BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are in advantages of easy collection and amplification in vitro, but the culture and induction in vitro still need to be modified. OBJECTIVE: To investigate the differentiated potency of BMSCs into ostcoblasts by modified in vitro culture methods and inductor matching. DESIGN: Observational study. SETTING: Department of Medical Laboratory, General Hospital of Guangzhou Military Area Command of Chinese PLA. MATERIALS: This study was performed in the Stem Cell Tissue Engineering Laboratory, Department of Medical Laboratory, Genera Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. Twenty adult SD rats of clean grade, irrespective of gender, weighing 140-180 g were provided by Animal Experimental Center, General Hospital of Guangzhou Military Area Command of Chinese PLA. The experimental animals were disposed according to ethical criteria. Β-sodium glycerophosphate, dexamethasone, and vitamin C were provided by Sigma Company, USA; goat-anti-rat osteocalcin antibody by DSL Company, USA; S-P immunohistochemical kit by Maixin Biotechnology Developing Co., Ltd., Fujian. METHODS: Cells were cultured and induced into osteoblasts by modified methods. Separation and culture of BMSCs: By anesthesia, bone marrow of bilateral femur and tibia was separated to prepare single cell suspension; subsequently, the suspension was inoculated in culture bottle, and the culture media was semi-quantitatively changed after 48 and 96 hours in order to get rid of non-adherent hematopoietic cells. The liquid was changed every three days to further get rid of non-adherent cells. At 80% cell confluence, the medium was digested with 0.25% trypsin and cells were subcultured in the ratio of 1:2. MSCs in the second generation were inoculated in 6-well culture plate and glass fiat plate; after 48 hours, basic culture fluid was removed. Differentiation of BMSCs into osteoblasts: Subcultured BMSCs differentiated into osteoblasts induced by dexamethasone (10 mmol/L), β-sodium glycerophosphate (10-7 mol/L), and vitamin C (50 mg/L). MAIN OUTCOME MEASURES: Ten days after differentiation by modified culture methods and inductor matching, alkaline phosphatase activity was measured with Ca-Co technique. Twelve days after culture, osteocalcin secretion was detected with immunohistochemical method. Two weeks after culture, cell mineralization was detected with Von kossa staining. RESULTS: Alkaline phosphatase activity: Alkaline phosphatase staining of cells was apparent; gray-black particles or massive precipitations were observed in cytoplasm after positive reaction; regions expressing alkaline phosphatase activity were brown-black. Osteocalcin secretion: Osteocalcin was apparently positive; nucleus was blue; cytoplasm was brown. Cell mineralization: Induced cells grew layer by layer, and adiaphanous nodus was observed at the same time. Black mineralized nodus granules in various sizes were observed after Von kossa staining, and this suggested that mineralized apposition occurred. CONCLUSION: BMSCs may be successfully cultured and induced into osteoblasts by modified culture method.

BACKGROUND:With the potential of multi-directional differentiation and high proliferation, bone marrow mesenchymal stem cells (BMSCs) have wide application prospect in tissue engineering. OBJECTIVE:To investigate changes in cells and bioactivity in the differentiation from BMSCs into osteoblasts. DESIGN, TIME AND SETTING:A randomized control experiment was performed at the Laboratory of Stem Cells and Tissue Engineering, Department of Medical Experiment, General Hospital of Guangzhou Military Area Command of Chinese PLA from July 2004 to June 2006. MATERIALS:Twenty adult SD rats of both genders were used for bone marrow collection. METHODS:Rats were equally and randomly divided into an experimental group and a control group. BMSCs were isolated from adult rats by modified bone marrow culture method. BMSCs were inoculated in basic medium containing 10% fetal bovine serum, high glucose DMEM, 100 U/L penicillin, 100 U/L streptomycin and 2 mmol/L glutamine in the control group. BMSCs were inoculated in conditioned medium (basic medium, 10 mmol/L β-glycerophosphoric sodium, 10-7 mol/L dexamethasone, 50 mg/L vitamin C). Cell slide was prepared. MAIN OUTCOME MEASURES:Inverted microscope and transmission electron microscope were used to observe cell appearance. Gomori modified calcium-cobalt method was applied to do alkaline phosphatase staining. Immunocytochemistry was employed to measure osteocalcin expression. RESULTS:Forty-eight hours after inoculation, a mass of BMSCs adhered to a wall. Seventy-two hours later, BMSCs proliferated and well grew. Seven to nine days later, cells were grown to 80% confluency. Transmission electron microscope showed BMSCs with big cell nucleus and immature appearance. After in vitro osteoblast induction, BMSCs proliferated, aggregated, had node and mineralized. One week later, alkaline phosphatase activities were expressed in BMSCs. Two weeks later, osteocalcin expression was detected. CONCLUSION:After one week of in vitro osteogenic induction, BMSCs enter the process of osteoblast transformation, remain proliferative activities, and can be used as seed cells in bone tissue engineering

