BACKGROUND: The study of isolation, purification, culture, cell labeling, inducing factors, effects of gene transfection on cytobiology, cell carrier construction, and time window for back transplantation of cell compound pertaining to marrow stromal cells (MSCs) is still in its infancy. OBJECTIVE: To search for an in vitro culture method that can be simply and effectively obtained. DESIGN, TIME AND SETTING: The present cytological in vitro experiment was performed at the Beijing Institute of Genome, Chinese Academy of Sciences between June 2006 and July 2007. MATERIALS: Eight specific pathogen-free New Zealand rabbits, aged 6 weeks, were provided by the Laboratory Animal Center, Institute of Genetics and Development, Chinese Academy of Sciences. METHODS: Under sterile condition, 1 mL rabbit bone marrow was taken and diluted with D-Hanks solution. Following centrifugation and subsequent supernatant removal, bone marrow was re-suspended using dulbecco's modified eagle's medium (DMEM) for single cell suspension. Next, single cell suspension was dropped onto the liquid surface of equal-volume lymphocyte separation medium (density: 1.077). Subsequent to centrifugation, cloudlike mononuclear cell layer was collected and re-suspended with DMEM containing 20% fetal bovine serum. The cells were inoculated at lxl0/cm2 and purified by adherent method. When 70%-80% of flask bottom was covered, cell digestion and passage was performed.MAIN OUTCOME MEASURES: Cell growth was observed with an operating microscope. Surviving cells were counted by Trypan blue viability test. Cell identification was performed by hematoxylin-eosin staining. Through the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, cell viability was detected to observe the cellular resuscitation of the cultured cells following cryopreservation. RESULTS: Twenty-four hours after inoculation, cells began to adhere to the wall, exhibiting short shuttle- or triangle-shaped appearance with different sizes of cellular processes. Three days later, adherent cells began to divide, and cell clusters could be found in some areas; One week later, most of cells exhibited scattered fibroblast-like growth; After passage, cells were evenly distributed with long shuttle-shaped appearance and arranged orderly. Following 3 passages, there wound be 5×106-1×107 adherent cells in 1 mL MSC suspension. Approximately 90% of MSCs survived and identified as mononuclear cells. Cells vigorously grew at days 1-6 after inoculation and reached a peak level at day 8, followed by a viability decline. After 56 days of resuscitation, frozen cells displayed a good and rapid growth. CONCLUSION: Highly purified MSCs can be acquired by gradient centrifugation of lymphocyte separation medium. Enough seeded cells can be obtained by in vitro culture and the cellular viability does not change after frozen preservation and resuscitation.

BACKGROUND: SOX-9 plays an important role in occurrence and development of cartilaginous tissues,enhances agglutination of mesenchymal cells,has structural domains of transcriptional activation,and can directly regulate the transcription,OBJECTIVE: To construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells (BMSCs) using SOX-9 gene,and to investigate the effects of SOX-9 gene on growth characteristics of BMSCs and product expression. DESIGN,TIME AND SETTING: The cell gene engineering in vitro experiment was performe,d at the Beijing Institute of Genome, Chinese Academy of Scienees from June 2006 to January 2007.MATERIALS: Eight SPF New Zealand rabbits aged 6 weeks were used for culture of BMSCs.METHODS: pDC316-SOX-9 plasmid was used for transfection of rabbit BMSCs by liposome mediated method,Cell transfection included a SOX-9 plasmid transfection group,a blank plasmid group and a blank control group (only treatment of liposome). MAIN OUTCOME MEASURES: Cell morphology; growth activity; The SOX-9 protein expression in rabbit BMSCs were detected by immunohistochemistry,hematoxylin-eosin staining,reserve transcriptase-polymcrase chain reaction (RT-PCR) and enzyme-labeled immunosorbent assay (ELISA). RESULTS: Some cell colonies were detected at 4 days after pDC316-SOX-9 plasmid transfection.Spindle-shaped cells were collected after clone amplification.Cells in the blank control group gradually died over time.Cell activities in the SOX-9 plasmid transfeetion group and the blank plasmid group significantly prolonged,reached a peak at 2 weeks of transfection,and then gradually decreased.At 6 days,BMSCs were yellow in the SOX-9 plasmid transfection group following immunohistochemistry,expressing SOX-9 protein.Hematoxyliu-eosin staining showed many dikaryocytes,rich cell proliferation,similar to chondroblasts.No SOX-9 protein expression and unproductive cell proliferation in the blank plasmid group.SOX-9 mRNA was detected in the SOX-9 plasmid transfection group,but SOX-9 mRNA was not detected in the blank plasmid group and blank control group.SOX-9 levels were significantly higher in the SOX-9 plasmid transfection group than in the blank plasmid group and blank control group at 24,48 and 72 hours,1,2 weeks (P＜ 0.01).CONCLUSION: Rabbit BMSCs were successfully transfected with pDC316-SOX-9 plasmid to enhance cell growth activity and to persistently stably secrete SOX-9 protein,resulting in the differentiation of BMSCs into cartilages.

