Objective To investigate the effect of different doses of ketamine on inflammatory cytokines in rabbits with severe burn at early stage and preliminarily approach its regulatory action on early stage of inflammatory reaction due to stress of trauma.Methods Forty healthy male New Zealand rabbits were randomly divided into four groups in accord with the random number table method: normal control group, scald model group, ketamine analgesia group and ketamine anesthesia group. Before scald, pentobarbital sodium was used for anesthesia, afterwards catheters were inserted into internal jugular vein and internal carotid artery respectively ready for use, and 24 hours later, Ⅲ degree scald at the animal back and buttocks occupying 30% total body surface area (TBSA) was performed as the scald model for all the rabbits except those in normal control group. In ketamine analgesia group, after scald for 0.5 hour, 0.5 mg/kg ketamine intravenous injection was given to the rabbits as the loading dosage and then persistent intravenous pump infusion of 9μg·kg-1·min-1 ketamine was applied for all together 24 hours. In ketamine anesthesia group, after scald for 0.5 hour, 1.5 mg/kg ketamine intravenous injection was given to the rabbits, and then persistent intravenous pump infusion of 45μg·kg-1·min-1 ketamine was applied for 4 hours to maintain systemic anesthesia. In normal control and scald model groups, only intravenous infusion of equal amount of normal saline was given to the rabbits. The amount of intravenous transfusion in each group and the total dosages of ketamine used in ketamine analgesia group and ketamine anesthesia group were recorded. Before scald and 0.5, 6, 12, 24 hours after scald, arterial blood gas analyses were made, and the levels of serum interleukins (IL-1, IL-6) and tumor necrosis factor-α (TNF-α) were determined.Results Although the indexes of blood gas analysis were changed in the four groups, they were all in the normal range, showing that the respiratory function was in the normal range and indirectly reflecting that the circulatory function was also in the normal range, thus the effects on cytokines by factors of respiratory and circulatory functions were ruled out. The levels of IL-1, IL-6 and TNF-α before scald showed no statistically significant differencesamong the four groups (allP > 0.05). From 0.5 hour after scald, the levels of IL-1, IL-6 and TNF-α were markedly higher in model group than those of normal control group [IL-1 (ng/L): 30.27±0.93 vs. 13.79±1.11, IL-6 (ng/L): 47.22±1.49 vs. 46.31±4.12, TNF-α (ng/L): 243.39±20.85 vs. 190.95±14.97, allP < 0.05], and the situation continued until 24 hours after scald; the levels of IL-1, IL-6 and TNF-α from 6 hours after scald were significantly decreased in ketamine analgesia and ketamine anesthesia groups compared with those in the model group, and from 12 hours after scald, the degrees of descent in levels of the above indexes in ketamine analgesia group were more obvious than those in ketamine anesthesia group [IL-1 (ng/L): 19.28±2.51 vs. 40.12±10.31, IL-6 (ng/L): 52.10±4.23 vs. 72.20±10.11, TNF-α (ng/L): 246.03±20.74 vs. 313.71±27.34, allP < 0.05].Conclusion The low-dose ketamine analgesia and ketamine anesthesia have certain degree of inhibitory effect on the expression and release of inflammatory cytokines at the early stage in rabbits with severe burn, the effect of long-term low-dose ketamine analgesia being more significant.

