BACKGROUND: At present, many problems deserve to be solved before clinical utilization of adipose tissue-derived mesenchymal stem cells (ADMSCs) such as complete differentiation from ADMSCs into cardiomyocytes and ADMSCs with or without specific function of cardiomyocytes. It is of significance to solve the problems including how to elevate the differentiation rate of ADMSCs into cardiomyocytes and how to elevate the homing and survival rate after transplantation for clinical utilization of stem cells.OBJECTIVE: To observe the differentiation of ADMSCs into cardiomyocytes after in vitro culture and induction.DESIGN: Randomized controlled observation.SETTING: Laboratory of Cardiology, General Hospital of Chinese PLA.MATERIALS: Adipose tissue was collected from abdominal operative patients at Department of General Surgery of General Hospital of Chinese PLA with the agreement of patients. Iscove's Modified Dulbecco's Medium (IMDM), type Ⅰ collagenase, 5-azacytidine (5-aza) and polyclonal antibody of specific anti-myocardial Troponin T (TnT) were purchased from Hyclon company, Gibco company, Sigma and Fujian Maixin Biotechnology Company, respectively.Monoclone antibodies of CD44, CD45, CD34, HLA2DR and factor-Ⅷ, Desmin, anti-α-striated muscle actin and anti-myosin heavy chain (MHC) were bought from Beijing Zhongshan Golden Bridge Biotechnology Limited Corporation. DAB stain, reverse transcription-polymerase chain reaction (RT-PCR) kit and atrial natriuretic peptide (ANP) radioimmunity kit were purchased from Beijing Zhongshan Golden Bridge Biotechnology Limited Corporation,Invitrogen and Radioimmunity Research Institute of Science and Technology Development Center of General Hospital of Chinese PLA, respectively.METHODS: The experiment was performed at the Laboratory of Cardiology, General Hospital of Chinese PLA from March 2005 to April 2006. ADMSCs were isolated and cultured by digestion and attachment culture method. The third generation of cells were determined by immunocytochemical method for surface molecule CD44, CD13, CD105, CD45,CD34, HLA-DR, factor Ⅷ and induced by addition of 5-aza into culture medium. On the 7th, 14th, 21st and 28th days, gene of GATA4 and Nkx2.5 were tested by reverse transcription-polymerase chain reaction (RT-PCR), and the concentration of atrial natriuretic polypeptide (ANP) were also examined by radioimmunoassay.MAIN OUTCOME MEASURES: Determination of cell surface marker, gene of GATA4 and Nkx2.5, and ANP concentration.and factor Ⅷ negative for the cultured cells. After induction for 7 days with 5-aza, no cells expressed Desmin,α-Sarcomeric Actin, Myocin heavy chain (MHC) and TnT, most cells were positive stained for CD44. After induction for 14 days, small amounts of cells displayed positive for Desmin,α-Sarcomeric Actin and MHC but still negative for TnT,while partial cells expressed positive CD44. After induction for 21 days, most cells were positive for Desmin,α-Sarcomeric after induction and no significant difference was found compared with that at the 7th day [(0.022±0.01) ng/L (P＞0.05].ANP concentration was respectively (7.92±0.21) and (8.12±0.50) ng/L at the 21st day and the 28th day (P＞0.05), but there was significant difference compared with that at the 7th day (P＜0.05).