BACKGROUND:There is lack of an objective and standardized animal model for assessing the therapeutic effect of physical and medication treatment on bone defoct, the effectiveness of operation, as well as the role of bone substitute in the repairing of bone defects.DESIGN:Verified study on the experimental model of bone nonunion in rabbitsSETTING: Department of Orthopaedics in Shenzhen people' s Hospital MATERIALS:Twenty common grade pure New Zealand rabbits of either gender were selected with body mass of (2.5±0.5)kg,aged 6 to 8 months.METHODS :This experiment was carried out at the experimental animal center of Shenzhen people's Hospital between May and August 1999. 1.5cm bone segment (including periosteum)was cut off in the middle of forearm radius in 20 common grade pure NewZealand rabbits,the broken ends were covered with bone wax, 10 weeks later, the bone nonunion status was assessed by macropathological observation, pathohistological and X-ray examination.MAIN OUTCOME MEASURES:Observations on rabbit forearm radius defects by macropathological observation, pathohistological and X-ray examination.RESULTS :Twenty rabbits(40 side radius)were enrolled in this study and weeks later, bone defect region was found filled with fibrous cicatricial tissue without osseous connection ,bone wax was not absorbed, capitulum was ossified with medullary cavity blocked,a small amount of callus formed at both broken ends of fractural bone ,length of bone defect ranged from 0.8 to blocked under optical microscope,chondrocyte and osteocyte could be observed arranging disorderly and covered with fibrous membrane,defect reosseous connection could be detected at defect region at week 10,broken end was ossified and medullary cavity was blocked ,there was small amount of callus appeared at both broken ends displaying irregular shape.CONCLUSION:Bone nonunion experimental animal was successfully established on rabbits in this study, with pathological changes meeting the need of bone nonunion and displaying typical properties,which can be used as reliable and feasible experimental animal model.

Objective To study the effect of Yuzhenxifeng Decoction on the brain ferritin in PD mouse models .Methods All ex‐perimental animals were devided into 4 groups:the control group ,the model group ,the positive drug group and TCM group .Prepare PD model mice with MPTP ,then use the immunohistochemical staining technique to observe the change of expression of Fn .Results the results showed that compared with the model group ,movement coordination disorder of rats in TCM group were relieved ;the Fn level of the model group on 6th ,13th ,20th were higher than control group(P<0 .05) ,and were 2 .21 times ,1 .15 times and 0 .36 times higher respectively .Compared with model group mice ,expression of Fn were enhanced in the treatment group .Conclusion Yuzhenxifeng Decoction can improve the expression of Fn in the brain ,which provide the basis for further study on mechanism of the treatment of Parkinson′s disease .

