BACKGROUND: Multipotency of hepatic stem cells is of important value in liver transplantation. Stem cells have been successfully identified and isolated from the animal livers. However, reports on whether stem cells exist in human hepatic tissue and how to isolate and identify them ars few.OBJECTIVE: This study was in attempt to isolate hepatic stem cells from human para-cancerous tissues of hepatoma and in vitro culture them, also to identify the stem cell surface marker, in order to find a new source of heptatic stem cells.DESIGN: Cell observation experiment.SETTING: Department of Common Surgery, First Hospital, Jilin University; Department of Common Surgery,Dongfeng Hospital of Traditional Chinese Medicine.PARTICIPANTS: Samples were harvested from 10 patients with hepatoma admitted to Department of hepatobiliary surgery, First Clinical College, Jilin University between October 2005 and June 2006, with age of 45 to 58 years.Hepatic tissue 2 cm away from cancer nest was cut when patients underwent hepatectomy, and it was pathologically confirmed as carcinoma-free tissue. Written informed consents were obtained from each patient. DMEM/F12 dry powder used for cell culture was provided by Hyclone Company, USA. Fresh fetal bovine serum was prepared by Lianxing Biotech Co.,Ltd, Tianjin. Various cell growth factors were the products of Cytolay Company, USA.METHODS: Para-cancerous tissues of hepatoma was cut into pieces, rinsed with Hank's solution and digested with type Ⅳ collagenase. Then the isolated cells were re-suspended in the DMEM/F12 medium supplemented with 0.1 volume fraction of fetal bovine serum, and hepatocyte growth factors, epidermal growth factors and α- fibroblast growth factors of 25 μg/L each were added in the above medium. When the cultured cells covered 2/3 of bottom,they were digested with trypsinase for passage and inoculated at 2×107 L-1. When cells propagated to the 3rd and 4th generations, 2.60×109 L-1 cell suspension prepared with trypsinase was added, and subsequently, anti-human C-kit antibody, immunomagnetic beads and Buffer solution were added in order. C-kit+ cells were preliminarily isolated by immunomagnetic bead separation. Haematoxylin-eosin staining and immunofluorescent histochemical double-staining were used for detecting the hepatic stem cells in para-cancerous tissues.MAIN OUTCOME MEASURES: ① Observation of cell morphology. ② Identification of hepatic stem cells from para-cancerous tissues. ③ Identification of C-kit+ cells by immunofluorescent histochemical double-staining.RESULTS :① After primarily cultured for 2 weeks, the adherent cells grew in colony. After one half of culture medium was renewed, mature hepatocytes were gradually broken and disappeared. Small round cells propagated, and most of them were located in the center and arranged in cluster. Most cells were found with one big nucleus in each, less cytoplasm and clear cell boundary. When cells propagated to the 1st and 2nd generations, they still grew in colony, but fast. Each C-kit+ cell isolated by immunomagnetic bead separation presented a spherical cell body with a very big nucleus and less cytoplasm. After in vitro cultured for 1 week, it presented broken pieces and apoptotic symptoms.② After para-cancerous tissue was stained by haematoxylin-eosin, atypically proliferated biliary tracts with small round cells could be seen in the portal area. After para-cancerous tissue was stained by immunofluorescent histochemical double-staining, small round cells in the biliary tracts proliferated in the portal arsa co-expressed red fluorescence AFP and green fluorescence cytokeratin (CK) 19 with yellow superposition arsa. ③ After C-kit+ cells were stained by fluorescence immunocytochemisty, cytoplasm expressed alpha-fetoprotein (AFP) red granules and CK19 green granules. The superposition area of both presented yellow fluorescence of AFP+/CK19+-positive cells.CONCLUSION: Hepatic stem cells exist in human para-cancerous tissues of hepatoma. Therefore, expressions of C-kit+/AFP+/CK19+, the surface markers of hepatic stem cells, can be used for identifying and isolating hepatic stem cells. Small round cells obtained by in vitro isolation and culture, i.e. hepatic oval cells possess bipotential differentiation of hepatocyte and hepatobiliary epithelial cells.

The goal of this work was to explore the prospect of standardized application of an in-vitro bioactivity assay for recombinant human follicle-stimulating hormone based on a reporter gene. The relative accuracy, intermediate precision, linearity and applicable range of the method were validated according to the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Three laboratories used this method to determine the in-vitro biological activities of six batches of drug product and three batches of drug substance manufactured by two different companies. The consistency of the potency determined by three laboratories, the intra-laboratory precision and inter-laboratory precision were analyzed. The method was optimized during the collaborative validation. The results of method validation meet the requirements of the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Aiming to resolve the problems found in the collaborative validation, the medium for cell seeding, the pre-diluted buffer solution of standard and sample, and the means of removing and discarding supernatant after stimulation were optimized. After optimization, there was no significant difference in the bioactivity among the different laboratories (P > 0.05), indicating statistical equivalency. Intra-laboratory and inter-laboratory precision were good and the geometric coefficient of variation (GCV%) were both less than 15%. In conclusion, the reporter gene assay has good intra-laboratory repeatability and inter-laboratory reproducibility and is suitable for analyzing recombinant human follicle-stimulating hormone drug product and drug substance by different manufacturers. It is expected to be used as a standardized method for the determination of the in-vitro bioactivity of such products.