Objective To observe the effect of small interfering RNA(siRNA)expression plasmids targeting transforming growth factor p receptor(TαR)Ⅰ gene on the collagen synthesis of hepatic stellate cells(HSCs).Methods Three siRNA expression plasmids were designed and constructed according to TBR Ⅰ sequence.Then the plasmids were transfected into HSC-T6 using 1ipofectamine2000 reagent. The mRNA and protein expressions of TβR Ⅰ were analyzed by reverse transcription polymerase chain reaction(RT-PCR)and Western blot technique, respectively. The cell proliferation was detected using methylthiazo-lyldiphenyl-tetrazolium bromide(MTT)methods. Concentrations of haluronic acid and type Ⅲ pro-collagen in the supernatants were determined by radioimmunoassay. The data were analyzed using least significant difference(LSD).Results Three recombinant plasmids expressing siRNAs were successfully constructed and confirmed by restriction enzyme assay. Compared with the blank control,all the three recombinant plasmids could inhibit the expressions of TβR Ⅰ mRNA,of which plasmid expressing siRNA2 exhibited the strongest inhibitory effect(psiRNA1 group:t=7.354,P＜0.01;psiRNA2 group:t=9.214,P＜0.01;psiRNA3 group:t=5.967,P＜0.01).The expressions of TβR Ⅰ protein were also reduced by all the three recombinant plasmids,of which the plasmid expressing siRNA2 showed the strongest inhibitory effect(psiRNA1 group: t=6.324,P＜0.01;psiRNA2 group:t=8.741,P＜0.01;psiRNA3 group:t=4.128,P＜0.01).The proliferation activity and collagen synthesis of HSCs also decreased in all three HSC groups treated with recombinant plasmids, of which, again, plasmid expressing siRNA2 exhibited the strongest inhibitory effect. However, no significant change was observed in HSCs transfected with non-related siRNA. Conclusion Recombinant plasmids targeting TβR I can inhibit collagen synthesis, which suggests a novel target for gene therapy of liver fibrosis.

Objective To study prevention and treatment for patients during the intermittent period of maintaining hemodialysis complicated by acute left heart failure. Methods The treating process of 126 examples suffered from acute left heart failure during the intermittent period of 14 225 person-time maintaining hemodialysis in 143 patients were retrospectively analyzed. Results In this case group, 0.89% suffered from acute left heart failure during the intermittent period of hemodialysis and the successful rescuing rate was 88.9%. Conclusions Applying the treatment of emergency hemodialysis, quickly filtering body fluid, the curative effect is reliable and rapid. It is one of the best ways to cure patients suffered from acute left heart failure during the intermittent period of maintaining hemodialysis. Combined with other treatments, it will be able to improve the successful rate, and ensure the sufficiency of hemodialysis. United with other steps of reducing blood pressure, such as using angiotensin converting enzyme inhibitors, angiotensin II receptor blocker, and ? receptor blocker, it is an effective countermeasure to prevent patients suffering from acute left heart failure during the intermittent period of maintaining hemodialysis, and effectively reduces its appearance as well.

Objective To observe the dynamic expressions of kielin/chordin-like protein (kcp) in mouse model of hepatic fibrosis and the effects of bone morphogenetic protein-7 (BMP-7)intervention on the expressions,and to explore a new target for treatment of fibrosis.Methods A total of 50 healthy male ICR mice were divided into three groups:control group(n=10) ; model group (n=30) and BMP-7 treatment group (n=10).The model group was further divided into three subgroups according to different time points:subgroups of 4,8 and 12 weeks with 10 mice in each subgroup.The mouse model of hepatic fibrosis was established by hypodermic injection of carbon tetrachloride(CCl4 ).The mice in BMP-7 treatment group began to receive human recombmant BMP- 7via intraperitoneal injection after 8 weeks of the first administration of CC14 and lasted for 4 weeks.The serum levels of alanine aminotransferase (ALT),aspartate transaminase (AST) and albumin (Alb) were detected.The pathological changes of liver were observed under optical microscope after HE and Masson staining.The dynamic expressions of kcp mRNA and protein of each group were detected by reverse transcription-polymerase chain reaction,immunohistochemistry and Western blot.The comparison of means among groups was done by univariate ANOVA.Results In model group,ALT and AST levels increased,while Alb level gradually decreased,and peaked at week 12.BMP-7treatment could reduce the changes,and there were significant differences among groups (F=23.501,34.600 and 16.244,respectively; all P＜0.05).In normal control group,the expressions of kcp mRNA and protein were low,while those in model group were gradually increased.BMP-7 treatment could achieve remission and the changes were all significantly different among groups (F=30.362 and 10.727,respectively; P＜0.01 or 0.05).The expression of kcp mRNA was positively correlated with levels of transforming growth factor (TGF)-β1 mRNA and BMP-7 mRNA (r=0.760 and 0.769,respectively; both P＜0.05).Conclusions BMP 7 can improve the hepatic fibrosis in mice.kcp may play an important role in the occurrence and development of liver fibrosis which is a potential therapeutic target for hepatic fibrosis.