Objective To investigate the significance of acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) scoring system in evaluating elderly patients with hyperglycemic hyperosmolar state. Methods Elderly patients with hyperglycemic hyperosmolar state were enrolled in the study from January 2003 to May 2008. The patients were divided into two groups according to the outcome: death group and survival group. APACHE Ⅱ scoring system was used on the day of admission and the scores were compared in the two groups. The risk factors for death were evaluated by logistic regression analysis. Results Forty patients were registered including 10 cases in death group and 30 cases in survival group. The mortality rate was 25%. There were no differences in blood glucose, serum sodium, blood osmotic pressure, serum creatinine and hemoglobin between two groups before the treatment (t=-1.50～1.53, all P>0.05). The APACHE Ⅱ scores were significantly higher in death group than in survival group on the day of admission(30.1±5.4 vs. 22.9±3.9,Z=-4.08, P<0.01). Multiple logistic regression analysis showed that APACHE Ⅱ scores on the day of admission were the independent risk factor for death. Age, sex, blood glucose, effective blood osmotic pressure, serum creatinine and hemoglobin were not related to mortality rate. Conclusions APACHE Ⅱ scoring system is a helpful method for evaluating the severity and outcome of patients with hyperglycemic hyperosmolar state.

Objective To develop and validate a simple,rapid,sensitive,and reproducible HPLC method for simultaneous determination of baicalein and its metabolite baicalin in dog plasma and for the subsequent pharmacokinetic study after iv administration to dogs.Methods An accurate and reproducible HPLC-UV method was developed and validated for simultaneous determination of baicalein and baicalin in dog plasma,using luteolin as internal standard.The analytes were separated by an Agilent Zorbax SB-C<,18> column(250 mm×4.6 mm,5μm)and the column temperature was maintained at 40 ℃.The mobile phase was a binary mixture of acetonitrile and water(27:73),containing 0.05% phosphoric acid in water,with a flow rate of 1.0 mL/min.The UV detector was set al 276 nm.Results Linear relationships were validated over the range of 0.05-25 μg/mL for baicalein and 0.05-20 μg/mL for baicalin.The intra-and inter-day precision values for all samples were within 8.0%,using relative standard deviation.This method was successfully applied to the pharmacokinetic studies in dogs after iv administration of baicalein.Baicalein was converted to baicalin quickly.Cmax values were 21.13 μg/mL at 0.05 h for baicalein and 1.57 μg/mL at 0.5 h for baicalin,areas under the plasma concentration-time curve were 4.97 h.μg/mL for baicalein and 0.63 h.μtg/mL for baicalin,and the elimination half-life is 0.50 h for baicalein and 0.75 h for baicalin,respectively.Conclusion The method is able and sufficient to be used in drug metabolism and pharmacoklnetic studies of baicalein.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) can not only differentiate into multiple nonhematopoietic cell lineages, but seek out damaged tissues and repair them as well. Hence, they were largely studied for their potential clinical use. However, their biological characteristics have not been fully discovered. OBJECTIVE: To compare the biological characteristics of BMSCs of different species cultured in vitro, in order to provide basis for the clinical research of stem cell therapy.DESIGN: Randomized controlled observation was designed.SETTING: Medical Research Department of General Hospital of Guangzhou Military Area.MATERIALS: The experiment was performed in Medical Research Department of General Hospital of Guangzhou Military Area from June 2004 to July 2005. Thirty SD rats weighing (160±20) g, aged 35 to 40 days, 30 Kunming mice weighing (16.0±2.0) g, aged about 40 days, 8 New Zealand white rabbits weighing (2.0±0.2) kg, aged 80 to 90 days and 10 healthy volunteers (25-32 years old) were selected. All the animals were of clean grade, which were purchased from