BACKGROUND: Studies confirm that ischemia/reperfusion (I/R) injury can induce myocardial apoptosis. The loss of mitochondrial membrane potential (MMP) after reperfusion is the inevitable pathway of apoptosis. Protection of MMP may reduce apoptosis.OBJECTIVE: To observe the effect of naloxone on MMP of hypoxic myocardial cells and apoptosis in neonatal rats, and investigate the protective effect of naloxone on hypoxic myocardial cells.DESIGN: Observation and controlled trial.SETTING: Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS: Detection of apoptosis of myocardial cells was carried out in the Laboratory of Pathophysiology, General Hospital of Chinese PLA in December 2004. Ten neonatal rats were used, and detection of MMP of myocardial cells was carried out in the same laboratory in March 2006 and 20 rats were used. All the involved rats were provided by the Animal Center of the General Hospital of Chinese PLA on the day of birth, were involved in this trial. Reagents: Naloxone hydrochloride injection (0.4 g/L, Beijing Sihuan Pharmaceutical Factory, Batch No. 0206272); the lowest and essential medium (Dulbecco, DEME,GIBCO company); phosphate buffer solution and fetal bovine serum (PBS and FBS, SIGMA Company).METHODS: The neonate rats were cut open along the median line of chest bone after local sterilization with iodine tincture on the day of birth. 1/3 ventricular myocardium before cardiac apex was harvested. On the 4th day of the culture,culture flasks of cells in good growth status ( ＞ 106 cells/bottle) were selected and divided into 3 groups: control group (normal culture, n =3 bottles), hypoxia group (hypoxia/reoxygenation, n =15 bottles) and naloxone group (hypoxia/reoxygenation, and treated by naloxone, n =15 bottles). Three time points were set in hypoxia group and naloxone group according to different time of hypoxia and reoxygenation: hypoxia 2 hours/reoxygenation 0 hour; hypoxia 2 hours/reoxygenation 2 hours; hypoxia 2 hours/reoxygenation 4 hours, 5 bottles at each time point. In the hypoxia group, DEME medium, which was pre-filled with 0.95 volume fraction of N2 and 0.05 volume fraction of CO2, containing 0.01 volume fraction of FBS, was used and 0.95 volume fraction of N2 and 0.05 volume fraction of CO2 was also filled to replace the air in the culture flasks. The culture flasks were enveloped for incubation at 37 ℃. The cells in the hypoxia group were incubated at normal condition (0.95 volume fraction of air and 0.05 volume fraction of CO2) at set time. In the naxolone group, hypoxia/reoxygenation treatment was the same as above, and naloxone hydrochloride was added at the sametime and the final concentration of naloxone in culture flasks was 5 μmol/L (The volume for naloxone ≤ 0.5% of the total medium). In the control group, hypoxia/reoxygenation and naloxone treatment were not given, but the same volume of normal saline was added, and this time served as time point of hypoxia 0 hour/reoxygenation 0 hour. After intervention,myocardial MMP changes and apoptosis were detected with fluorescent staining-flow cytometer at different time after hypoxia/reoxygenation.MAIN OUTCOME MEASURES: ① MMP changes of hypoxia group and naloxone group at different time points; ② Comparison of survival, apoptosis and necrosis of cells at hypoxia 2 hours/reoxygenation 4 hours in each group.RESULTS: ① After hypoxia, MMPs of the cells in hypoxia group and naloxone group were decreased. MMP of the cells in the naloxone group was higher than that in the hypoxia group at each time point (P ＜ 0.01). The great amplitude of decrease of MMP occurred in hypoxia period, but not in reoxygenation period. ② At hypoxia 2 hours/reoxygenation 4 hours, the apoptotic and necrotic rates in the hypoxia group were significantly higher than those in the naloxone group [(9.88±0.98)% vs. (2.41±0.52)%; (5.10±0.29)% vs. (3.56±0.56)%, both P ＜ 0.01]. The apoptotic rate was significantly higher than the necrotic rate in the hypoxia group (P ＜ 0.05).CONCLUSION: Early application of naloxone can significantly alleviate and postpone the decrease of myocardial MMP after I/R, and reduce apoptosis and necrosis.

Objective To explore the hereditary susceptibility of children with asthma through studying the relationship between erythroeyte CR1 genomic density polymorphism and erythrocyte immune function. Methods The rates of RBC-C3_(3b)RR and RBC-ICRR were detected to the asthma group consisted of 65 children with asthma and the control group consisted of 28 normal children. The CR1 activity and genomic density polymorphism of erythrocyte from the two groups were detected by Hind Ⅲ restriction enzyme digestion, polymerase chain reaction restriction fragment length polymorphism. Results Frequencies of high expression gene (HH), mid expression gene (HL) and low expression gene (LL) genotypes were 43.08% (28/65), 36.92% (24/65) and 20.00% (13/65) in asthma group, and 78.57% (22/28), 17.86% (5/28) and 3.57%(1/28) in control group respectively. A significant difference was found in the distribution frequency of CR1 genotype between the two groups(P< 0.01).The rates of RBC-C_(3b)RR were significant lower and the rates of RBC-ICRR were significant higher in asthma group than those in control group (P < 0.01). The rates of RBC-C_(3b) RR in HH, HL and LL were decreased in order (P < 0.01),while the rates of RBC-ICRR in HH,HL and LL were increased in order (P < 0.01). Conclusion It suggests that CR1 gene polymorphism may play an important role in determining susceptibility to asthma.