Objective To set up a system in vitro to rapidly recover plasma proteins lost during artificial-liver treatment.Methods The polyprotein precipitation was obtained by all proteins whose isoelectric point pH value were between 7.3 and 5.1,which collided with each other and aggregated using gradient pH co-precipitation(adding 1 mol/L citric acid slowly in the plasma solution to change the pH values gradually from 7.3 to 5.1 in 5 h)combined with salting out(degree of saturation of NaCl is 33%,reacted for 5.5 h at 4℃)or low-temperature ethanol precipitation(40% ethanol, reacted for 5.5 h at -7℃)so that to get rid of toxicants by discarding the supernatant.Results In the range of pH 5.1-7.3,50%(29g/57g)of the total plasma proteins had been recovered by the gradient pH salting out and 41%(25 g/61g)by the gradient pH low-temperature ethanol co-precipi- tation.The protein remained in the supernatant was mostly albumin and its combined bilirubin.The levels of total bilirubin decreased to 0.07% and 0.06% of the original levels by these two methods respectively and the serum HBV DNA level decreased to be undetected(quantitative PCR).Conclu- sions The proteins with close isoelectric point can co-precipitated with the presence of high concen- tration of NaCl or low-temperature ethanol and by changing the pH value gradually.The total protein in the discarded plasma during artificial-liver treatment can be recovered rapidly using the gradient pH coprecipitation.

Objective To explore the dynamic expressions and the significance of Notch/Jagged signal pathway in rat model of hepatic fibrosis. Methods A total of 42 healthy male SD rats were randomly divided into normal control group (n=6) and model group (n= 36). The model group was further divided into six subgroup according to different time points: subgroups of 4 days, 1, 2, 4, 6 and 8 weeks with six rats in each subgroup. The rat model of hepatic fibrosis was induced by dimethylnitrosamine (DMN). The serum levels of alanine aminotransferase (ALT), aspertate aminotransferase (AST), albumin (Alb) and hyaluronic acid (HA) were detected dynamically after 4 days, 1,2,4,6 and 8 weeks of injection. The liver tissues were observed under optical microscope after HE and Masson staining. Notch-1, Jagged-1 mRNA and protein in liver were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The comparison of means among groups was done by univariate ANOVA. Results The hepatic fibrosis model was successfully induced by DMN injection and pseudolobules were found after 4 weeks of injection. The serum levels of ALT, AST, Alb and HA were all increased after 4 day of injection and peaked at week 4 which were all significantly higher than those in control group (F=83.10, 104.63, 54.24, 203.81,respectively; all P＜0.05). The expressions of Notch-1, Jagged-1 mRNA and protein in model group were all significantly increased than those in control group (F=282. 44, 369.14, 374.17, 256. 14,respectively;P＜0. 01). And the expressions of Notch-1, Jagged-1 were closely correlated with the hepatic fibrosis stages and transforming growth factor β1 (TGFβ1) expression (r=0. 821, 0. 917,0. 767,0. 844, respectively; P＜0. 01 ). Conclusions The Notch/Jagged pathway may participate in the development of hepatic fibrosis, which is closely correlated with the progression and severity of liver fibrosis.

