BACKGROUND: Mesenchymal stem cells are few in human bone marrow, and their number will decrease with aging or body weakening, so a large amount of amplification is necessary. However, the biological characteristics of human mesenchymal stem cells of each passage remain poorly understood.OBJECTIVE: To analyze and compare the biological characteristics of each passage of bone marrow-derived mesenchymal stem cells (MSCs) so as to provide a basis for clinical demands of tissue engineering.DESIGN,TIME AND SETTING: Cytological observation in vitro. The experiment was performed at the Department of Orthopedics and Central Laboratory, Dongzhimen Hospital, Beijing University of Chinese Medicine from March to October 2008.MATERIALS: From bone marrow of patients with non-hematopoietic disease, MSCs were provided by Department of Orthopedics, Dongzhimen Hospital, Beijing University of Chinese Medicine.METHODS: Bone marrow was collected form posterior superior iliac spine of patient, MSCs were isolated and cultured by Percoll method. When the cells were confluent at 90%, they were trypsinized and observed by inverted miscroscopy. The second passage of cells were collected for index detection.MAIN OUTCOME MEASURES: Cell morphological characteristics and immunophenotype; cell activity was detected by MTT; cell division and apoptosis in the proportion of necrosis were analyzed by flow cytometry analysis.RESULTS: The passaged MSCs exhibited uniform appearance in fusiform shape, and their growth was slowed down after 9 passages, exhibiting cytoplasm vacuolization and body enlarging. The second passage of MSCs was positive for CD44, CD106,and CD105, but negative for CD34 and CD45. MTT values peaked at passage 9, and gradually decreased since passage 10. At passage 11, the number of MSCs at division stage was increased, but from the sixth passage, the number of apoptotic cells increased significantly, reaching more than 60% at passage 8.CONCLUSION: According to biological characteristics analysis of MSCs at each passage, the third to the sixth passage cells are recommend for clinical therapy.

ObjectiveTo investigate the extracorporeal clearance rate of imipenem in severe infection patients in the mode of continuous vena-venous hemofiltration (CVVH) during continuous renal replacement therapy (CRRT), in order to approach if the concentration of imipenem in plasma could achieve effective levels of anti-infection, and to explore the effect of time and anticoagulation measure on imipenem clearance during CRRT treatment.Methods A prospective observational study was conducted. All adult severe infection patients complicating acute kidney injury (AKI) in the Department of Critical Care Medicine of the Fourth Hospital of Hebei Medical University from March 2013 to September 2014, who were prescribed imipenem as part of their required medical care, and CRRT for treatment of AKI were enrolled. 0.5 g doses of imipenem was administered intravenously every 6 hours or 8 hours according to random number table, and infused over 0.5 hour. The unfractionated heparin was used for anticoagulation in the patients without contraindications, and no anticoagulation strategy was used in the patients with high risk of bleeding. At 24 hours after first time of administration, postfilter venous blood and ultrafiltrate samples were collected at 0, 0.25, 0.5, 0.75, 1, 2, 5, 6, and 8 hours after imipenem administration. The concentration of imipenem in above samples was determined with liquid chromatography-mass spectrometer/mass spectrometer (LC-MS/MS).Results A total of 25 patients were enrolled. Thirteen patients received imipenem intravenously every 6 hours, and 12 patients, every 8 hours. The anticoagulation was conducted with heparin in 13 cases, and 12 cases without anticoagulation. The intra-day precision, inter-day precision, matrix effect, and recovery rate in low, medium, and high concentration of plasma and ultrafiltrate, and the stability of samples under different conditions showed a good result, the error of accuracy was controlled in the range of±15%. With the application of Prismaflex blood filtration system and AN69-M100 filter, under the mode with CVVH, the total clearance rate of imipenem was (8.874±2.828) L/h when the actual dose of replacement fluid was (31.63±1.48) mL·kg-1·h-1, the total CRRT clearance rate of imipenem in vitro was (2.211±0.539) L/h, which accounting for (30.1±15.7)% of the total drug clearance. In 6 hours interval dosage regimen, the percentages of the time> 4×minimum inhibitory concentration (MIC) at specific 4×MIC of 2, 4, 6, and 8μg/mL of imipenem were more than 40% of the dosing interval. But in the 8 hours interval dosage regimen, when the level was above the 4×MIC of 4μg/mL, maintaining time would drop below 40% of the dosing interval, with significant differences compared with that in 6 hours interval dosage regimen [4×MIC = 2μg/mL: (60.84±20.25)%vs. (94.01±12.46)%,t = 4.977,P = 0.001; 4×MIC = 4μg/mL: (39.85±15.88)% vs. (68.74±9.57)%,t = 5.562, P = 0.000; 4×MIC = 6μg/mL: (27.58±13.70)% vs. (53.97±8.36)%,t = 5.867,P = 0.000; 4×MIC = 8μg/mL:(18.87±12.43)% vs. (43.48±7.83)%,t = 5.976,P = 0.000]. No significant change in sieving coefficient of imipenem was found within a short time (6 hours), which indicated that there was no effect of anticoagulation on clearance of imipenem by AN69-M100 filter, and no statistical significance was found with repeated measure analysis (F = 0.186, P> 0.05).ConclusionsThe clearance rate of imipenem is increased significantly in vitro under the mode of CVVH with the actual dose of replacement fluid was (31.63±1.48) mL·kg-1·h-1 in severe infective patients with severe sepsis complicating AKI, affecting the level of plasma drug concentration, need to adjust the dosage regimen. When the time of the dosing interval was shortened, the concentration of imipenem in patients' plasma could be increased significantly. In a short period of time, the sieving coefficient of imipenem through AN69 filter is not affected by anticoagulation measures and time cleaning efficiency will not decline.

