BACKGROUND:In vitro isolation and purity technique of stem cells mostly depends on the identification of cell surface marker,such as monoclonal antibody adherent spreading method,flow cell sorting method and immunomagnetic beads sorting method,but the operation was complicated and the price was high.OBJECTIVE:To observe the biological characteristics of human amniotic fluid-derived embryonic mesenchymal stem cells,which were isolated and cultured using the two-step method.DESIGN,TIME AND SETTING:The opening study was conducted at the Stem Cell Research Room of Xinjiang Medical University from March 2008 to March 2009.MATERIALS:Totally 10 amniotic fluid specimens were obtained from pregnant women who underwent prenatal diagnosis following 16-22 weeks of gestation or voluntarily induced abortion.With ultrasonic guidance,amniocentesis was performed to collect 20-40 mL amniotic fluid.METHODS:Human amniotic fluid-derived embryonic mesenchymal stem cells were isolated and cultured using the two-step method.Amniotic fluid was first centrifuged and incubated till spindle-shape cells were seen,with the presence of flbroblast-tike cell colonies.Supematant was moved to a new 25 cm~2 culture flask for further culture till spindle-shape fibroblast-like mesenchymal stem cell colonies.When 70% confluence,cells were digested,and incubated in α-MEM,supplemented with basic fibroblast growth factor,served as the first passage.MAIN OUTCOME MEASURES:Morphological changes in human amniotic fluid-derived embryonic mesenchymal stem cells of primary culture and subculture were measured.Karyotype,cycle,growth curve and colony formation ability of human amniotic fluid-derived embryonic mesenchymal stem cells were measured.Surface antigen and cytokine were examined using flow cytometry,immunofluorescence and RT-PCR.RESULTS:Human amniotic fluid-derived embryonic mesenchymal stem cells were successfully isolated and subcultured.During metaphase,primarily cultured amniotic fluid cells presented scattered spindle cells and flbroblast-like mesenchymal stem cell colonies every 7 days.Passaged cells completely adhered in 12 hours.Following 1 or 2 days of latent period,cells proliferated rapidly.About 90% confluence was observed following 6 or 7 days of culture.Cell arranged regularly,showing whirlpool-shape,radiated shape.Cells were spindle-shape,with unclear boundary.Chromosome karyotype of human amniotic fluid-derived embryonic mesenchymal stem cells was normal diploid.Growth curve showed "S" shape,but the two-step method reached a peak at (6.1±0.5) days,which was significantly rapid compared with the one-step method (7.2±0.6) days (P=0.035).Flow cytometry analyses showed that P3 cells at S phase took up (14±2.3)% using the two-step method,which was more than the one-step method (9.0±1.4)% (P=0.031).Low-density human amniotic fluid-derived embryonic mesenchymal stem cells were incubated for 7 days prior to cells formed scattered cell colonies.However,colony forming efficiency using the two-step method (15.0±2.3)% were significantly more than the one-step method (10.0±1.8)% (P=0.021).Flow cytometry results showed that human amniotic fluid-derived embryonic mesenchymal stem cells expressed CD44,CD29 and CD105,but were negatively for CD45,CD34,HLA-DR.Immunofluorescence suggested that Oct-4-positive cells were observed in amniotic fluid.However,the proportion of Oct-4-positive cells using two-step method (1.2±0.3)% was significantly greater than the one-step method (0.9±0.2)% (P=0.041).RT-PCR suggested that human amniotic fluid-derived embryonic mesenchymal stem cells obtained using the two methods expressed Oct-4.CONCLUSION:Human multipotent mesenchymal stem cells are present in human amniotic fluid.The two-step culture protocol could be a kind of high performance and simple protocol which may not interfere with the normal prenatal diagnosis procedure.

Objective: To analyze the association between hepatitis B virus (HBV) infection and pancreatic cancer. Methods: Retrospective analysis was performed to explore the positive rate of serum hepatitis B virus surface antigen (HBsAg) in patients with pancreatic cancer, lung cancer, diabetes mellitus and general population. Z test was used to compare the rate of HBV infection between the samples and general population. The rates among the samples were compared by Chi-square test. Results: A total of 3,701 registered patients seen in our hospital between January 1st 2003 and March 31st 2009 were collected. There were 230 pancreatic cancer patients with a positive rate of serum HBsAg of 16.1%, 1,188 lung cancer patients with a positive rate of serum HBsAg of 10.7%, and 2,283 patients with diabetes mellitus with a positive rate of serum HBsAg of 11.6%. There was no statistical significance in Z-test results between lung cancer patients and general population (Z=1.104, P=0.163), but the Z-test results between patients with diabetes mellitus and general population showed a statistical significance (Z=2.98, P=0.002). The positive rate of HBsAg was higher in pancreatic cancer patients than that in lung cancer patients (OR=1.60, 95% Cl: 1.077-2.382, r=5.487, P=0.019). Similar results were found between pancreatic cancer patients and diabetic patients (OR=1.46, 95% CI: 1.004-2.123, r=3.965, P=0.046). Conclusion: The positive rate of HBsAg is high in pancreatic cancer patients. There might be an association between HBV infection and pancreatic cancer.

