of abdominal distention and belching showed more effective in mosapride than that in controls (P < 0.05). Decrease of symptoms score was more in mosapride than that in controls (P < 0.05). Mosapride was less effective in controls in improving the gastric empty in terms of proportion (46.2% vs. 25.9%, P = 0.020) and range (46.2% vs. 24.0%, P = 0.003). Side effects would include diarrhea, constipation, headache, dizziness, insomnia, skin scare and the like. There was no significant difference between the two groups (9.6% in mosapride vs. 14.0% in controls).</p><p><b>CONCLUSION</b>Mosapride was safe and effective in improving the symptoms and gastric empty of functional dyspepsia.</p>

Cost-effective analysis of preventive treatment on diabetes / 中华流行病学杂志

<p><b>OBJECTIVE</b>To evaluate the cost-effectiveness of preventive treatment on diabetes, using metformin or acarbose among patients with impaired glucose tolerance.</p><p><b>METHODS</b>Using data from diabetes prevention program (DPP) and STOP-NIDDM study, we evaluated the cost of preventing one new onset of diabetes in Shanghai, and to compare its cost with the current treatment cost.</p><p><b>RESULTS</b>If metformin was used for preventive treatment as in DPP study, a total cost of 69 122.95RMB was needed for preventing one new onset of diabetes in three years period. If acarbose was used for preventive treatment as in STOP-NIDDM, then 154 116.05RMB was the cost to prevent one diabetes in 3.3 years of treatment. However, if the generic metformin was used, the total cost was only 21 666.63RMB for the 3-years treatment. Data showed that the average cost for treating diabetes per year was 9143.70RMB in Shanghai.</p><p><b>CONCLUSION</b>The total cost of diabetes prevention was formidable, although generic metformin showed the trend of cost-effective. The cost of drugs took the biggest part of the total cost. To choose the cheap but effective drug for treatment might save a large part of the cost. Further clinical research concerning the prevention of complications might provide us with more information on the cost-effectiveness of preventive treatment on diabetes.</p>

Oxymatrine in the treatment of chronic hepatitis B for one year: a multicenter random double-blind placebo-controlled trial / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of oxymatrine in the treatment of chronic hepatitis B.</p><p><b>METHODS</b>A multicenter randomized double-blind placebo-controlled trial was conducted. A total of 144 patients with chronic hepatitis B entered the study for 52 weeks; of them 72 received oxymatrine, and 72 received a placebo. Before and after the treatment, clinical symptoms, liver function, serum hepatitis B virus markers, and adverse drug reactions were observed.</p><p><b>RESULTS</b>In 144 patients, 14 were dropped and excluded due to inconsistencies in the included standard. Therefore, the efficacy and safety of 130 patients were analyzed. After being treated for 52 weeks, 70.77% of the patients in the study group had a normal ALT level, and in 43.08% and 33.33% their HBV DNA and HBeAg became negative. In the placebo group, 39.68% had normal ALT level, and 12.31% and 3.33% had their HBV DNA and HBeAg become negative. The rates of complete response and partial response in the oxymatrine group were 23.08% and 58.46%, and in the placebo group they were 3.08% and 44.62%. They were significantly higher in the oxymatrine group than in the placebo group. In the oxymatrine treated patients, 12 weeks after its withdrawal, 60.00% had a normal ALT level, 41.54% and 23.33% had both HBV DNA and HBeAg negative. In the placebo group, 31.75% had a normal ALT level, 3.08% and 1.67% had both HBV DNA and HBeAg negative. The rates of complete response and partial response in the oxymatrine group were 21.54% and 47.69%, and in the placebo group they were 0 and 41.54%. They were significantly higher in the study group than in the placebo group. The adverse reaction rates of oxymatrine in the study and the placebo group were 7.69% and 6.15%, respectively, but there was no statistical significant difference between them.</p><p><b>CONCLUSION</b>Oxymatrine is an effective and safe agent for the treatment of chronic hepatitis B.</p>

Effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of antisense transforming growth factor beta receptor-II (TGFbetaRII) expressing plasmid on experimental liver fibrosis.</p><p><b>METHODS</b>RT-Nest-PCR and gene recombinant techniques were used to construct the rat antisense TGFbetaRII recombinant plasmid which can be expressed in eukaryotic cells. Thirty-six male SD rats were randomly distributed into five groups: 10 in experimental liver fibrosis model induced by pig-serum as disease control group; 10 in antisense TGFbetaRII transfection as treatment group; 10 in pCDNA3 transfection as treatment control group and 6 in normal control group. The recombinant plasmid and empty vector (pCDNA3) were encapsulated by glycosyl-poly-L-lysine and then transducted into rats of pig serum-induced liver fibrosis model respectively. Expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR and Western blot. We also tested ELISA of serum TGF-beta1, the contents of hepatic hydroxyproline, immunohistochemistry of type I and III collagen, and VG staining for pathological study.</p><p><b>RESULTS</b>The antisense TGFbetaRII expressing plasmid could be well expressed in vivo, and could block the mRNA and protein expression of TGFbetaRII in the fibrotic liver induced by pig serum. Its expression also reduced the level of TGF-beta1 [antisense treatment group (23.16+/-3.13) ng/ml, disease control group (32.96+/-3.79) ng/ml; F=36.73, 0.01]. Compared with the disease control group, the contents of hepatic hydroxyproline [antisense treatment group (0.17+/-0.01) mg/g liver, disease control group (0.30+/-0.03) mg/g liver; F=15.48, 0.01] and the deposition of collagens type I and type III decreased in the antisense group (antisense treatment group collagen type I 650.26+/-51.51, collagen type III 661.58+/-55.28; disease control group type I 1209.44+/-116.60, collagen type III 1175.14+/-121.44; F values are 69.87, 70.46, 0.01). And its expression also improved the pathologic classification of liver fibrosis models (0.01).</p><p><b>CONCLUSION</b>The results demonstrate that TGF-beta plays a key role in liver fibrogenesis and the prevention of liver fibrosis by antisense TGFbetaRII recombinant plasmid intervention may be therapeutically useful.</p>

Anti-neoplastic effect of chitosan-polyaspartic acid-5 fluorouracil nanoparticles on tumor growth of the implanted gastric cancer in nude mice / 中华消化杂志

Objective To prepare chitosan-polyaspartic acid-5 fluorouracil (CTSPasp-5FU) nanoparticles and to investigate its anti-neoplastic effect and toxicity.Methods CTS-Pasly5FU nanopartieles were synthesized by ion gelatifieation.BALB/C nude mice were injected with gastric carcinoma cell line SGC- 7901 mass subcutaneously near nape to establish human gastric carcinoma model.Then they were randomly al- located into chitosan-polyaspartie acid -5fluorouracil(CTS-Pasp-SFU,containing 5-FU 1.25mg/kg) group, 5-FU (1.25mg/kg) group and normal saline group.Tumor weight was measured and the colony forming unit- granulocyte and maerophage (CFU-GM) was investigated.Results The drug content of CTS-Pasp-5FU was 40.2% and the encapsulation efficiency was 34.9%.Compared with normal saline group,tumor volume of 5-FU group and CTS-pasp-5-FU group were significantly decreased 21 days after treatment (P

Down-regulation of osteopontin by siRNA on the biological behavior of gastric cancer cell lines / 中华消化杂志

Objective To explore the effects of down regulation of osteopontin(OPN)on the bio- logical behavior of MKN28 and SGC7901 cell lines.Methods OPN siRNA was designed according to the relevant literature and was transfected into the two cell lines.Fluorescent labeling was used to test the transfected efficiency.The down-regulation of OPN protein was measured by Western blot.Real- time PCR was used to test the ratio and time difference of down-regulation of OPN mRNA after siRNA transfection.The biological changes before and after OPN siRNA transfected into these two cell lines were tested by flow cytometry(to test cell cycle and apoptosis)and MTT method(to test the prolifera- tion for the consecutive seven days)and the difference between OPN siRNA transfected or non-transfect- ed cells was compared using mixed model.The capability of moving and invasion of cancer cells were tested by Transwell method and analyzed by t-test.Results The transfected efficiency of OPN siRNA were more than 90% in the two cell lines.OPN mRNA down-regulated to 47% at the 72th hour in SGC7901,while 40% at the 48th hour in MKN28.The expression of OPN protein was both down- regulated after siRNA transfection in the two cell lines.The proliferation decreased after transfected with OPN siRNA both in MKN28 andSGC7901(P