Child, Preschool ; Female ; Humans ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; pathology ; Rett Syndrome ; pathology

Objective To compare the effect of perioperative intravenous injection of Analgecine on the analgesic efficacy and complications of patient-controlled intravenous analgesia ( PCIA ) of different doses of fentsnyl in postoperative lumhotomy patients.Methods 200 patients underweat hmbotomy in general anesthesia were randomly divided into four groups with fifty cases each.Fentanyl 1.0mg in group A,fentanyl 0.5mg in group B,fentanyl 1.0mg in group C,fentanyl 0.5mg in group D.The drugs in each group were diluted to 100ml and infused by pumps.Besides,the patients in group C and D were injected with analgecine 3.6u and 7.2u at the night before the operation,preoperation and postoperation respectively.The visual analog scale(VAS),times of PCA and incidence of side effects were recorded during the period of postoperative 24 hours.Results The VAS of group B at 2h after operation was (5.2 ± 1.9 ) points,which was significandy higher than that of group A,C and D( P ＜ 0.05 ),VAS became similar 4h later( P ＞0.05).The demanding times for supplemental bolus in group B were also significantly higher than that of A,C and D( P ＜ 0.05 ).The incidence of nausea,vomiting,itching,somnolence in group B and D were significantly less than those in group A and C( P ＜0.05 ).No respiratory depression or abnormal bleeding occurred in the four groups.Conclusion Perioperative intravenous injection of analgecine had a better effect on PCIA of fentanyl and could reduce fentanyl requirement and its side effects in lumbotomy patients.

Cell Line, Tumor ; Enzyme Inhibitors ; pharmacology ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Proteasome Endopeptidase Complex ; drug effects

Objective To investigate the effects of ketamine on neuronal nitric oxide synthase (nNOS) activity and carboxy-terminal PDZ ligand of nNOS (CAPON) expression in the prefrontal lobe of mentally depressed rats.Methods Adult male Sprague-Dawley rats,aged 2.5-3.0 months,weighing 210-260 g,were used in the study.Menial depression was induced by exposing the rats to chronic unpredictable mild stress.Twenty-four animals in which mental depression was successfully induced were randomly divided into 2 groups (n =12 each):mental depression group (group D) and ketamine group (group K).Another 12 rats were chosen and served as control group (group C).Group K received intraperitoneal ketamine 10 mg/kg once a day for 7 consecutive days,while groups C and D received intraperitoneal normal saline 10 ml/kg instead of ketamine.Sucrose preference test and open field test were performed before administration and at 1 day after the end of administration.The total distance,number of rearing and sucrose preference percentage (SPP) were recorded.The rats were sacrificed 1 day after the last test for determination of the expression of nNOS and CAPON protein (using immuno-histochemistry)and mRNA (by RT-PCR) in the prefrontal lobe.Results Compared with group C,the total distance was shortened,the number of rearing and SPP were significantly decreased,the expression of nNOS protein and mRNA was up-regulated and the expression of CAPON protein and mRNA was down-regulated in groups D and K (P ＜ 0.05).Compared with group D,the total distance was prolonged,the number of rearing and SPP were significantly increased,the expression of nNOS and mRNA was down-regulated and the expression of CAPON protein and mRNA was up-regulated in group K (P ＜ 0.05).Conclusion Ketamine can improve the depressive state through promoting the expression of CAPON and inhibiting nNOS activity in the prefrontal lobe of mentally depressed rats.

AIMTo evaluate the effects of dipfluzine on the expressions of E-selectin, P-selectin, and ICAM-1 and the infiltration of polymorphonuclear leukocytes in brain ischemia-reperfusion rats.

METHODSThe model of focal cerebral ischemia was established with the Zea-Longa occluding suture. Dipfluzine (0.25, 0.5 and 1 mg x kg(-1)), flunarizine 0.5 mg x kg(-1) and solvent were injected separately into lingual vein at 30 min after ischemia. The occluding suture was slowly taken away to cause reperfusion at 1 h after ischemia. Rats were decapitated under anesthesia at 24 h after ischemia-reperfusion and brains were immediately removed to do the following procedures. Effects of dipfluzine on morphology of the brain tissue were observed through hematoxylin-eosin (HE) staining. By immunohistochemistry and flow cytometry technique and biochemical method, effects of dipfluzine on P-selectin, E-selectin, ICAM-1 and myeloperoxidase (MPO) were observed.