.036;r=-0.968,P=0.032).Conclusions Epo might decrease the chemo-sensitivity of SW1990 to gemcitabine possibly by up-regulating the expressions of MDR-1 and RRM1.

Objective To investigate the nerve recanalization and the motor function of hind legs after transplantation of peripheral nerve grafts treated with microsurgical technique at chronic spinal cord injury (SCI) in rats.Methods The SD rats were established into SCI model with improved Allen method.The rats were divided into two parts 12 weeks after the injury.In experimental group:by microsurgieal technique. the sural nerves were removed epineufium and transplanted into SCI lesion,control group rats were treated without any operation.Retrograde HRP tracing through sciatic nerve were practiced at 1 month,2 month,3 month after transplantation of peripheral nerve grafts.The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rat were detected.Results The morphology of the injured spinal cord sections turned better.Retrograde HRP tracing through sciatic nerve showed some HRP positive markers at the site of near rostral end of the nearly injured part at one month after transplantation and increased with the time going by.Motor function of hind legs of rats recovered significantly in transplantation groups.Conclusion Peripheral nerve grafts treated with mierosurgical technique have repairing effect on chronic spinal cord injury in rats.

Objective To explore the risk factors in elderly type 2 diabetes mellitus with cerebral infarction. Methods Retrospective investigarion was performed on 148 elderly hospitalized patients with type 2 diabetes.The patients were classified based on the presence or absence of cerebral infarction and compared with 60 controls.Logis- tic regression analysis was used to reveal the risk factors for cerebral infarction.Results The levels of systolic blood pressure(SBP),body mass index (BMI),fasting blood glucose (FBG),total cholesterol (TC),triglyceride (TG) and plasma fibrinogen(Fg) were higher in the patients with cerebral infarction[141.15?17.46)mmHg,(23.81?3.53)kg/m~2,(8.82?2.81)mmol/L,(5.69?1.15)mmol/L,(2.08?0.75)mmol/L and (4.08?0.65)g/L] than those without cerebral infarction[(129.78?14.65) mmHg,(22.18?3.22)kg/m~2,(7.06?1.72 )mmol/L,(5.09?1.12)mmol/L,(1.62?0.43)mmol/L and (3.48?0.58)g/L].The logistic analysis showed COUR,SBP,FBG, TC,TG and Fg were the independent risk factors for cerebral infarction.Conclusion Early intervention of the inde- pendent risk factors including SBP,FBG,TC,TG and Fg in elderly patients with type 2 diabetes was important for reduction and postponement of cerebral infarction.

Objective To study the relationship between the levels of uric acid in type 2 diabetic patients aged over forty years and lower extremity arterial disease(LEAD).Methods Lower extremities of 212 patients with type 2 diabetes were detected by high resolution color Doppler ultrasonography,and the levels of uric acid,fasting blood glucose and serum lipid were examined,which were applied to anylaze the relationship with diversity process of lower extremity arterial disease.Results The uric acid,low-density lipoproteins cholesterol(LDL-C),body mass in- dex(BMI),age and course in lower extremity arterial disease group was significantly higher than those without LEAD group(P

OBJECTIVETo determine bergapten's concentration in plasma and observe its pharmacokinetics in rats using a combined LC-MS/MS analytical method.

METHODBlood samples were separated on a Hypersil ODS column (4.6 mm x 250 mm, 5 mm) at a temperature of 30 degrees C, and mobile phase consisted of water and methanol (22.5: 77.5) at a flow rate of 0.8 mL x min(-1).

RESULTThe methodological study showed a good linear relationship of 8.12-162.4 g x L(-1) (r = 0.9999). The inner and inter-days relative standard deviations were both less than 10% , indicating legitimate precise and accuracy to the requirement of biological sample analysis.

CONCLUSIONThe method is suitable for in vivo quantitative analysis for bergapten due to its accuracy, sensitivity and specificity. The pharmacokinetic process in rats forms a two-compartment model with first-order absorption.

Animals ; Chromatography, Liquid ; Male ; Methoxsalen ; analogs & derivatives ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Reference Values ; Reproducibility of Results ; Sensitivity and Specificity ; Tandem Mass Spectrometry ; Time Factors

OBJECTIVETo explore the pathological mechanism in the repair of chronic spinal cord injury with free grafting of autoperipheral nerve tissues in rats.

