BACKGROUND: Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a kind of cytokine which can stimulate the differentiation and proliferation of haematopoictic precursor cells and plays an important role in the process of immunoloregulation. Escherichia coli (E.coli) expression system has advantages,such as low cost and high output, over other systems.Therefore, E.coli is still the most commonly used exogenous gene expression system.OBJECTIVE: To observe the expression of hGM-CSF in the E.coli swain DH5α.DESIGN, TIME AND SETTING: The present observational experiment was performed at the Central Laboratory of Biotechnology Research,China Three Gorges University between February and June 2007. MATERIALS: Plasmid pBR322hGM-CSF was purchased from American Type Culture Collection, USA.hGM-CSF antibody was sourced from Sigma Company, USA.IPTG, X-gal,and vector pMGT-18 were purchased from Shanghai Bioengineering Technology Company, China. METHODS: Primer was designed according to hGM-CSF gene. Taking cloning vector plasmid pBR322-hGM-CSF as template, hGM-CSF gene was acquired and recombined into prokaryotic expression vector pGEX-4T-1. Recombinant plasmid confirmed by enzyme digestion and sequencing was transformed into E.coli strain DH5α and induced by isopropy-β-D-thiogalactoside (IPTG). Expression product was identified by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and detected by Western blotting assay. MAIN OUTCOME MEASURES: hGM-CSF expression in the E.coli strain DH5α. RESULTS: SDS-PAGE results revealed that the recombinant hGM-CSF protein with relative molecular weight of 40 500 was produced up to approximately 17.9% of total protein. Western blotting detection results indicated that there was a 40 500 bp brand. CONCLUSION: The present study reconstructed prokaryotic expression vector pGEX-hGM-CSF, induced its expression in the E.coli strain DH5α, and obtained hGM-CSF fusion protein.

In this study, the effect of prophylactic anti-inflammation on the development of smoke-induced emphysema was investigated. Young male guinea-pigs aged 1.5-2 months (weighing 198.3+/-26.9 g) were randomly divided into 4 groups: group A (cigarette smoke exposure only), group B (cigarette smoke exposure plus pentoxifylline-rich (PTX, 10 mg/d) forage feeding), group C (cigarette smoke exposure plus intermittent cortical steroid injection (Triamcinolone acetonide, 3 mg, i.m., every three weeks) and control group (group D: animals with sham smoke exposure, raised under the same conditions). Animals in group A, B and C were exposed to smoke of cigarettes for 1 to 1.5 h twice a day, 5 days a week. All animals were killed at the 16th week and followed by morphometrical analysis of the midsagittal sectioned lung slices. Smoke exposure of 16 weeks resulted in visible emphysematous development in Group A but not in Group B and C. It was evidenced by the indicator of air-space size, mean linear intercept (Lm): 120.6+/-16.0 microm in Group A; 89.8+/-9.2 microm in Group B and 102.4+/-17.7 microm in Group C. The average Lm in either group B or group C was shorter than that in Group A (ANOVA and Newman-Keuls test, F=8.80, P=0.0002) but comparable to that (94.8+/-13.2 microm) in group D (P>0.05). It is concluded that long-term prophylactic anti-inflammation inhibits pulmonary emphysema induced by cigarette smoking in the guinea pigs.
Anti-Inflammatory Agents/*pharmacology ; Pentoxifylline/pharmacology ; Pulmonary Emphysema/etiology ; Pulmonary Emphysema/pathology ; Pulmonary Emphysema/*prevention & control ; Random Allocation ; Smoking/*adverse effects ; Triamcinolone Acetonide/*pharmacology

Objective To discuss risk factor and operation guard in dissection of cholecyat from its bed during laparoseopic cholecystectomy.Methods The clinical data of 490 cases of laparoscopic cholecystectomy dissect cholecyst from its bed by traditional method during were retrospectively analyzed and simultaneously perspective study on 500 cases of improved laparoscopic cholecyatectomy dissecting cholecyst from its bed.Results There are 3 cases in hemorrhea due to injuring bed brancha of arteria cystica posterior branch,2 cases in bed brancha of arteria cystica posterior branch haemorrhage,5 cases in postoperative bile leak,2 cases in variant right hepatic portal vein injury,1 case in variant right hepatic duct in the 490 cases of laparoscopic choleeystectomy by traditiorhI method.Using our new technique exposed,and then ligated 39.6%(198/500)bed brancha of arteria cystica posterior branch,11.2% (56/500)bed brancha of venae hepaticae intermediate almost in fight side of cholecyst bed,2.2%(11/500)dilated aberrant bile-duct duct.We separated and then protected 0.6%(3/500)unusual right hepatic duct being exposed,0.4%(2/500)right liver branch of portsl vein.Conclusion There are some larvate risk in isolation of choleeyst from its bed during laparoseopic cholecystectomy.Unexpected injury happen during LC,and it ought tO be open cholecysteetomy The improved method provides a safe,efficiency operative procedure.