The correlation of osteopontin and outcom of gastric cancer / 中华消化杂志

0.05].In 48 patients without lymph node metastasis,there was no significant difference between the OPN-positive and OPN-negative patients in survival times.Multivariate analysis revealed that survival times was associated with patients′sex(P=0.032)and TNM stage(P

The effect of endoplasmic reticulum stress in hyperlipidemia rats with acute pancreatitis / 中华消化杂志

Objective To investigate the effect of endoplasmic reticulum stress in hyperlipidemia rats with acute pancreatitis(AP).Methods Forty-eight SD rats were divided into AP group(n=18,in- duced by injection of 40?tg/kg caerulein in normal rats twice with 2 hrs interval),normal control(n=6), byperlipidemia group(n=6,fed with a high-fat diet for 8 weeks)and hyperlipidemia AP group(n=18,in- duced by injection of 40?g/kg caerulein in hyperlipidemia rats twice with 2 hrs interval).The rats were sacrificed at 9,12,24 hrs(6 of each),respectively.Serum level of amylase was tested and the pathologi- cal changes of the pancreatic tissues were observed.The index of pancreatic apoptosis was assessed by TUNEL method.The expression of GRP78/Bip(glucose regulated protein)protein was determined by immunochemistry,the endoplasmic reticulum stress related molecules of XBP-1 splicing(X box binding protein),CHOP/GADD153(C/EBP-homologous protein or growth arrest and DNA-damage-inducible gene 153),caspase-12 were analyzed by RT-PCR.The dynamic expressions of GRP78/Bip and caspase- 12 were determine,d by Western blot.Results Artier 8 weeks of fat diet in hyperlipidemia rats,the ser- um levels of triglyceride[(0.99?0.38)mmol/L]and cholesterol[(3.17?0.18)mmol/L] were signifi- cantly increased(P

Effect of RNAi on the expression of COX-2 in human rheumatoid arthritis synovial fibroblasts / 中华风湿病学杂志

Objective To design,synthesize and screen high efficient small interfering RNA(siRNA) targeting to cyclooxygenase-2(COX-2)on rheumatoid arthritis synovial fibroblasts(RASF).To further study the effect of specific COX-2 siRNA interfering on mediators of inflammatory cytokines.Methods Four pairs of siRNA for human COX-2 mRNA were synthesized by utilizing RNA design software,while another random sequence was designed as control.They were divided into group A to H.Among them,group A was used as the negative control(CTL),and group B to F were transfected as random siRNA(NC),1#～4#siRNA in order. These siRNAs were transferred into RASF by LipofectAMINE2000 package and PMA(phorbol-12-myristate- 13-acetate)was added into each culture and with a final concentration of 100 nmol/l.RASF was collected 48 hours after transfection.The expression of hCOX-2 at mRNA level was determined by reverse transcription- polymerase chain reaction(RT-PCR)and hCOX-2 protein level by Western Blot.The supernatant levels of PGE_2,IL-1?,IL-6,TNF-?and vascular endothelial growth factor(VEGF)of the above groups were detected by ELISA.Results The levels of hCOX mRNA and protein in RASF treated with 4-#siRNA were significantly lower than those of the negative control and other groups.The level of PGE_2 and cytokines like IL-1?,IL-6, TNF-?and VEGF in the supernatant were lower in the 4#siRNA group than in other groups.Conclusion 4#siRNA can effectively inhibit the expression of COX-2 mRNA and the synthesis of the COX-2 protein in human synovial fibroblasts.The level of PGE_2,IL-1?,IL-6,TNF-?and VEGF is the lowest in the super- natant.Thus 4#siRNA has been confirmed to specifically block the COX-2 in human synovial fibroblasts.