METHODSThe SD rats were used to establish SCI model with modified Allen method. The rats were divided into two groups at 12 weeks after the injury, each group had 20 rats. In the experimental group, the sural nerves were removed epineurium and transplanted into SCI lesion by using microsurgical technique; and in the control group, the rats were treated without any operation. The survival and differentiation of the grafts, and the ability of repairing host spinal cord were observed under the light microscope at the postoperative 4th and 12th week. Regeneration rates of nerve tracts in spinal cord were evaluated by using HRP tracing technique at the postoperative 4th and 12th week. The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rats were detected.

RESULTSIn the control group, spinal cord exhibited degeneration with cicatrices and cavitates. In the experimental group, peripheral nerve was almost survived, fused with the spinal tissue and axons could regrow into or span the place of injured spinal cord. Higher number of labeled nerve tracts in spinal cord were observed in experimental group, there was significant difference when compared with the control group. Motor function of hind legs of rats recovered significantly in the treatment group.

CONCLUSIONAutoperipheral nerve graft tissues transplantation could survive and integrate with the host and have repairing effects on chronic spinal cord injury in rats.

Animals ; Female ; Male ; Peripheral Nerves ; transplantation ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; physiopathology ; surgery ; Transplantation, Autologous

OBJECTIVETo develop a LC-MS method for the determination of senkyunolide I (SI) in rat plasma, in order to observe whether there is significant change in the pharmacokinetics parameters of complex prescriptions of Huoluoxiaolingdan (HLXL) and single herbal extracts from Ligusticum chuanxiong Hort. in rats, and assess the effect of other components in HLXL on the pharmacokinetics of SI.

METHODTwelve male Sprague-Dawley (SD) rats were randomly divided into two groups, and orally administered with extract from HLXL and L. chuanxiong (both equal to SI 4.53 mg x kg(-1)). Their blood was collected at different time points for LC-MS, in order to detect the plasma concentration of SI. The pharmacokinetic parameters of SI were calculated by DAS 2.0 software. SPSS 16.0 software was used for independent-sample T-test and Nonparametric T-test.

RESULTA linear relationship of SI ranged from 6.750 to 675.0 microg x L(-1), and with the lowest limit of detection being 6.750 microg L(-1). Both of the plasma concentration-time curves of SI were fitted with the two-compartment model for extract of HLXL and L. chuanxiong. The detected AUC and Cmax of SI showed significant difference, with no significant difference in other parameters.

CONCLUSIONThe LC-MS determination method established in this experiment was so exclusive, accurate and sensitive that it is suitable for pharmacokinetic studies on extracts of HLXL and SI from L. chuanxion. The experiment results show that other ingredients of HLXL have noticeable effect on the absorption of SI in rat plasma.

Administration, Oral ; Animals ; Area Under Curve ; Benzofurans ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Male ; Metabolic Clearance Rate ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective: To investigate the effect and mechanism of hydroxyfasudi (HF), a specific Rho kinase inhibitor, on lipopolysaccharide(LPS)induced endothelial dysfunction. Methods: A total of 24 male Sprague Dawley rats were randomly divided into control group(n=6), HF group(n=6), LPS group(n=6) and LPS + HF group(n=6) with random number table method. There was no special treatment in control group. HF (30 mg/kg) was injected intraperitoneally in HF group. LPS (1 mg/kg) were injected intravenously in LPS group. In LPS+ HF group, HF (30 mg/kg) was injected intraperitoneally, followed by intravenous LPS injection (1 mg/kg) 30 minutes later. All rats were sacrificed after 8 hours, and aortic tissue was extracted. RT-PCR was performed to detect mRNA levels of Rho-associated coiled-coil protein kinase (ROCK)1, connexin (Cx)43 and caveolin (Cav)1. The protein levers of ROCK1, Cx43 and Cav-1 were assessed by Western blot and immunohistochemical staining respectively. Results: (1) RT-PCR experiments showed that mRNA levels of ROCK1(2.67±0.03 vs. 1.00±0.04), Cx43(1.73±0.03 vs. 1.00±0.08), and Cav1(1.85±0.04 vs. 1.0±0.03) in LPS group were significantly higher than in control group(all P<0.05). mRNA levels of ROCK1(0.38±0.02), Cx43(0.58±0.02), and Cav1(0.27±0.01) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (2)Western blot analysis showed that protein levels of ROCK1(3.46±0.82 vs. 2.19±0.56), Cx43(0.33±0.09 vs.0.11±0.06), and Cav1(3.45±0.74 vs. 2.25±0.91) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(1.09±0.52), Cx43(0.01±0.06), and Cav1(2.06±0.40) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (3) Immunohistochemical staining showed that protein levels of ROCK1(84.1±0.9比53.7±2.9), Cx43(99.1±2.1 vs. 46.2±0.8), and Cav1(167.0±6.4 vs. 84.9±1.0) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(30.4±0.6), Cx43(21.4±1.3), and Cav1(55.8±2.8) in LPS + HF group were significantly lower than in LPS group(all P<0.05). Conclusion HF attenuates LPS induced endothelial dysfunction probably via suppressing the expression of ROCK1, Cx43 and Cav1.