RESULTSDipfluzine could relieve pathological damages in the brain tissue after ischemia-reperfusion, and reduce the expressions of E-selectin, P-selectin and ICAM-1 and activities of MPO in dose-dependent manner.

CONCLUSIONDipfluzine depresses the expressions of P-selectin, E-selectin, and ICAM-1, which are correlated with their effects on the activities of MPO, suggesting that dipfluzine has anti-inflammation effect in certain extent and could protect brain tissue from ischemia-reperfusion injury.

Animals ; Brain Ischemia ; etiology ; Cerebral Cortex ; metabolism ; pathology ; Cinnarizine ; administration & dosage ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; E-Selectin ; metabolism ; Infarction, Middle Cerebral Artery ; complications ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Neuroprotective Agents ; administration & dosage ; pharmacology ; P-Selectin ; metabolism ; Peroxidase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; etiology ; metabolism ; pathology

Objective T o explore the relationship betw een the change rules of volatile organic com pounds (V O C s) in rat m uscle and postm ortem interval (PM I). Methods A total of 120 healthy rats w ere divided random ly into 12 groups (10 for each group). A fter the rats w ere sacrificed by cervical dislocation, the bodies w ere kept at (25±1)℃. R at m uscle sam ples w ere separately obtained at 12 PM I points, including 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 d. T he V O C s in rat m uscles w ere collected, detected and ana-lyzed by headspace solid-phase m icroextraction (H S-SPM E ) coupled to gas chrom atography-m ass spec-trom eter (G C-M S ). Results In total, 15 species of V O C s w ere identified, including 9 arom atic com-pounds, 3 sulfur com pounds, 2 aliphatic acids and 1 heterocyclic com pound. T he species of V O C s in-creased w ith PM I: no species w ere detected w ithin 1 day, 3 species w ere detected on day 2, 9 on day 3, 11 on day 4, 14 from day 5 to 7, and 15 from day 8 to 10. T otal peak area of 15 species of V O C s w as significantly correlated to PM I (adjusted R2=0.15-0.96): the regression function w as y=-17.05 x2+164.36 x-246.36 (adjusted R2=0.96) from day 2 to 5, and y=2.24 x+101.13 (adjusted R2=0.97) from day 6 to 10. Conclusion T he change rules of V O C s in rat m uscle are helpful for PM I estim ation.

Objective To observe the different expression of somatomedin-receptor in cell membrane of prostate epithelial cells at anoxic or normoxic condition.Methods Human prostate epithelial cells line RWPE-1 were cultured in vitro.At 4,8,12,24,48 h after cells had been seeded,the gene and protein expression of epidermal growth factor receptor (EGFR),fibroblast growth factor receptor (FGFR),transforming growth factor β1 receptor (TGF- β 1R),insulin-like growth factor-1 receptor (IGF-1 R) and vascular endothelial growth factor receptor (VEGFR) in prostate epithelial cells were tested by RT-PCR and immunohistochem-istry methods,respectively.Results The expression of mRNA and protein of EGFR,FGFR,IGF-1R,TGF- β1R,VEGFR were significantly increased in anoxic and normoxic prostate epithelial cells (P ＜ 0.01 ).At different time point,the expression of mRNA and protein of EGFR,FGFR,IGF-1R,TGF- β1R,VEGFR significantly higher in anoxic than those in normoxic prostate epithelial cells (P＜ 0.01 )besides 4 h EGFR mRNA,12 h EGFR protein,4 h IGF-1R mRNA,4 and 8 h IGF-1R protein,4 and 8 h TGF-β 1R mRNA,4 and 8 h TGF-β 1R protein,4 h VEGFR mRNA (P ＞ 0.05).Conclusion Anoxic prostate epithelial cell can up-regulate the expression of somatomedin-receptor.

Objective To determine the risk factors for emergence agitation (EA) during the recovery period after general anesthesia.Methods One thousand and thirty-four patients of both sexes aged 18-89 yr undergoing general anesthesia were divided into EA group and non-EA group.EA occurring during recovery from general anesthesia was assessed by using Riker sedation-agitation scale.Age,sex,complication,education,medical history,ASA physical status,type and duration of anesthesia and operation,volume of blood loss,fluid replacement,urine volume,duration of stay in PACU,number of drainage tubes and so forth were recorded.Multivariate logistic regression was used to analyze the risk factors for the occurrence of EA.Results Thirty-six patients developed EA during recovery from anesthesia.The incidence of EA was 3.5 ％.Logistic regression indicated that high risk operation,premedication with diazepam,induction of anesthesia without midazolom and fluid replacement during operation were the risk factors for EA (P ＜ 0.05).Conclusion High-risk operation,premedication with diazepam,induction of anesthesia without midazolom and fluid replacement during operation are the risk factors for EA during recovery from general anesthesia.