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 clinical and serological manifestations of the interstitial lung disease(ILD) resulting from the dermatomyositis(DM) or the polymyodsitis(PM),and the factors affecting the resulting of treatment for the purpose of providing necessary reference for clinical treatment and diagnosis.Methods The clinical manifestations and the findings of serological tests of 18 patients compared with only DM or PM.Results ILD was likely to strike 42.9 percent of patients with DM or PM,who were higher than those without ILD in the rate of positive Jo-1,LDH and Reynolds disease.Conclusion The patients with DM or PM,with their high probability to be stricken by ILD,need HR-CT scanning in order to be diagnosed as early as possible.

Objective To explore the relationship between TGF-?, P21ras, c-fos and the apoptosis of human embryonic pancreatic islet cells. Methods Semi-quantitative RT-PCR and immunohistochemistry (SABC method) were used to detect TGF-?, P21ras, c-fos expression at different growth periods of the primary human embryonic pancreatic islet cells and human fibroblast cells. Results P21ras expression was weak in every period of the pancreatic islet cells and significantly weaker than the fibroblast cells at exponential growth period and was stronger than in incubation period and retention period (P

Objective:To explore blood type distribution of newborns hemolytic disease ( HDN ) caused by maternal and neonatal blood type incompatibility and analyze the value of hemolysis three trials in the diagnosis of HDN.Methods:Hemolysis three trials of type O or Rh negative maternal cord blood samples and hyperbilirubinemia of the newborn blood samples from January 2014 to 2016 were detected by micro-column gel test cards.Then the results were statistically analyzed.Results:(1) There were 918 cases of maternal and neonatal blood type incompatibility in all 1350 cases.569 cases were detected HDN positive with the rate of 62%( 569/918).Among 569 cases,the positive rate of direct anti-globulin test,free antibody test and antibody released test were 27.9%(159/569),86.5%(492/569) and 100% respectively.There was statistical difference of the combination of direct anti-globulin test negative,free antibody test positive and antibody released test positive compared with other combinations ( P<0.05 ).( 2 ) There was statistical difference of HDN positive rate between ABO 73.8%(551/747) and Rh 10.5%(18/171)in 918 cases of blood type incom-patibility.(3)There was statistical difference between A positive rate of 80%(280/350) and B positive rate of 68.3%(271/397) in 747 cases of ABO incompatibility.(4)There was statistical difference among RhD positive rate of 17.7%(14/79),RhE positive rate of 6.8%(4/59) and RhC positive rate of 0(0/33).Conclusion: Antibody released test was the most sensitive test in hemolysis three trials to diagnose HDN.The probability of HDN positive caused by maternal and neonatal ABO blood type incompatibility was significantly higher than Rh.The probability of HDN positive with type A newborns was significantly higher than type B.The probability of HDN positive caused by RhD blood type incompatibility was significantly higher than RhE and RhC.

Objective To study the inhibitory effects of Probucol on the expressions of serum promatrix metalloproteinase-2(pro-MMP-2) and matrix metalloproteinase-2(MMP-2) in patients with coronary heart disease(CHD).Methods The levels of serum Pro-MMP-2 and MMP-2 were detected by zymograph and total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C)were measured by enzyme method in 30 CHD patients before and after treatment with Probucol.Results After treatment of Probucol,the expression levels of pro-MMP-2,MMP-2,TC and LDL-C in serum were decreased than those before treatment,respectively;there were obvious differences of various indexes mentioned above between before and after treatment(P0.05).Conclusion Probucol can inhibit the expressions of pro-MMP-2 and MMP-2 in serum as same as TC and LDL-C.Probucol,as one of TIMPs,may be applied to stabilize plaque..

OBJECTIVE:To establish the GC fingerpriont for Lingyang ganmao tablets.METHODS:The determination was performed on DB-5 ms capillary column.Flame ionization detector was adopted with temperature of 250 ℃ (temperature programming).The temperature of injector was 240 ℃.Carrier gas was nitrogen with flow rate of 1.0 mL/min;the sample size was 1 μL by split sampling with split ratio of 20 ∶ 1.Using peppermint ketone as reference,Similarity Evaluation Software for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 edition) was used for the similarity analysis of 10 batches of Lingyang ganmao tablets.RESULTS:There were 14 common peaks in the batches of Lingyang ganmao tablets,similarity degrees were higher than 0.98.It was proved that the GC profiles and control fingerprint of 10 batches of samples had good consistency.CONCLUSIONS:The established fingerprint can provide reference for the identification and quality evaluation of Lingyang ganmao tablets.