Objective To investigate the influence of rehmannia glutinosa oligosaccharide(RGOs)on apoptosis of human adipose tissue-derived mesenchymal stromal cells(ADMSCs)induced by hydrogen peroxide(H2O2).Methods Cultured human ADMSCs were randomly divided into three groups as the normal group(group N,without any treatment),H2O2 group(group H,treated with 0.1 mmol/L H2O2),and RGOs group(group R,treated with 0.2g/L RGOs plus 0.1 mmol/L H2O2).After 1 h,6 h and 24 h,morphological changes of ADMSCs apoptotic were observed,the apoptotic rate was determined by flow cytometry.Results After 1 h,6 h and 24 h,the apoptotic rate of group H was significantly higher than that of the group N and group R(P<0.05),and the apoptotic rate of group H was significantly higher than that of the group R(P<0.05).Conclusion RGOs can attenuate apoptosis of human ADMSCs induced by H2O2.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ADMSCs) have the multilineage differentiation potential, and are relatively easier to be obtained, thus they have attracted more and more attention as a new seed cell for cell engineering.OBJECTIVE: To observe the in vitro culture conditions of ADMSCs isolated from rat's subcutaneous adipose tissue, and identify them using immunohistochemical staining.DESIGN: An animal experiment.SETTING; Department of Cardiology, the General Hospital of Chinese PLA.MATERIALS: One healthy male Wistar rat of clean degree, 4 months old, weighing 200 g, was used. DMEM, fetal bovine serum were from GIBCO; Monoclone antibodies of rabbit-anti-rat CD13, CD34, CD44, CD45, CD105, D-related human leucocyte antigen (HLA-DR), factor-Ⅷ, vov Willebrand factor (VWF), Myosin, SABC kits and DAB staining kit from Wuhan Booster Biological Engineering, Co.,Ltd; Adeno-associated virus encoding green fluorescent protein from Vector Gene Technology Company Limited (Beijing).METHODS: The experiments were carried out in the Department of Internal Medicine, the General Hospital of Chinese PLA in October 2006. ① Cell isolation and culture: 0.3 g adipose tissue was cut from subcutaneous adipose tissue of Wistar rat's groin under aseptic condition, then minced and digested before culture, DMEM was changed at 2-3 days after plenty of fusiform-shap ed attached cells were observed under microscope, and the cell growth was observed. The cell concentration was adjusted to 2×107 L-1, then seeded into 96-well plate, and 100 μL for each well. From the second day, 3 wells were randomly selected every day, the cells were released with tripsin, and counted with blood cell counting chamber under inverted microscope. ② Cell viability assay: ADMSCs of passages 3 to 8 were added to DMSO freeze medium, and thawed after 2-4 weeks, and the cell viability was assessed by trypan blue dye exclusion. ③Immunohistochemical staining and identification: 2 ×107 L -1 cells were seeded to culture plate, then the immunohistochemical (SABC method) identification and Oil red O staining were performed to determine the cell surface antibodies of CD13, CD34, CD44, CD45, CD105, HLA-DR, factor-Ⅷ, HLA-DR and VWF. ④Lineage-specific differentiation and identification: The ADMSCs were plated on multi-well chamber and induced with lineage-specific media supplementation at least two weeks and identified by histologic/immunohistochemical assay of Oil red O for adipogenisis, alkaline phosphatase (ALP) stain for osteogenisis and Myosin monoclonal antibody for myogenisis. ⑤Transfected adenovirus carried green fluorescence protein (AD-GFP) medium: The fourth generation of ADMSCs were seeded on 96-well plate, 3 000 cells for each well, serum-free DMEM was changed after 24 hours, and added by AD-GFP at the same time, then transfected with different multiplicity of infection (MOI) of 1∶50, 1∶100, 1∶150 and 1∶200respectively, and then the transfection was observed.MAIN OUTCOME MEASURES: ① Results of cell isolation and culture; ② Cell viability after freezing and thawing; ③Results of immunohistochemical staining and