Effects of Rho GTPase expression and migration induced by transform growth factor?1 in hepatic stellate cells / 中华消化杂志

Objective To investigate the effects of Rho GTPase expression,actin cytoskeleton re- organization and cell migration in hepatic stellate cells after transform growth factor(TGF)?_1 stimulated. Methods Primary rat hepatic stellate cells were isolated and cultured.A Transwell Chamber system was used to observe the changes of serum starved HSCs haptotactic migration (direct stimulation) and chem- otactie migration(indirect stimulation) after different concentration of TGF?_1 treatment.Changes in ac- tin cytoskeletal organization were visualized by fluorescence staining using FITC labeled phalloidin and fluorescence images were recorded using confocal microscopy.The activation of GTP-loaded GTPases were evaluated by GST pull-down assays with GST-fusion proteins of GTPase hinging domains of differ- ent effectors for Rho GTPases.Results TGF?_1 treatment of hepatic stellate cells resulted in the en- hancement of migration in response to haptotactic and chemotactic stimuli,especially after 5ng/ml TGF?_1 stimulation(haptotactic migration cells :130.90?7.64 vs 102.93?1.01,P＜0.05;chemotactie migration cells:205.17?10.78 vs 102.93?1.01,P＜0.05).Serum-starved,untreated cells had a rounded-up morphology.Stimulation of hepatic stellate cells with 5 ng/ml TGF?_1 induced a rapid mor- phological change concomitant with a robust reorganization of actin cytoskeleton.Five minutes after TGF?_1 stimulation,the cells flattened out and the formation of lamellipodia occurred.Five minutes after TGF?_1 was added,stress fibers became visible,and after 15-30 minutes,the cells became well spread with fully developed stress fibers.TGF?_1 stimulation did not alter the amount of GTP-bound Racl.In contrast,Cdc42 and RhoA GTPase activity was significantly augmented after more than 5ng/ml TGF?_1 stimulation(GTP-Cdc42:0.273?0.024 vs 0.176?0.001,P＜0.05;GTP-RhoA:0.176?0.005 vs 0.096?0.004,P＜0.05).Conclusion The stimulation of TGF?_1 can induce the actin cytoskeleton re- organization,which appears to be mediated through the activity of the Rho GTPases signaling pathway. The ability of TGF?_1 to trigger activation of Cdc42 and RhoA GTPase along with actin cytoskeleton reor- ganization might well play a crucial role in hepatic stellate cells migration.

Effects of imatinib mesylate on rat hepatic fibrogenesis and the expression of transforming growth factor-?1 / 中华消化杂志

Objective To investigate the anti-fibrogenesis property of imatinib mesylate in a rat model of liver fibrosis induced by carbon tetrachloride/olive oil and its effect on the expression of trans- forming growth factor(TGF)-?1.Methods Rat liver fibrosis was induced by intraperitoneal administra- tion of carbon tetrachloride and olive oil mixture twice a week for eight weeks.Imatinib mesylate was given 20 mg/kg daily by oral lavage.The control rats received saline by oral iavage.Liver collagen depo- sition was evaluated by immunohistochemistry with Masson staining.The activation of hepatic stellate cells was detemined by the immunohistoehemistry staining of?-smooth muscle actin.The mRNA expres- sions of TGF-?1,c-Abl and TIMP-1 were measured by RT-PCR.While protein expressions of TGF-?1, phosphorylated platelet-derived growth factor receptor and c-Abl were detected by Western blot and im- munohistochemical staining.Hepatic hydroxyproline content was also quantified.Results The collagen deposition[(16.23?1.01)%vs(25.61?0.92)%]and the number of activated HSCs(10.52?1.33vs 13.10?1.21)were reduced in the imatinib mesylate treatment group compared with the control group by 35% and 20%,respectively(P