Aim To investigate the anticoagulant efficacy and mechanism of a semi-synthesized sodiumβ-1,4-glucan sulfate (Na-MCS). Methods Anticoagulant activity was evaluated by means of coagulation assays in comparison with heparin. The anticoagulant mechanism of Na-MCS was disclosed by inhibitory analysis of the activities of coagulation factors using chromogenic substrates. Results 0. 6concentration. The dosage of Na-MCS required to double APTT of normal human plasma was 0.7represented a potent anticoagulation activity in vitro, which matched the efficacy of heparin in a certain range of concentrations. Na-MCS exhibited anticoagulant activity due to inhibition of the coagulation factors Ⅱa and Xa by the mediation of anti-thrombin AT-Ⅲ.

Objective To investigate the effect of lateral ventricle transplantation of neurotrophic factor-transfected cells derived from Glia cell line on vascular dementia in rats and gene expression of Drebrin in hippocampal region.Methods By using gene clone technique,the GDNF gene was transfected into SH-SY5Y cell lines.104 adult male Sprague-Dawley rats weighing (200± 20) gram were divided into groups:transplanted group,injected group,control group,all of which accepted operation by permanent ligation of left common carotid artery and clipping right common carotid artery repeatedly to build up model of vascular dementia,and sham operation group which accepted no ligation or clipping.6 rats from each group were decapitated on the third day,seventh day and tenth day after transplanting treatment were for fluorescence detection.The rest 20 rats in each group were used to detect learning and memory functions by Morris water maze on the third day and decapitated on the fourth day after transplanting treatment.Then GDNF level in temporal lobe were detected by enzyme-linked immunosorbent assay (ELISA),while Drebrin mRNA and protein levels in hippocampal region were detected by real time-PCR and Westernblot respectively.Results There was strong fluorescent light detected around lateral ventricle of rats in transplanted group on the third day after transplantation,which faded on the seventh day and disappeared on the tenth day.The learning and memory functions of rats in transplanted group were improved significantly.The escape latency was shorter in transplanted group than in injected group and control group [(34.89±4.15) s vs.(43.86±6.95) s,(50.89±3.66) s,both P＜0.05],while shuttle times through the third quadrant were more often in transplanted group than in injected group and control group [(11.00±1.49) vs.(9.26 ±1.38),(8.04 ± 1.12),both P＜0.05].GDNF level and Drebrin mRNA and protein levels were higher in transplanted group than in injected group and control group [GDNF:(315.71±27.43) vs.(256.26±19.90),(141.95±21.33),Drebrin mRNA:(5.54±0.35) vs.(3.10±0.33),(1.32±0.23),Drebrinprotein:(0.55±0.05) vs.(0.43±0.06),(0.26±0.06),all P＜0.05].Conclusions GDNF-transfected cells could survive in the lateral cerebral ventricle of rats for about seven days.The method for treating vascular dementia through the technique of transplanting GDNF-transfected cells is certain feasible,which has a better therapeutic effect than GDNF-injection directly into lateral cerebral ventricle.The therapeutic effect of GDNF on vascular dementia may be related to its action of regulating neural plasticity.

Objective To investigate hippocampal neuronal damage and dynamic change of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR2 in status epilepticus, to find out whether GluR2/glyceral dehyde-3-phosphate dehydrogenase (GAPDH) interaction has any change.Methods Male Wistar rats (62 cases) were induced to status epilepticus by using LiC1-pilocarpine.The 62 rats were divided into 1 h (6 cases),6 h (12 cases), 24 h (12 cases),72 h (12 cases)and 7 d (20 cases)after status epilepticus.At the same time, the healthy control group (12 cases) was established.Morphologic changes of hippocampus and the amount of apoptotic cells in healthy control and SE model groups at different time points (6 h, 24 h, 72 h, 7 d) (6 cases each group) after status epilepticus were quantified by adopting Nissl staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining respectively.Expressions of GluR2 in healthy control and SE model groups at 1 h,6 h,24 h,72 h and 7 d (6 cases each group) after status epilepticus were detected by using Western blot.Co-precipitation and Western blot techniques were used to investigate whether the GluR2/GAPDH interaction in the hippocampus was increased.Results Compared with the healthy control group, the number of nerve cells in the hippocampal CA1 and CA3 regions was significantly reduced at all studied time points(F =30.866,24.043, all P ＜0.05).Apoptotic cells in hippocampal CA1 and CA3 regions were significantly increased at 24 h,72 h and 7 d after status epilepticus (F =84.762,52.574, all P ＜ 0.01).GluR2 at 1 h,6 h after status epilepticus was equal to that of the control group (P ＞ 0.05), but it was shown to be significantly down-regulated at other studied time points (F =76.506,P ＜ 0.01);when compared with the healthy control group,the GluR2/GAPDH interaction was significantly up-regulated in the hippocampus at 72 h after status epilepticus (t =7.029, P ＜ 0.05).Conclusions Status epilepticus can lead to neuronal damage in the hippocampus.Down-regulation of GluR2 and increase of the GluR2/GAPDH complex formation might be one of the mechanisms involved in hippocampal neuronal damage.