Objective To explore the difference in Z-score reference range for aorta measurements, and pulmonary artery(PA)/aortic(AO)valve annular diameter ratio(PA/AO ratio)between normal and aortic stenosis(AS)fetuses and to evaluate the diagnostic value of Z-score of aorta in fetuses with AS. Methods A retrospective cross-sectional study of fetal color Doppler echocardiograms were undertook from 300 singleton normal fetuses with gestational age ranged from 16 to 40 weeks.Standardized fetal echocardiographic measurements included AO valve annular diameter and PA valve annular diameter at end-systole,and PA/AO ratio were obtained.With statistical analysis,best model was established for predicting AO and PA using biparietal diameter (BPD)and femoral length (FL)as independent variables.The correlations between standard deviation (SD)and two independent variables (BPD,FL)were analyzed,and the AO Z-score and PA Z-score of normal fetuses were calculated.One hundred and six singleton fetuses with AS were studied whose gestational age ranged from 1 5 to 40 weeks.The AO Z-score and PA Z-score of AS fetuses obtained by using above regression equation and PA/AO ratio were subsequently calculated.The AO and PA Z-scores and PA/AO ratio between normal fetuses and AS fetuses were compared.And the detection rate of AS according to AO Z-score,PA/AO ratio and AO percentile range were assessed.Results Compared with normal fetuses,the Z-score of AO and the PA/AO ratio in AS fetuses were statistically different (P <0.01).While there was no statistical difference of Z-score of PA between two groups (P >0.05).AO Z-score in 96.2% of AS fetuses were found < -3,PA/AO ratio in 95.3% of AS fetuses were found outside 95% CI.The method using AO Z-score had the highest detection rate of AS,the positive predictive value was up to 89.5%,the negative predictive value was up to 98.6%,the sensitivity,specificity and Youden index were 96.2%,96%,and 0.9223,respectively.PA/AO ratio had good but slightly lower diagnostic accuracy than AO Z-score,while AO percentile range performed poor.Conclusions Fetuses whose AO Z-score are outside ±3 and the PA/AO ratio is greater than 1 .4 at the same time are more likely to suffured from AS.Quantification of AO Z-score has important clinical value in the diagnosis of AS,and is more accurate than the percentile range in quantitative assessment of congenital heart growth and development of the fetal pathology of AS.

Objective To develop Z-score reference ranges for mitral valve-tricuspid valve distance (MTD) in normal fetuses from the measurements of femur length (FL),biparietal diameter (BPD) or gestational age (GA) using fetal echocardiography.Methods A retrospective cross-sectional study of 1216singleton normal fetuses was performed.The gestation age ranged from 20 to 40 weeks.Non-cardiac fetal biometric parameters included FL and BPD and GA based on menstrual age.MTD was measured in a standard apical four-chamber view.Normal Z-score ranges were developed for MTD using GA,BPD and FL as independent variables.These were accomplished by using first standard regression analysis and then weighted regression of absolute residual values for each parameter in order to adjust for inconstant variance.Results Linear regression model was the best description of the data in each case and correlations between MTD and independent variables (FL,BPD,GA) were excellent.The linear regression equations between MTD and FL,BPD,GA were:Y =0.04629 × FL-0.02868 (r =0.827) ; Y =0.04018 × BPD-0.08293(r =0.767) ; Y =0.0098 × GA-0.07075 (r =0.721),respectively.Heteroscedasticity of standard deviation(SD) with increasing independent variables also could be modeled with a simple linear regression.Linear regression equations between SD of MTD and FL,BPD,GA were:Y =0.0149 × FL-0.0095 (r =0.9947),Y =0.0126 × BPD-0.02793 (r =0.9931),Y =0.00305 × GA-0.021 (r =0.991),respectively.According to these equations,MTD Z-score =(the actual measurement of MTD-predicted MTD)/prediction SD.Conclusions Normal reference ranges and Z-scores for MTD have been provided.These normative data may be useful tools for assessment of fetal MTD,especially has potential applications in Ebstein anomaly or other malformations involved atrioventricular valves.

Objective To measure mitral annual displacement (MAD) by M-mode echocardiography in normal second and third trimester fetuses,and assess relationships between MAD and gestational age and routine echocardiographic parameters for evaluation of left ventricular function.Methods One hundred fifty-five normal fetuses from 19 to 38 weeks of gestation were recruited in the study.MVD and tricuspid annual displacement (TAD) were measured.Early diastolic inflow velocities (E) of the atrioventricular valves were assessed by pulsed-wave Doppler(PW),and early diastolic velocities (Em) of the mitral annular were estimated by pulsed-wave tissue Doppler imaging (TDI).The ratio of E/Em was calculated.Results In normal fetuses,the MAD was (7.05 ± 1.17) mm.There was significant positive correlation between the fetal MAD and advancing gestational age ( r =0.82,P ＜0.01 ),and between MAD and E,A,Em,Am or Sm ( r =0.25,0.24,0.32,0.29 and 0.40 respectively,P ＜0.01).There was no correlation between fetal MAD and E/A,E/Em,LVEF or LVFS.The left MAD was significantly lower than the right TAD ( P ＜0.01 ).Conclusions MAD of fetuses from 19 to 38 weeks of gestation can be quantitatively measured by Mmode echocardiogram.The measurement of MAD provides a reliable quantitative standard to estimate long axis ventricular function of fetuses,which may be clinically useful in prenatal detecting early cardiac failure.