Background and purpose: High-dose chemotherapy followed by autologous stem cell transplantation (auto-HSCT) is considered as the ifrst line treatment for patients with relapse/refractory lymphoma after conventional chemotherapy. However, most of these patients still relapse the second time. The purpose of this study was to analyze the efifcacy of the consolidation chemotherapy after autologous stem cell transplantation (HSCT) refractory/relapse lymphoma in high risk. Methods:A total of 38 patients with relapsed/refractory lymphoma including Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) were included, who were underwent auto-HSCT in our transplan-tation department from Jan. 2010 to Dec. 2013. In treatment group, 19 patients received 2 courses of consolidation che-motherapy after auto-HSCT every 2 to 3 months, with the regimen of mini-BEAM or modiifed mini-CBV. Another 19 patients had no chemotherapy after auto-HSCT as control group. Results:The median follow-up duration was 17.2 and 7.5 months in the treatment and control group respectively. The follow-up data demonstrated prolonged progression-free survival (PFS) in the treatment group than the control group [24.7 months vs 7.8 months, P=0.029 under intend-to-treat analysis ITT;24.7 months vs 5.2 months, P=0.01 under per protocol analysis(pp)]. There is also a trend of improved overall survival (OS) in the treatment group (P=0.055, ITT). Conclusion:Consolidation chemotherapy after auto-HSCT for refractory/relapsed lymphoma patients delay the relapse and tend to improve the overall relapse rate.

Objective To study the clinical features of children with rotavirus enteritis complicated with respiratory infection.Methods The clinical features of 32 children with rotavirus enteritis were evaluated retrospectively complicated with respiratory infection (respiratory infection group) and 37 children with rotavirus enteritis complicated without parenteral infection (control group).Results 1. The respiratory symptoms became alleviative as the alimentary symptoms changed for better. 2. Duration of diarrhea weve(7.06?1.50)d in respiratory group was significantly longer than that in control group (4.73?1.31)d (t=6.90 P