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase (PI3K) signaling pathway in nerve growth factor-beta (NGF-β)-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9c2 cells were seeded in 96-well plates at a density of 5× 105 cells/ml (100 μl/well).The wells were randomly divided into 5 groups (n =15 each) using a random number table: control group (group C);group H/R;NGF-β group (group N);NGF-β+NGF receptor trkA antagonist K252a group (group N+K);NGF-β+ PI3K inhibitor LY294002 group (group N+L).The cells were exposed to 95％ N2-5％ CO2 for 4 h in an anaerobic incubator, followed by reoxygenation in a standard incubator for 4 h in H/R, N, N+K and N+L groups.In addition, the cells in N, N + K and N + L groups were incubated in a standard incubator containing NGF-β, the mixture of NGF-β and K252a and the mixture of NGF-β and LY294002, respectively, during reoxygenation, and the final concentrations of NGF-β , K252a and LY294002 were 50 ng/ml, 100 nmol/L and 50 μmol/L, respectively.The cell viability was detected by using CCK-8 assay, and the cell survival rate was calculated.The cell apoptosis was examined by flow cytometry, and apoptosis rate was calculated.The expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), caspase-12, Akt and phosphorylated Akt (p-Akt) was detected by Western blot.The ratio of p-Akt to Akt was calculated.Results Compared with group C, the cell survival rate was significantly decreased, and the apoptosis rate was increased in H/R and N groups, and the expression of GRP78, CHOP and caspase-12 was significantly up-regulated in group H/R, and p-Akt/Akt was significantly increased in group N (P＜0.05).Compared with group H/R, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was increased in group N (P＜0.05), and no significant change was found in the parameters mentioned above in N+K and N+L groups (P＞0.05).Compared with group N, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was decreased in N+K and N+L groups (P＜0.05).Conclusion NGF-β can mitigate the cell apoptosis induced by endoplasmic reticulum stress during H/R, and activation of PI3K signaling pathway is involved in the mechanism.

Objective To evaluate the effects of nerve growth factor-beta (NGF-β) pretreatment on cell apoptosis induced by endoplasmic reticulum stress during ischemia-reperfusion in isolated rat hearts.Methods Male Sprague-Dawley rats,weighing 180-220 g,were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.The hearts were excised and perfused in a Langendorff apparatus with K-H solution aerated with 95％ O2 and 5％ CO2 at 37 ℃.Thirty-two isolated rat hearts were randomly divided into 4 groups (n =8 each) using a random number table:control group (group C),I/R group,NGF-β pretreatment group (group N) and NGF-β combined with K252a (trkA receptor antagonist) pretreatment group (group N + K).In group C,the hearts were continuously perfused with K-H solution for 195 min.The hearts were perfused with K-H solution for 45 min in group I/R.In N and N + K groups,the hearts were perfused with K-H solution for 15 min,and then with K-H solution containing 0.1 μg/ml NGF-β and 0.1 μg/ml NGF-β mixed with 100 nmol/L K252a,respectively,for 30 min.The perfusion was suspended for 30 min followed by 120 min of reperfusion with K-H solution in I/R,N and N + K groups.HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and + dp/dtmax were measured at the end of 15 min equilibration (baseline) and at 5,30,60 and 120 min of reperfusion.Myocardial specimens were obtained at 120 min of reperfusion for detection of myocardial apoptosis (by TUNEL) and expression of glucose-related protein 78 (GRP78),CCAAT/enhancer-binding protein homologous protein (CHOP),and caspase-12 (by Western blot analysis).Apoptosis index was calculated.Results Compared with group C,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index and expression of GRP78 and CHOP were increased in I/R and N groups,and the expression of caspase-12 was upregulated in I/R group.Compared with group I/R,HR,LVDP,and + dp/dtmax were significantly increased,and LVEDP,apoptosis index and expression of GRP78,CHOP and caspase-12 were decreased in group N,and the expression of GRP78 was down-regulated in group N + K.There was no significant difference in cardiac function indexes between group I/R and N + K.Compared with group N,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index,and expression of GRP78,CHOP and caspase-12 were increased in group N + K.Conclusion NGF-β pretreatment can protect the isolated rat hearts against ischemia-reperfusion injury,and inhibition of the endoplasmic reticulum stress-triggered cell apoptosis after activating trkA receptors is involved in the mechanism.

