Objective To isolate and purify pancreatic ductal epithelial cells in adult rats,and induce differentiation of pancreatic ductal epithelial cells to islets in vitro.Methods By retrograde in- jection of collagenase into biliary-pancreatic tract,pancreatic tissues were digested and different types of pancreatic cells including islets,duct and degranulated aicni cells were separated by means of density gradient centrifugation.Ductal cells were purified by adhering method and identified by immunocyto- chemistry stain of ductal epithelial cells maker antigen(Cytokeratin 19,CK-19).Ductal cells were ex- panded in RMPI 1640 with 10% FBS.About one week when most adherent ceils were of monolayer, the medium was changed to serum-free DMEM/F12 supplemented with keratinocyte growth factor (KGF)to further expand ductal epithelial cells.When ceils reached 80% confluence,nicotinamide and high concentration of glucose were added to promote differentiation of pancreatic ductal epithelial cells.Islets like-structure was stained by Dithizone.Results Irnmunocytochemistry stain of CK-19 re- vealed that most isolated ceils were ductal epithelial ceils.The cultured ductal epithelial cells began to adhere at day 1,reached 80% confluence and cell clones were formed at day 14-21.At day 28,islets- like-structure appeared and was positive for Dithizone staining.Conclusions Ductal epithelial cells of rats can be isolated by means of density gradient centrifugation and purified by adhering method.Duc- tal epithelial cells can differentiate into islets-like-structure in vitro.

Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Female ; Fetal Blood ; Humans ; Infant, Newborn ; Lipopolysaccharides ; Lymphocytes ; metabolism ; Male ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Suppressor of Cytokine Signaling 1 Protein ; Suppressor of Cytokine Signaling 3 Protein ; Suppressor of Cytokine Signaling Proteins ; genetics ; metabolism ; Time Factors ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

The main treatment strategies for chronic pancreatitis in young patients include therapeutic endoscopic retrograde cholangio-pancreatography (ERCP) intervention and surgical intervention. Therapeutic ERCP intervention is performed much more extensively for its minimally invasive nature, but a part of patients are referred to surgery at last. Historical and follow-up data of 21 young patients with chronic pancreatitis undergoing duodenum-preserving total pancreatic head resection were analyzed to evaluate the outcomes of therapeutic ERCP intervention and surgical intervention in this study. The surgical complications of repeated therapeutic ERCP intervention and surgical intervention were 38% and 19% respectively. During the first therapeutic ERCP intervention to surgical intervention, 2 patients developed diabetes, 5 patients developed steatorrhea, and 5 patients developed pancreatic type B pain. During the follow-up of surgical intervention, 1 new case of diabetes occurred, 1 case of steatorrhea recovered, and 4 cases of pancreatic type B pain were completely relieved. In a part of young patients with chronic pancreatitis, surgical intervention was more effective than therapeutic ERCP intervention on delaying the progression of the disease and relieving the symptoms.

Objective: To evaluate the service competence of 99 smoking cessation clinics in Zhejiang Province, so as to provide reference for the improvement. Methods: The questionnaire, prepared by Tobacco Control Office of Chinese Center for Disease Control and Prevention, was used to investigate all the smoking cessation clinics in Zhejiang Province, to score the basic and developmental indicators, and to assess the qualified rates ( basic indicators >50 points and total scores >60 points ). Results: There were 99 smoking cessation clinics, with 52 in secondary hospitals and 47 in tertiary hospitals. The overall assessment scored from 11 to 96 points, with an average of ( 53.99±16.56 ) points. The smoking cessation clinics in secondary and tertiary hospitals scored ( 53.92±15.88 ) points and ( 54.06±17.45 ) points. The scores of basic and developmental indicators were ( 45.66±12.16 ) points and ( 8.33±5.39 ) points, with the scoring rates of 65.23% and 27.77%. The overall assessment of 43 smoking cessation clinics were qualified and the rate was 43.43%, which was 44.23% in secondary hospitals and 42.55% in tertiary hospitals. Conclusion The qualified rate of smoking cessation clinics in Zhejiang Province was 43.43%, which was similar between second hospitals and tertiary hospitals.