Objective To study the morphological characteristics of pituitary glands in healthy adult of the Han nationality on the High-resolution MRI and provide morphological data of pituitary glands for the construction of database for Chinese Standard Brain.Methods This is a clinical multi-center study.Nine hundred and seventy eight Chinese healthy volunteers (age range = 18 to 70) recruited from 16 hospitals were divided into 5 groups, i.e., Group A ( age range = 18 to 30), B ( age range = 31 to 40), C ( age range = 41 to 50), D ( age range = 51 to 60 ), and E ( age range = 61 to 70).All of the volunteers were scanned by MR using T, weighted three-dimensional magnetization prepared rapid acquisition gradient echo sequence.By Multiplan reconstruction with 3D software, the pituitary gland's volume, size, and inclination of pituitary stalk were measured and the differences between sexualities and among age groups were compared by analysis of variance using SPSS13.0; those among five age groups were compared pair-wised by Tukey analysis; and the differences between male and female were analyzed by independent t test, and the differences of measurements of typing of pituitary glands were analyzed by Chi-square analysis.Results( 1 ) Volume of pituitary glands: the mean volume of male and female among 5 age groups were ( 1142±290), (996±223), (979 ±178), (971 ±174), (930 ±189) and (1247 ±210), (1199 ±216),( 1108 ± 196), ( 1059 ± 212 ), (984 ± 177 ) mm3 respectively.There were significant differences among the age groups(F = 13.811,27.091 ,P <0.01), the volume tended to decrease after 30 years old in male and 40 years old in female.Female has bigger volume than man in the same age group (P < 0.05).There were statistically correlation between sexes, age, typing, heights in middle sagittal width in coronal MR and anteroposterior diameter in middle sagittal MR.(2) The size of pituitary giands:the mean heights on middle sagittal MR images of male and female among 5 age groups were(6.6±1.5), (6.0±1.4), (5.6±1.1 ),(5.0±1.2), (4.9±1.4)and (7.9±1.6), (6.4±2.0), (5.6±1.7), (5.1±1.7), (4.4±1.4)mm respectively.There were significant difference among the age groups ( F = 24.582,62.978, P < 0.01 ).Females demonstrated larger pituitary glands than men in the age from 18 to 30 year old groups ( P < 0.01 ).The pituitary height tended to decline after 30 years old, minimum after 50 years old group in male and 40 years old in female.The heights were statistically correlated with sexes and age.The mean width in coronal MR images of male and female among 5 age groups were( 14.8± 1.8), ( 14.7 ± 1.4), ( 14.2±1.9), (14.3 ±2.1), (13.8 ±2.4) and (14.9±1.8), (15.8±1.7), (15.5±2.1), (15.6±2.2),( 14.9±2.1 ) mm respectively.There were significant difference among the age groups( F = 4.566,4.233,P<0.05),The width of pituitary before 30 years old and after 60 years old were smaller than the other groups (P <0.05 ), but man did not show this relationship.The widths were statistically correlation with sexes, age and weight.The mean anteroposterior diameter in middle sagittal images of male and female among 5 age groups were (11.2±1.2), (11.8±1.3), (11.8±1.5), (12.3±1.8), (12.2±2.0) and (11.9±1.3), (12.5±1.5), (12.4±1.5), (12.9±1.6), (12.7±1.6)mm respectively.There were significant difference among the age groups ( F = 7.270, 5.903, P < 0.01 ).Before 60 year old, the anteroposterior diameter was smaller in male than all female groups (P < 0.05 ), and there was a tendency to grow downwards with age.The anteroposterior diameter was statistically correlated with sexes and age.(3)The angle of inclination of pituitary stalks: on reconstructed coronal MRI imaging, the angle of inclination of pituitary stalk were in the middle, no significant differences in the angle measurements were found between male and female among 5 age groups ( P > 0.05).(4) The typing of pituitary glands in middle sagittal MR images : there were significant differences between each typing ( χ2 = 44.212, 107.518, P < 0.01 ), there was a tendency of pituitary upper border to depressed when age increased.Conclusions With highresolution MRI and 3D volumetric analytic software, volume and size of pituitary glands and the angle of inclination of pituitary stalks were accurately measured, and that provide morphological data of pituitary glands for the construction of database for Chinese Standard Brain.