Objective To study the feature of clinical and pathological type of Henoch-Sch(o)nlein purpura nephritis (HSPN) in children.Methods Clinical and pathological data of HSPN in 279 children were collected and retrospectively analyzed.Results Clinical manifestation of HSPN in 279 children with haematuria and proteinuria (107 cases,38.4％) was the most common type,followed by nephritic syndrome type (69 cases,24.7％),isolated proteinuria type (40 cases,14.3％),isolated hematuria type (29 cases,10.4 ％),acute glomerulonephritis type (21 cases,9.3 ％),rapidly progressive glomerulonephritis type (8 cases,2.9％),chronic glomerulonephritis type (5 cases,1.8％).According to the International Study of Kidney Disease in Children,the majority renal pathological type of HSPN were grade Ⅱ (133 cases,47.7％)and grade Ⅲ (109 cases,39.1％).The pathological changes of hematuria and proteinuria type were mainly grade Ⅱ (61 cases,57.0％) and grade Ⅲ (35 cases,32.7％),and the pathological change of nephritic syndrome type was grade Ⅲ (41 cases,59.4％).All of renal pathological changes,38.7％ (108/279) had codeposition of immunoglobulins A and M,30.8％ (86/279) had co-deposition of immunoglobulins A,G and M.The pathological change of nephritic syndrome type was more serious (x2 =35.989,P ＜ 0.05).Immune complex deposition was not correlated with renal pathologic classification (P ＞ 0.05).Conclusion The HSPN patients mainly show the type of hematuria and proteinuria and the type of nephritic syndrome.The majority renal pathological type of HSPN are grade Ⅱ and grade Ⅲ.In children with HSPN,the severity of the clinic symptoms is not completely consistent with the pathological changes.The pathological changes of nephritic syndrome type are more serious.To improve theprognosis of HSPN,we should make the treatment planning according to the clinical types and pathologic classification.

Objective:To investigate the effect of enteral nutrition support one week before surgery and postoperative periods on the postoperative immune function and nutritional status in old patients with gastric cancer. Methods:A retrospective analysis was con-ducted on 77 stomach cancer patients who were admitted and received surgical treatment in our hospital. Among the patients, 38 re-ceived enteral nutrition support one week before surgery and early period of postoperation, and were labeled as the experimental group. The remaining 39 patients did not receive enteral nutrition support one week before surgery and early period of postoperation, and were named as the control group. The levels of prealbumin, albumin, transferrin, CD4+T (%), CD8+T (%), and CD4+/CD8+cells were also an-alyzed before surgery and 1 d and 8 d after surgery. Results:No statistical differences in the gender, age, site of tumor, TNM staging, and differentiated degree were found between the two groups. All operations were successful and the patients recovered well after sur-gery. In the nutritional status, the levels of prealbumin, albumin, and transferrin in the patients of the two groups had no significant dif-ference (P>0.05). On the first day after the surgery, the levels of prealbumin, albumin, and transferrin were significantly low in both groups;the control group had lower levels than the experimental group (P<0.05). No differences in the levels of CD4+T (%), CD8+T (%), and CD4+T/CD8+T cells were found between the two groups (P>0.05). On the first day after surgery, the levels of CD4+T (%), CD8+T (%), and CD4+T/CD8+T cells were significantly low in both groups;the control group had lower levels than the experimental group (P<0.05). Conclusion:Elderly patients with gastric cancer who receive appropriate enteral nutrition support one week before sur-gery would have better immune function and nutritional status after surgery than those who did not, and the recovery of old patients im-proved.