This study aimed to analyze the etiology of the encephalitis outbreak in Longyan, Fujian Province, China in 2010, in order to provide valuable information for this prevention and control of this disease. Pathogens were confirmed from cerebrospinal fluid samples with fluorescent RT-PCR, virus isolation (RD cells), and neutralization tests. Then, the VP1 fragments or whole genome nucleotide sequences were determined for four virus strains using PCR. Homology was assessed using the MegAlign software, and a phylogenetic evolutionary tree was drawn using Mega 4.0 software. The results confirmed that the etiology of the outbreak was the ECHO6 intestinal virus, and the nucleotide sequence of the VP1 segment indicated that the C2 subtype was responsible. The genome sequence consisted of 7407 nucleotides, and resembled the genome of other ECHO and CoxB viruses with homology levels of 78.5%-87.3%. The encephalitis outbreak in Longyan in 2010 was caused by the ECHO6 C2 subtype intestinal virus, and its complete genome sequence length is similar to the standard strain (U16283) with a sequence homology of 80.4%.
Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Echovirus 6, Human ; classification ; genetics ; isolation & purification ; Echovirus Infections ; epidemiology ; virology ; Encephalitis ; epidemiology ; virology ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny

Objective To discuss the value and experience of the percutaneous vertebroplasty (PVP)in the treatment of vertebral body compression fracture(VCF)in aged osteoperosis.Methods PVP was performed in 44 cases with VCF including 28 with single vertebral compressed fracture,12 with double compressed fracture and four with triple compressed fracture,with 67 vertebrae,for clinical and radiologieal evaluation.Results The mean follow-up was 15 months(4-23 months).There could be seen immediate relief of pain in 40 cases,out-of-bed activities at operation day in 19 and out-of-bed activ- ities at second day after operation in 25.Postoperative X-ray showed uniformly distributed bone cement in the vertebral,without leakage.Conclusion PVP is a recommendable method for VCF,for it has ad- vantages of pain relief,vertebrae stabilization,minimal invasion and minor complications.

ObjectiveTo observe the effect of early rehabilitation on patients with closed traumatic brain injury (TBI).Methods80 TBI patients were divided into rehabilitation group and control group with 40 cases in each group. The control group was treated with routine surgical and other treatments. The rehabilitation group was treated with limb exercise and daily living training, besides treatments mentioned above. The scores of degrees of neural damage, Fugl-Meyer Assessment (FMA), modified Barthel index (MBI) and disability rating scale, and complications within 3 months of two groups were compared.ResultsThe scores of degree of neural damage, FMA, MBI and disability rating scale, and rate of complication of the rehabilitation group were significantly different from that of the control group (P<0.05～0.01).ConclusionEarly rehabilitation can improve brain function and reduce complication incidence of TBI patients.

OBJECTIVEN-Acetylcysteine (NAC) is a sulfhydryl donor molecule with antioxidant and antiinflammatory effects. A major role has been described for inducible nitric oxide (NO) synthase in several inflammatory liver diseases. NAC attenuates NO generation following lipopolysaccharide injection in rats. The purpose of this study was to investigate the effect of NAC against lipopolysaccharide injury in hepatocytes of neonatal mice and the molecular mechanisms by which NAC influences inflammatory responses of the hepatocytes.

METHODSThe liver of neonatal mouse was digested by collagenase to dissociate the hepatocytes. The hepatocytes were cultured and isolated. After 7 days of culture the normal hepatocytes were divided into two groups: LPS group and NAC group. In LPS group, 10 microg/ml LPS was added into the culture medium. In NAC group, 5 mmol/L NAC was added into the culture medium firstly, 10 microg/ml LPS was added after 1 h of culture. There were 12 mice in each group. The cell supernatants and the hepatocytes were collected at 0, 6 and 12 hours after adding LPS. The cell supernatants were taken to measure the alanine aminotransferase (ALT) level and nitric oxide (NO) production by the biochemical methods. The cells were taken to analyze the gene expression of induced nitric oxide synthase (iNOS) by the RT-PCR.

RESULTSIn LPS group, the levels of ALT, NO and iNOS mRNA increased significantly at the time points 6 h and 12 h compared with the time point 0, (P < 0.01). Compared with the LPS group, the levels of ALT, NO and iNOS mRNA of NAC group were lower at the time points 6 h and 12 h (P < 0.01).

CONCLUSIONSNAC may play a protective role in the hepatocytes injury caused by LPS in the neonatal mice. The protective mechanism works partially through the inhibition of iNOS activation by LPS.

Acetylcysteine ; pharmacology ; Alanine Transaminase ; metabolism ; Animals ; Animals, Newborn ; Anti-Inflammatory Agents ; pharmacology ; Cells, Cultured ; Hepatocytes ; drug effects ; Lipopolysaccharides ; Mice ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism

OBJECTIVETo investigate the expression of TLR4/2 mRNA in neonatal cord blood mononuclear cells (MNC).