Four salts of ticagrelor, ticagrelor-3,5-dinitrobenzoic acid, ticagrelor-pyrazinamide, ticagrelor-D-proline and ticagrelor-L-proline were prepared by solvent suspension and liquid-assisted grinding to improve the solubility of ticagrelor. The compounds were characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, differential scanning calorimetry, nuclear magnetic resonance spectroscopy, elemental analysis, and the intermolecular salt-bonding forces were analyzed. The equilibrium solubility of salts and pure drug in hydrochloride buffer pH 1.2 and phosphate buffer pH 6.8 were measured by high-performance liquid chromatography. Ticagrelor was salted with 3,5-dinitrobenzoic acid, pyrazinamide, D-proline, L-proline all in a stoichiometric ratio of 1∶1; with the exception of ticagrelor-D-proline, the solubility of the other three salts provided significantly improved solubility in hydrochloride buffer pH 1.2, and the equilibrium solubility of ticagrelor-3,5-dinitrobenzoic acid was increased by approximately 1.7 folds as compared to pure drug. Salt-forming technology is convenient and can improve the solubility of ticagrelor.

Objective To study the correlation between the coagulation factor Ⅶ (F Ⅶ) and progressive hemorrhage after brain contusion in mice and provide the experimental evidence for the clinical application of recombinant human FⅦa.Methods Twelve male BALB/c mice were given liposomeencapsulated FⅦsiRNA via tail vein at doses of 1,3,5 and 10 mg/kg with 3 mice per dosage.The other 3 mice received equivalent volume of normal saline as controls.Two days after the injection,mice blood sampling was used to detect FⅦ mRNA expression in liver using real-time PCR,level of plasma FⅦ using ELISA method,and activity of plasma FⅦ using chromogenic substrate assay.The optimal dose at which F Ⅶ expression was inhibited was determined.Thirty BALB/c male mice were assigned to two groups (n =15 per group) according to the random number table:FⅦ-suppressing group,mice were injected with FⅦsiRNA at the optimal dose and control group,mice were injected with same volume of negative control vector.The model of brain contusion was established in both groups.Volume of hemorrhage following brain contusion was measured at 3,24 and 72 h postinjury,and hematoma volume at 24 and 48 h postinjury.Results Liposome-encapsulated siRNA delivery down-regulated FⅦ expression in the mouse liver.Level and activity of plasma FⅦ were also reduced significantly.The optimal siRNA dose was 3 mg/kg.At 3,24 and 72 h postinjury,relative volume of brain hemorrhage in FⅦ-suppressing group was 1.46 ± 0.10,1.82 ± 0.23 and 2.28 ± 0.15 respectively,significantly higher than that in control group (1.00 ± 0.25,1.20 ± 0.31 and 1.20 ± 0.22 respectively) (P ＜ 0.05).At 24 and 48 h postinju-ry,volume of hematoma in FⅦ-suppressing group was (6.7 ± 1.5)mm3 and (9.8 ± 1.0) mm3,significantly higher than that in control group [(5.2 ± 1.2) mm3 and (5.5 ± 1.5) mm3] (P ＜0.01).Conclusions Level of FⅦ in vivo relates closely to the progressive hemorrhage of brain contusion in mice.Administration of FⅦ is effective to reduce the incidence of progressive hemorrhage.

OBJECTIVETo investigate the migration of fluorescent dye PKH26-labeled BM-MSC in the Alzheimer's model rats.

METHODSNormal human bone marrow extracted for isolation of BM-MSC was cultured in vitro. The 5th passaged BM-MSC was labeled with PKH26, and observed under a fluorescence microscope for PKH26 labeling efficiency, and using flow cytometry BM-MSC surface markers was checked. The PKH26 labeled BM-MSC injected into the tail vein of the normal control group and AD animal model group, 14 days after finding the PKH26-labeled BM-MSC cells in the rat hippocampus using fluorescence microscopy. Using the Morris water maze experiment comparison of AD model and BM-MSC transplantation group of spatial learning and memory ability.