Objective To investigate the effect of sevoflurane intravenous inhalational anesthesia and remifentanil intravenous anesthesia for single lung ventilation in the department of thoracic surgery operation .Methods 80 patients using thoracic surgery were randomly divided into the two groups with 40 cases in each group according to the number table methods ,the study group were treated with sevoflurane inhalation anesthesia while the control group were treated with remifentanil intravenous anesthesia .HR,MAP and SBP of the two groups before induction of anes-thesia(T0),1min before intubation(T1),10min after ventilation(T2),30min after ventilation(T3) and 1min before extubation(T4) were compared,and recovery time,extubation time,directional force the recovery time were recorded of the two group.Results The HR in the two groups at T1,T2,T3,T4 time did not change significantly than T0 time, while MAP decreased significantly than T0 time,with significant difference (t =8.12,7.82,8.14,8.05,all P <0.05),and there was no statistically significant difference between the two groups (P >0.05);The SBP in the study group did not change obviously at each time while the SBP in the control group at T 1 decreased significantly than T0 time,while it increased significantly at T 2 time than T1 time,with a significant difference ( t=10.51,5.34, 5.05,5.06,all P<0.05);The patients in study group were elevated blood pressure during anesthesia or decreased more than 30% occurred in 8 cases while the control group occurred in 24 cases,there was significant differences between the two groups (χ2 =13.33,P<0.05);The postoperative recovery time ,extubation time and orientation recovery time of the study group patients were (11.4 ±5.1)min,(13.4 ±6.8)min,(18.9 ±5.8)min,which were significantly lower than (19.5 ±5.2)min,(26.5 ±7.6)min,(21.9 ±7.8)min of the control group(t=5.34,6.12, 6.15,all P<0.05).Conclusion Sevoflurane intravenous inhalational anesthesia has a better anesthetic effect for single lung ventilation in the department of thoracic surgery operation than remifentanil intravenous anesthesia ,and it can effectively maintain hemodynamics stability .

Objective To explore the clinical phenotype of 22q11.2 deletion syndrome with imperforate anus. Methods The clinical diagnosis and treatment of one case of 22q11.2 deletion syndrome complicated with anal atresia were retrospectively analyzed. Gene and phenotype of this disease were analyzed, and the related literature was reviewed. Results An elder male test tube infant of twins, born to a G2P2 mother were found to have special facial features, cleft palate, congenital heart disease, and imperforate anus after birth. A genome-wide microarray scan revealed 22q11.2 deletion syndrome. In related literatures, 22q11.2 deletion syndrome could be affected by TBX1 gene, histone modification, Ranbp1 gene, and even microRNA; while imperforate anus were influenced by multiple elements such as genetic, maternal, and environmental factors and multiple embryonic developments related genes could be involved in its pathogenesis. Conclusions Congenital imperforate anus and 22q11.2 deletion syndrome are mostly sporadic cases in epidemiology and are incidental accidents in development. Whether there is a common interaction factor between them needs to be further studied and defined.

Objective To investigate effects of inhalation of sevoflurane at different time on pulmonary function and oxidative stress in patients with one lung ventilation.Methods 80 patients who needed external surgery according to the random grouping principle,were divided into sevoflurane preconditioning group(OLV for 30min after inhalation of sevoflurane),sevoflurane treatment group(OLV inhalation of sevoflurane),sevoflurane inhalation group(inhalation of sevoflurane),total intravenous group(propofol and remifentanil anesthesia) four groups,20 patients in each group,each group was treated accordingly.The differences of serum malondialdehyde(MDA),superoxide dismutase(SOD),lactate dehydrogenase(LDH) and intrapulmonary shunt(Qs/Qt) in the four groups were observed and compared at different time points.Results Compared with the other three groups,the intrapulmonary shunt fraction of the sevoflurane preconditioning group were significantly lower[(4.9±0.6)% vs.(6.1±0.5)%,(6.3±0.6)%,(5.9±0.4)%](x2=5.415,5,882,4.977,all P<0.05).The serum levels of MDA,SOD,LDH in the sevoflurane pretreatment group were significantly lower than those in the other three groups[(5.06±2.10)nmol/mL vs.(13.04±3.27)nmol/mL,(12.40±4.28)nmol/mL,(13.13±2.30)nmol/ml],[(58.7±5.9)U/L vs.(84.9±13.8)U/L,(93.9±21.1)U/L,(84.2±11.2)U/L],[(16.3±2.1)U/L vs.(43.1±7.2)U/L,(45.4±6.7)U/L,(35.8±5.1)U/L](t=8.076,7.448,8.277;12.917,15.506,12.586;11.375,12.239,8.220,all P<0.05).Conclusion Thoracic surgery in patients with one lung ventilation during anesthesia,using sevoflurane preconditioning anesthesia surgery can effectively improve the patients with intrapulmonary shunt rate,reduce the level of oxidative stress and improve the prognosis,it is worthy of further clinical application.