Objective To evaluate right atrial function in patients with idiopathic dilated cardiomyopathy (IDCM) and ischemic cardiomyopathy (ICM) by using two-dimensional speckle tracking imaging (2D-STI).Methods Study population consisted of 31 patients with IDCM,30 with ICM and 30 healthy subjects.High frame rate two-dimensional images were recorded from the apical four chamber view.Right atrial global longitudinal strain (GLS) was measured using two-dimensional strain soft ware.Results Compared with the controls,left ventricular ejection fraction (LVEF),tricuspid annular plane systolic excursion (TAPSE),right ventricular fractional area change (RVFAC),right ventricular fractional shortening (RVFS) and tricuspid annular peak systolic velocity(S') decreased (P ＜0.05),while right ventricular myocardial performance index (MPI) increased in IDCM and ICM group.There were no significant differences for all above echocardiographic parameters between IDCM and ICM patients.Compared with the controls,right atrial GLS decreased significantly in patients with IDCM and ICM,even much lower in patients with IDCM (P ＜0.001).Conclusions Measurement of right atrial strain using 2DSTI could be used for the assessment of right atrial dysfunction in patients with ICDM and ICM.

Objective To observe the effects of different interventions on the body mass index (BMI) and body fat content in overweight and obese adolescents, with an attempt to design reasonable weight relief program.Methods Totally 67 overweight or obese adolescents from a middle school of Beijing were randomly divided into three groups: control group ( n = 16 ), diet intervention group ( n= 22), and combined interventions group ( n =29). The changes of BMI and body fat rate (BFR) were observed. Results BMI significantly decreased after interventions ( diet intervention group: P = 0. 000; combined interventions group: P = 0. 018 ); the change of BMI in diet intervention group was significantly larger than that in control group ( P = 0. 035 ). The trunk and body BFRs also significantly decreased after interventions (diet intervention group: P=0. 000, P = 0. 013; combined interventions group: P = 0. 000, P = 0. 000 ); the changes of trunk and body BFRs were significantly larger in combined interventions group than those in control group ( P = 0.005, P = 0. 003 ). Conclusion Diet intervention and combined interventions are both effective in achieving weight loss in adolescents, and combined interventions have superior effectiveness.

Objective To explore the effects of diet and/or exercise intervention on oxidative stress of overweight or obese adolescents.Methods Thirty-one adolescents with normal body weight(normal control group)and 93 overweight or obese adolescents(average age(13.6 ±0.7)years; body mass index (BMI)22.4 to 34.1 kg/m2)were enrolled in this study.The overweight or obese participants were then randomly assigned to the diet group(group A),exercise group(group B),diet plus exercise group(group C),and non-intervention group(group D).Individualized caloric intake was carried out.One-hour afterschool exercise was performed once per day,4 days per week for 10 weeks.Changes of anthropometry,body composition,and metabolic biomarkers were determined.Results Compared to the normal control group,serum levels of fasting glucose,total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and malondialdehyde(M DA)of the in overweight group were significant increased(all P ＜ 0.05),although the level of SOD was significant decreased(P ＜ 0.05).Serum MDA level was positively correlated with bodyweight,BMI,TC,and LDL-C(R values were 0.209,0.228,0.274,and 0.263,respectively ; all P ＜0.05),and serum SOD level was negatively correlated with bodyweight and BMI(R values were-0.334 and-0.362,respectively ; both P ＜ 0.05).Group A,B and C showed a significant decrease in BMI,waist circumference and body fat after the intervention(all P ＜ 0.05).The level of MDA was significantly decreased after the intervention in group A and B(both P ＜ 0.05); the levels of TC and LDL-C were significantly decreased in group B and C(both P ＜ 0.05).Conclusion:Oxidative stress could have existed in overweight adolescents even when their serum glucose and lipid profiles are at a normal level.Ten-week diet and/or exercise intervention may contribute to improved body weight control,lipid metabolism,and oxidative stress response in this population.