Objective To investigate the therapeutic effect and possible mechanism of adenosine A2A receptor agonist (CGS21680) combined with bone marrow mesenchymal stem cells (BMMSC) transplantation in acute liver failure (ALF).Methods Fifty male C57BL/6 mice, 6-8 weeks old, were fed with standard diet for 1 week and randomly divided into 5 groups according to random number table: healthy control group (n=6), model group (n=11), BMMSC group (n=11), CGS21680/BMMSC group (n=11) and CGS21680 group (n=11).Except healthy control group, the other mice were injected with D-GalN and lipopolysaccharide (LPS) to establish ALF model.Ten hours later, CGS21680/BMMSC group and CGS21680 group were injected intraperitoneally with adenosine A2A receptor agonist CGS21680 (2.1 mg/kg).In addition, the BMMSC group and CGS21680/BMMSC group were injected BMMSC (1×10.6) through tail vein.After 24 hours, pathological changes of liver tissue was observed by hematoxylin and eosin staining.The change of proportion of mouse splenic Treg among CD4+T lymphocytes was detected by flow cytometry.Toll-like receptor (TLR)4 and nuclear factor (NF)-κB expression levels in liver tissue were detected by real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blot.One-way analysis of variance (one-way ANOVA) and SNK-q test was conducted for data analysis.Results Serum IL-6 levels were (23.67±2.97) pg/mL in healthy control group, (151.47±6.03) pg/mL in model control group, (72.10±3.74) pg/mL in BMMSC group, (53.35±2.50) pg/mL in CGS21680/BMMSC group and (84.85±3.25) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.02, 25.51, 19.58 and 34.03, respectively, all P<0.01).Serum TNF-ɑ levels were (24.62±3.19) pg/mL in healthy control group, (102.25±2.10) pg/mL in model control group, (54.71±2.23) pg/mL in BMMSC group, (42.20±4.72) pg/mL in CGS21680/BMMSC group and (81.76±3.50) pg/mL in CGS21680 group.The differences between healthy control group and the other 4 groups were all statistically significant (t=46.49, 19.97, 7.72 and 29.57, respectively, all P<0.01).The differences of spleen Treg proportion in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=51.67, 12.22, 5.91 and 18.21, respectively, all P<0.01).The differences of TLR4 mRNA levels of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=26.31, 21.33, 13.24 and 27.14, respectively, all P<0.05).The differences of NF-κB mRNA level of liver tissue in healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=16.56, 16.34, 7.83 and 13.11, respectively, all P<0.05).The differences of TLR4 protein level in liver tissue of healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=35.60, 10.38, 6.05 and 18.02, respectively, all P<0.05).The differences of liver NF-κB protein level in the healthy control group were statistically significant compared with model control group, BMMSC group, CGS21680/BMMSC group and CGS21680 group (t=10.80, 7.30, 4.61 and 13.24, respectively, all P<0.05).Conclusions Adenosine A2A receptor agonist combined with BMMSC can significantly up-regulate the proportion of Treg cells in acute liver failure mice and inhibit the TLR4/NF-κB pathway activation, with both coordinated regulation, and further inhibit the liver inflammation.

ObjectiveTo investigate the inhibitory effect of bone morphogenetic protein 7 (BMP7) on proliferation of rat hepatic stellate cells (HSC).MethodsHSC-T6 cells were cultured in vitro,and divided into four groups:control group,transforming growth factor (TGF) β1 group,TGFβ1 + BMP7 group,and TGFβ1 + BMP7 + Noggin group. The mRNA expressions of smad1,α-smooth muscle actin (α-SMA),gremlin and fibronectin (FN) were determined by semi-quantitative reverse transcriptasepolymerasechainreaction(RT-PCR).Theproteinexpressionsof phosphorylated-smad1/5/8 (P-smad1/5/8),α-SMA and gremlin were detected by Western blot.The statistical analysis were done using one-factor analysis of variance,LSD-t test,Dunnett's T3 test and Pearson linear correlation analysis.ResultsPhosphorylated-smad1/5/8,α-SMA,gremlin and FN were expressed on HSC-T6 cells in control group,which were significantly up-regulated after TGFβ1 stimulation (P＜0.05).The expressions of α-SMA,gremlin and FN were significantly down-regulated in TGFβ1 + BMP7 group compared with TGFβ1group, while P-smad1/5/8 expression was upregulated (1.613±0.031 vs.2.503±0.014; t=34.89,P＜0.05).The expressions of α-SMA,gremlin and FN in TGFβ1 + BMP7 + Noggin group were up-regulated than those in TGFβ1 + BMP7 group,while P-smad1/5/8 was down-regulated (t=34.05,P＜0.05).There was no difference of total smad1 mRNA expression among each group.ConclusionThese results collectively suggest that BMP7 strongly inhibits the expressions of α-SMA,gremlin and FN.

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.