RESULTSTFlow cytometry showed BM-MSC surface markers CD73 and CD105 were positive. In vitro, PKH26-labeled rate of BM-MSC was 100 %. The Morris water maze experiment comparison of BM-MSC transplantation group and AD group of animals, BM-MSC transplantation group at 13, 14 days of spatial learning and memory ability than AD animal group had significantly improved. 14 days after BM-MSCs in rat hippocampus could be found which were PKH26-positive, consistent with DAPI staining. PKH26-positive cells in animal models of AD were significantly more than those in the normal control group.

CONCLUSIONBM-MSC in AD rats not only migrates through the blood-brain barrier, but also mainly survives in the hippocampus of AD rats, and it can improve AD rat model of learning disabilities.

Alzheimer Disease ; pathology ; Animals ; Bone Marrow Cells ; cytology ; Cell Movement ; Cells, Cultured ; Disease Models, Animal ; Humans ; Injections, Intravenous ; Male ; Mesenchymal Stromal Cells ; cytology ; Organic Chemicals ; Rats ; Rats, Sprague-Dawley

AIM: Cystostomy is the traditionary method for detecting urodynamic indexes in mice, which de-stroys the continuity of the bladder, and there are significant differences between this method and the clinically used trans-urethral method.This study aims to develop an appropriate urethral catheter to investigate the advantages and application val-ue of transurethral method for urodynamic test.METHODS:A pediatric intravenous catheter was used for urethral catheter-ization on 8 female mice, and linked to connect the catheter to baroreceptor and micropump.The epidural catheter was also used as manometry tube.RESULTS:Using this method, the following urodynamic indicators has been successfully cap-tured:basal bladder pressure (BBP), bladder leak point pressure (BLPP), maximum voiding pressure (MVP), maxi-mum bladder capacity ( MBC ) , post-void residual urine volume ( PVR ) , voiding volume ( VV ) , efficiency of voiding ( EV) and bladder compliance ( BC) .CONCLUSION:This is the first successful simulation used in human body to a-chieve mouse urodynamic testing through the urethra catheter, which avoids the impact of cystostomy on urodynamics in mice, and the mice are able to keep long-term survival after tests for the follow-up molecular and genetic experiments.

Adult ; Fracture Fixation, Internal ; Fractures, Ununited ; surgery ; Humans ; Male ; Scaphoid Bone ; injuries ; surgery

Objective To investigate the feasibility of Ad-hTGF-β1 transfected bone marrow mesenchymal stem cell(BMSCs) into chondrocytes differentiation and the change of TAZ mRNA. Methods Rats BMSCs were obtained and cultured by whole bone marrow method, and then the third-generation cells were seeded into cell culture plate, and divided into three groups:Ad-hTGF-β1 transfected group,Ad-EGFP transfected group and the control group. The control group was added in common medium without any treatment while the other two groups were respectively added in serum-free medium containing Ad-hTGF-β1 or that containing Ad-EGFP. Seven days later, real-time fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1 ,while immunohistochemical and Western blot for the expression of collagen Ⅱ , and real-time fluorescent quantitation PCR for the expression of TAZ mRNA. Results Seven days after the transfection, real-time fluorescent quantitation PCR revealed that the average relative expression of TGF-β1 was:Ad-hTGF-β1 group 0.863, Ad-EGFP group 0.183, and the control group 0.180; The average relative expression of TAZ was:Ad-hTGF-β1 group 0.810, Ad-EGFP group 0.416, and the control group 0.366.The expression difference of TGF-β1 and TAZ were statistically significant (P ＜ 0.05). Western blot and immunohistochemical proved strong collagen Ⅱ expression in Ad-hTGF-β1 group while it was detected a little in the other two groups. Conclusion BMSCs could be successfully and stably induced into chondrocytes differentiation by Ad-hTGF-β1. Meanwhile, the mRNA of TAZ is up regulate during the differentiation,so it is suppose that TGF-β1 improve BMSCs into chondrocytes differentiation by TAZ.