the Animal Center of Southern Medical University.METHODS: The BMSCs of mice and rats were prepared according to the protocol developed in the Caplan laboratory, while those of rabbits and human were isolated from bone marrow suspension obtained by iliac puncture.The morphology of BMSCs was observed by light microscope and transmission electron microscope. Cell growth curve was tested by MTT. Expression of Stro-1 was analyzed by immunofluorescence cytochemistry and flow cytometry. To evaluate the specific response of BMSCs to osteogenic supplements(10 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate,and 50 mg/L ascorbic acid), the activity of alkaline phosphatase (AKP) activity was tested by a commercial kit. Expression of osteocalcin was examined by immunocytochemistry and hydroxyapatite crystals were shown by von Kossa staining. Adipogenic differentiation was evaluated by estimating percent of cells containing Oil Red-O- stained oil droplets.MAIN OUTCOME MEASURES: ① Morphological observation and growth situation of BMSCs. ② Expression of Stro-1: BMSC marker. ③ Differentiation in osteogenic medium.RESULTS: ①The morphology of adherent BMSCs ofthose four species observed by optic microscopy was obviously different. When they became mature or aged, the mouse cells turned into flat shape, irregularly polygonal, fell to pieces and deposited on the flask-bottom, while the rat, rabbit and human cells would enlarge and become polygonal, vacuoles would appear in their cytoplasm, finally, the cells were detached from the flask-bottom, floating off like cotton wool. The cultures of different species also had some commonness, such as poly-layer growing manner, without contact inhibition and consisting of two groups. Cells of one group grew into colonies from single cells and expanded quickly, while cells of the other group were sporadic and did not proliferate. Electron microscopy revealed that all of the primary cells had microvilli and that they could be divided into two subpopulations according to their ultrastructures. Some cells were rich in organelles and most chromatin was euchromatin, while the other subpopulation cells had much fewer organelles and more heterochromatin. Growth curves of BMSCs of different species were almost the same. ② The positive rate of human adherent bone marrow-derived cells for Stro-1: BMSC marker was (91.4±8.3) %, and that of mouse adherent cells was (83.5±6.2) % .③Treated with osteogenic supplements, mouse BMSCs differentiated into adipose tissue, rabbit ones died, while rat and human ones differentiated into osteocytes. BMSCs also demonstrated spontaneous differentiation in vitro.CONCLUSION: Mouse, rat, rabbit and human BMSCs can be easily expanded in vitro, although the harvest of the current method is a mixture of mesenchymal cells with various maturities, most of which are poor-differen-tiated cells. BMSCs of those species are different in morphology and response to the same inductive supplements. Therefore, in order to establish a kind of stem cell therapy, it is necessary not only for evidence from animal experiments but for that from human experiments as well.

Background and objectives Right ventricular apical (RVA) pacing has been reported impairing left ventricular (LV)performance. Alternative pacing sites in right ventricle (RV) has been explored to obtain better cardiac function. Our study was designed to compare the hemodynamic effects of right ventricular septal (RVS) pacing with RVA pacing. Methods Ten elderly patients with chronic atrial fibrillation (AF) and long RR interval or slow ventricular response (VR) received VVI pacing. The hemodynamic difference between RVS and RVA pacing were examined by transthoracic echocardiography (TTE). Results Pacing leads were implanted successfully at the RVA and then RVS in all patients without complication. The left ventricular (LV) parameters,measured during RVA pacing including left ventricular ejection fraction (LVEF), FS, stroke volume (SV) and peak E wave velocity (EV) were decreased significantly compared to baseline data, while during RVS pacing, they were significantly better than those during RVA pacing. However, after 3-6 weeks there was no statistical significant difference between pre- and post- RVS pacing.Conclusions The LV hemodynamic parameters during RVA pacing were significantly worse than baseline data. The short term LV hemodynamic parameters of RVS pacing were significantly better than those of RVA pacing; RVS pacing could improve the hemodynamic effect through maintaining normal ventricular activation sequence and biventricular contraction synchrony in patients with chronic AF and slow ventricular response.