identification; ④ Results of lineage-specific differentiation and identification;⑤ Results of transfected adenovirus carried AD-GFP.RESULTS: ① About 3.6×105 attached cells were obtained from 0.3 g subcutaneous adipose tissue, and these cells could be subcultured for passages in vitro with stable population doubling time. ② The cells were thawed after freezing for 2-3 weeks, and the trypan blue staining showed that the cell viability was above 90%. ③ The immunocytochemical staining showed that CD13, CD44, CD105 were positive and CD45, factor-Ⅷ, HLA-DR and VWF negative in different generations. ④ From the second generation, a few Oil red O positively stained cells were observed, which were obviously increased after prolonging the refreshing. After lineage-specific differentiation, the cells were all positive by Oil red O staining, ALP staining and Myosin immunohistochemical staining. ⑤ 72 hours after transfection, it was observed under fluorescence microscope that most cells were green fluorescence when the MOI value was 1∶200, the transfection was successful, and it was generally determined that the transfection rate was above 90%.CONCLUSION: A large number of ADMSCs with multilineage differentiation potential can be easily obtained from rat adipose tissue, osteoblast, myoblasts, they can be expanded in large quantity and stored in vitro for long time, AD-GFP were also successfully transfected.

Objective To evaluate the efficacy and safety of sorafenib in the treatment of advanced renal cell carcinoma.Methods The clinical date of 33 patients with advanced renal cell carcinoma from September 2007 to April 2012 was reviewed retrospectively.26 were males and 7 were females,with an average age of 69 years.Pathological diagnosis showed 30 clear cell RCCs,2 papillary RCCs,and 1 unclassified RCC.These patients were treated by sorafenib 400 mg twice a day until intolerable toxicity or disease progression.The primary end points were objective response rate,clinical benefit rate,median survival time,median progression-free survival and the incidence of adverse reaction.Results All patients were evaluable for response and toxicity,with 8 patients (24％) of partial remission,19 cases (58％) of stable disease,and 6 cases (18％) of disease progression.The disease control rate was 82％,the median progression-free survival was 10.2 months,while the median survival time was 16.5 months.The common adverse reactions included hand-foot skin reaction (61％),diarrhea (46％),hypertension (21％).Most adverse reactions occurred around the second week after drug therapy,with the duration unequal.The majority of adverse reactions could be released by symptomatic treatment,which did not affect the medication.Conclusion Sorafenib has good short term efficacy for patients with advanced renal cell carcinoma,and most adverse reactions were tolerable.

Objective To explore the diagnostic value of MR diffusion weighted imaging (DWI) in the differential diagnosis between encephalitis and acute cerebral infarction.Methods The MR DWI appearances of 23 cases of encephalitis including 14 cases with viral encephalitis, 9 cases with demyelinative encephalitis and 30 cases with acute cerebral infarction were retrospectively analyzed.Results The 30 cases of acute cerebral infarction appeared bright signal in DWI with a ADC values of (0.46?0.13)?10 -3mm2/s. 7 cases with viral encephalitis and 9 cases with demyelinative encephalitis appeared slightly high or high signal intensity in DWI with a ADC values of (0. 98?0.18)?10 -3mm2/s and(0. 89?0.07)?10 -3mm2/s respectively. The ADC values of viral encephalitis and demyelinative encephalitis were higher comparing to acute cerebral infarction(?0.05). However, the ADC values showed higher or lower in different areas in 4 cases, and lower ADC values appearing in 3 cases with viral encephalitis comparing normal parenchyma.Conclusion MR DWI is useful in the differential diagnosis between encephalitis and acute cerebral infarction.