METHODSForty-six neonates without asphyxia and 40 neonates with asphyxia were divided into groups depending on the gestational age. In the neonates without asphyxia, there were 18 full term infants (the gestational age > or = 37 weeks), 16 preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 12 preterm infants whose gestational age was < 32 weeks. In the neonates with asphyxia, 11 were full term infants, 15 were preterm infants whose gestational age was > or = 32 weeks but < 37 weeks and 14 were preterm infants at gestational age < 32 weeks. MNCs were separated and cultured with LPS (1 microg/ml) for 3 h. Cells were collected for analysis of gene expression of TLR4/2 by RT-PCR technique. Cell supernatants were taken to measure TNF-alpha production following the ELISA protocol. Fifteen healthy adults were enrolled into the control group. In addition, the Pearson correlation analyses were carried out between the levels of TLR4, TLR2 mRNA and the levels of TNF-alpha.

RESULTSIn the neonates without asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.75 +/- 0.12, 0.63 +/- 0.08, 2502.6 +/- 273.1 ng/L, separately, in the full term infants, 0.37 +/- 0.04, 0.32 +/- 0.03, 1218.8 +/- 145.7 ng/L, separately, in the preterm infants whose gestational ages were > or = 32 weeks but < 37 weeks, and 0.26 +/- 0.03, 0.20 +/- 0.03, 811.8 +/- 105.2 ng/L separately, in the preterm infants whose gestational ages were < 32 weeks. In the neonates with asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.58 +/- 0.07, 0.50 +/- 0.06, 1946.4 +/- 244.2 ng/L, separately, in the full term infants, 0.29 +/- 0.03, 0.26 +/- 0.03, 970.0 +/- 94.3 ng/L, separately, in the preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 0.17 +/- 0.02, 0.14 +/- 0.02, 652.6 +/- 60.3 ng/L, separately, in the preterm infants whose gestational age was < 32 weeks. The levels of TLR4, TLR2 mRNA and TNF-alpha in the adults were 2.71 +/- 0.75, 2.61 +/- 0.33, 9270.1 +/- 1098.3 ng/L, separately. In the preterm infants and full term infants, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower in comparison to the adults. The lower the gestational age, the lower the levels of TLR4, TLR2 mRNA and TNF-alpha. There were significant differences between the levels of TLR4, TLR2 mRNA and TNF-alpha of the neonates without asphyxia and those of the neonates with asphyxia. In the neonates with asphyxia, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower than those in the neonates without asphyxia (P < 0.01). Whether the neonates were asphyxic or not, the levels of TLR4, TLR2 were paralleled with the levels of TNF-alpha.

CONCLUSIONSThe expression of TLRs in the neonates, especially in the preterm infants was lower than that in the adults, which probably contributes to the susceptibility of neonates to infections.

Blood Cells ; metabolism ; Gene Expression ; Humans ; Infant ; Infant, Newborn ; RNA, Messenger ; genetics ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo measure the plasma levels of transforming growth factor beta1 (TGFbeta1), the protein expression of alpha-SMA in hepatic stellate cells and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), and study on the relationships between the plasma levels of TGFbeta1, the protein expression and the serum hyaluronic acid (HA) in patients with different grades of hepatic fibrosis.

METHODSThirty seven cases with hepatic fibrosis of different grades were classified according to HE and VG staining categories from 0 to 4, in which there were 8 cases in grade 1, 9 cases in grade 2, 7 cases in grade 3, 13 cases in grade 4. The plasma levels of TGFbeta1 and the serum levels of HA were detected by ELISA. The protein expressions of a-SMA, uPA and PAI-1 in fibrotic liver tissue were observed by immunohistochemistry.

RESULTSWith the progression of hepatic fibrosis, the plasma levels of TGFbeta1 and the protein expression of a-SMA, uPA and PAI-1 in fibrotic liver tissue were increased. In grade 3 and 4, the plasma levels of TGFbeta and the protein expression of a-SMA and PAI-1 in fibrotic liver tissue were significantly increased, but the protein expression of uPA in cirrhosis liver tissue did not increased.

CONCLUSIONTGFbeta1, a-SMA, uPA and PAI-1 play an important role in the progression of hepatic fibrosis. Inhibiting the early activation of latent TGFbeta1 or increasing uPA and inhibiting PAI-1 over express may contribute to matrix degradation and retard the progression of hepatic fibrosis.

Actins ; blood ; Adult ; Aged ; Female ; Hepatitis B, Chronic ; blood ; complications ; Humans ; Liver Cirrhosis ; blood ; etiology ; Male ; Middle Aged ; Plasminogen Activator Inhibitor 1 ; blood ; Transforming Growth Factor beta ; blood ; Transforming Growth Factor beta1 ; Urokinase-Type Plasminogen Activator ; blood