【Objective】 To study the effect of intravenous immunoglobulin(IVIG) on the detection of blood transfusion compatibility in patients. 【Methods】 56 patients, submitted to our Hospital from March 1, 2017 to December 31, 2020, were enrolled as the research objects. They had negative unexpected antibody screening, major crossmatch incompatibility with the same blood type donors, and had a history of IVIG infusion. ABO and RhD blood groups typing, unexpected antibodies screening, crossmatch, direct antiglobulin test, indirect antiglobulin test, and acid elution test were all conducted by microcolumn gel method. 【Results】 After IVIG infusion, the initially major crossmatch incompatibility with the same blood type donors turned into compatiblity with O-type donors. Among them, 2 patients had transient discrepancy in ABO forward and reverse blood typing due to the IVIG infusion. IgG anti-A were detected in the red blood cell elution of 37 A-type patients; IgG anti-B in 2 B-type patients; 3 cases of IgG anti-A+ anti-B and 14 cases of solo IgG anti-A in 17 AB-type patients. 3 batches of IVIG preparations were detected randomly, IgG anti-A titer was 32-64, and IgG anti-B titer was 8-16. 【Conclusion】 The discrepancy in ABO forward and reverse blood typing and major crossmatch incompatibility with the same blood type donors may occur after non-O type patients received IVIG, which contains IgG types of anti-A and anti-B. In this situation, it is recommended to prepare major crossmatched O-type washed red blood cells to ensure the safety and effectiveness of clinical blood transfusion.

【Objective】 To explore the influencing factors of perioperative red blood cell transfusion in patients underwent lung transplantation, so as to provide reference for perioperative blood management (PBM) of lung transplantation patients. 【Methods】 The clinical data of 173 lung transplant patients completed in China-Japan Friendship Hospital from March 2017 to June 2019 were retrospectively analyzed. The patients were divided into two groups according to perioperative red blood cell transfusion volume: large blood transfusion group (transfusion red blood cell volume ≥6 U, n=66) and non-large blood transfusion group (red blood cell transfusion volume <6 U, n=107). The basic information, preoperative laboratory test results, and surgical status of the two groups were statistically analyzed.The clinical data of the two groups were analyzed by univariate analysis. The factors of P<0.15 were included in the binary logistic regression analysis, and the independent influencing factors of perioperative massive blood transfusion in patients with lung transplantation were found. 【Results】 Univariate analysis of clinical data of the two groups of patients (large blood transfusion group vs. non-large blood transfusion group) showed that the differences of smoking history ratio [44(66.7%) vs 87(81.3%)], BMI(20.8±4.5 vs 22.5±4.0)(P<0.05), preoperative Hb [124(111, 138.8) vs 138(126, 149)], preoperative Hct [37.9(34.8, 42.5) vs 41.3(37.9, 44.6)], surgery duration(327.9±107.7 vs 238.4±77.0), intraoperative blood loss(1 108.6±1342.0 vs 341.8±270.8) and single lung transplantation [28(42.4%) vs 84(78.5%)] (P<0.01) were statistically significant. Logistic regression analysis showed that intraoperative blood loss (OR=1.001, P<0.05), surgery duration (OR=1.006, P<0.05), preoperative Hb (OR=0.973, P<0.01), lung transplantation type(single or double lung transplantation)( OR=0.247, P<0.05) and extracorporeal membrane oxygenation (ECMO) (OR=0.187, P<0.01) were independent factors influencing red blood cell transfusion during lung transplantation. 【Conclusion】 Intraoperative blood loss and surgery duration are risk factors for massive blood transfusion during the perioperative period. And the use of ECMO, preoperative Hb, single lung transplantation (compared to double lung transplantation) are protective factors for perioperative massive blood transfusion.