Objective To evaluate the effectiveness of wechat-based transitional care in the acoustic neuroma patients with postoperative facial palsy.Methods 90 acoustic neuroma patients with postoperative facial palsy were randomly enrolled into 2 groups by tossing coin,45 cases in each group.Control group received routine discharge education,while the experimental group received wechat-based transitional care for three months.The rehabilitation adherence,the level of facial palsy,the ocular infection and the quality of life at patients discharged and three months after operation were compared between the two groups.Results Three months after operation,the cases of high,middle,low level of rehabilitation in the experimental group were 28,12,5,which were more than the control group whose cases were 15,21,9 (x2=7.528,P＜ 0.05).Facial palsy and the quality of life of the experimental group were 66.7％ (30/45) and (71.62±6.36) points,which was significantly higher than 42.4％ (19/45) and (63.75±11.28) points in the control group (x2=5.421 and 4.073,P ＜ 0.01).The incidence of ocular infection in the experimental group was 8.9％ (4/45) which was significantly lower than 31.1％ (14/45) in the control group (x2=6.671,P ＜0.05).Conclusions Wechat-based transitional care achieves good effectiveness in patients with postoperative facial palsy,which could improve the level of rehabilitation,facial palsy and the quality of life,and reduce the incidence of ocular infection,is worthy of promotion.

Objective To study the content of monoamine neurotransmitters and neurotrophic factor in the hippocampus, amygdala and prefrontal cortex in anxious depression rats, and explore the possible pathogenesis.Methods 60 SD rats were randomly divided into normal group, vehicle group, anxiety group, depression group, and anxious depression group, 12 rats in each group.Chronic restraint stress combined with corticosterone injection was used to establish anxiety and depression model, the modeling time was 21 d.After modeling, elevated plus maze test, open field test, and forced swimming test were used to evaluate the anxiety and depression-like behavior, HPLC-ECD was used to detect the content of 5-HT, NE, and DA in the hippocampus, amygdala, and prefrontal cortex of rats.Western-blotting was used to detect the expression of BDNF and NT-3 in rats.Results Rats in anxious depression model group were comparable to the anxiety group in time and frequency entering open arm time, and number of locomotor activity in open field, and it had a significant difference when compared with the control and depression groups (P<0.01 or P<0.05).Immobile time in anxious depression model rats was increased significantly when compared with the control and anxiety groups (P<0.01).Meanwhile, compared with the control group, 5-HT in hippocampus and 5-HT, NE in amygdala or prefrontal cortex were significantly decreased in the depressive rats with anxiety (P<0.01 or P<0.05).Moreover, the content of BDNF and NT-3 was significantly decreased in each brain regions compared with the control group (P<0.01 or P<0.05), and BDNF levels were obviously decreased compared with the anxiety group (P<0.05).Conclusions Rats of anxious depression have significant anxiety and depression-like behaviors.Its mechanism may be associated with the down-regulation of monoamine neurotransmitters and neurotrophic factors BDNF and NT-3 in hippocampus, amygdala, and prefrontal cortex region.

OBJECTIVE To investigate drug resistant genes associated with beta-lactamases,aminoglycosides and sulfonamides in Pseudomonas aeruginosa isolated from burned patients.METHODS Common drug resistant genes associated with beta-lactamases,aminoglycosides and sulfonamides of 20 strains of P.aeruginosa isolated from burned patients were detected by PCR.RESULTS The positive rates of genes of TEM,aac(6′)-Ⅰb,sul1 and ant(3″)-Ⅰwere 85%,85%,90% and 10%,respectively and genes of CARB and aac(6′)-Ⅱ were both positive(100%).Genes of oprD2 were all absent and genes of SHV,GES,IMP,VIM,DHA and armA were all negative.Furthermore,drug resistant genes of strains isolated from different sites of one patient on different time could be different.CONCLUSIONS Drug resistant genes of CARB and aac(6′)-Ⅱprevail in P.aeruginosa isolated from burned patients.Detections of drug resistant genes in P.aeruginosa isolated,especially continuously isolated from burned patients are essential.