AIM:Targeting of focal adhesion kinase (FAK) gene,we aim to construct FAK shRNA lentiviral vector and to identify its function on the growth of leukemic cells.METHODS:FAK shRNA was chemically synthesized,and inserted into a GFP-lentiviral plasmid through molecular biology methods.After packaged and concentrated,the lentiviral-FAK-shRNA-vector was transduced into a human leukemic cell line.FAK gene expression was detected by reverse transcriptional PCR and Western blotting.Cell apoptosis was measured by Annexin V labeling.RESULTS:The results showed that FAK shRNA was successfully inserted into the lentival vector,and the infection efficiency varied from 10% to 25%.Compared to the control vector (lentival vector without FAK shRNA),FAK shRNA inhibited the expression of FAK mRNA and protein by 40% and 70%,respectively.Moreover,the results of apoptosis experiment showed that the percentages of Annexin V+ cells in control vector group and FAK shRNA group were (4.19 ? 0.36) % and (8.48 ? 0.58) % respectively,the difference was statistically significant (P

OBJECTIVETo investigate the additive effects of uncoupling protein 2 (UCP2) gene Ala55Val variation and ADR beta(3) gene Trp64Arg variation on the obesity in Chinese Han population.

METHODSThe UCP2 gene Ala55Val variation and ADR beta(3) gene Trp64Arg variation were examined by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) in 119 obese subject with mean BMI (27.9+/-2.98)kg/m(2) and in 177 control subjects with mean BMI(21.9+/-1.9)kg/m(2). The additive effects of the two gene mutations were analyzed.

RESULTS(1) The frequency of ADR beta(3) gene Trp64Arg variation in obese subjects was not significantly different from that in control subjects. In control subjects, the Trp64Arg variation carriers had higher fasting glucose level and 2-hour-post-prandial glucose level than did non-carriers. (2) The frequency of homozygote of UCP2 gene Ala55Val variation in obese subjects was higher than that in the control subjects (OR=3.71, P=0.001). In control subjects the Ala55Val variation carriers had higher BMI. (3) When there was only UCP2 gene or ADR beta(3) gene mutation, the frequency of gene mutation in obese subjects was not significantly different from that in control subjects (P>0.05). But when there were simultaneously two gene mutations, the frequency of gene mutations was higher in obese subjects than in control subjects (OR=2.57, P=0.009). (4) The genotype carriers with Val/Val+ Trp/Arg were the greatest relation to obese obesity (OR=8.58, P=0.002).

CONCLUSIONThe homozygote of UCP2 gene Ala55Val mutation increases the risk of obesity. Though the UCP2 gene mutation alone or the ADR beta(3) gene mutation alone is not associated with obesity, the possible additive effects of the two micro-genes increase the occurring of obesity.

Adult ; Aged ; Female ; Humans ; Ion Channels ; Male ; Membrane Transport Proteins ; genetics ; Middle Aged ; Mitochondrial Proteins ; genetics ; Mutation ; Obesity ; genetics ; Receptors, Adrenergic, beta-3 ; genetics ; Uncoupling Protein 2

OBJECTIVETo investigate whether Notch signaling is activated in hepatic stellate cells (HSCs), and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs.

METHODSThe expression of Notch signaling components in unactivated or TGF-b1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis. Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis. Notch3-mediated expression of the myofibroblastic markers, a-SMA and collagen I, was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting.

RESULTSNotch signaling components were expressed in both unactivated and activated HSC-T6 cells, but the TGF-b1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold, F = 2543.482), Notch3 (4.2-fold, F = 287.982), and HES1 (3.2-fold, F = 1719.851). Transfection-mediated over-expression of Notch3 led to significantly increased expression of a-SMA (6.8-fold, t = 13.157) and collagen I (5.5-fold, t = 9.810) (both P less than 0.01). Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (a-SMA by approximately 90%, t = 19.863 and collagen I by 84%, t = 10.376; both, P less than 0.01). Moreover, knock-down of Notch3 antagonized the TGF-b1-induced expression of a-SMA and collagen I.

CONCLUSIONNotch signaling may participate in liver fibrogenesis by regulating HSC activation. Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.

Animals ; Calcium-Binding Proteins ; genetics ; metabolism ; Cell Line ; Hepatic Stellate Cells ; metabolism ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Jagged-1 Protein ; Membrane Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Rats ; Receptor, Notch3 ; Receptors, Notch ; genetics ; metabolism ; Serrate-Jagged Proteins ; Signal Transduction

Objective To explore the influence to nursing efficiency using exposed wound care ( EWC) at different time point after finger replantation. Methods 60S patients after finger replantation were recruited according to selection criteria and were randomly divided into 4 groups. 136 cases in group A were treated with EWC without gauze dressing cover 1 to 2 hours after operation. 183 cases in group B were treated with EWC 6 to 8 hours after operation. 159 cases group C were treated with EWC 12 to 24 hours after operation. 127 cases in group D were treated with gauze dresssing cover all the time until taking the stiches out ( 14 days). Blood circulation, wound infection, finger survival rate, medical costs were observed and compared among 4 groups. Results Compared with the other 3 groups, group B had better outcomes including blood circulation, wound infection, survival rate and medical costs of replanted finger. The EWC therapy could decrease expenditures of inpatient, length of stay, and incidence of complications. Conclusions The EWC therapy should be used 6 to 8 hours after operation when dressing oozing did not dry completely. That therapy can decrease the incidence of blood vessel crisis of replanted finger, reduce medical expenditures and improve survival rate of replanted finger.

OBJECTIVETo detect the differences in subcortical structures between patients with paroxysmal kinesigenic dyskinesia (PKD) and normal subjects during movement preparation and execution.

METHODSThe PKD patients performed a movement task, in which a CUE signal (preparation) indicated the movement sequence prior to the appearance of an imperative GO signal (execution). Event-related functional magnetic resonance imaging (fMRI) and 3dDeconvolve program of AFNI were used to estimate the hemodynamic response function and to generate activation maps.

RESULTDuring movement preparation, the activated brain areas in PKD patients were less than those of normal subject, and there was no activation in basal ganglia in PKD patients. During execution, the activation was also less in PKD patients except in bilateral M1.

CONCLUSIONDuring intermission, abnormalities of the brain still exist in PKD patients when during preparing or performing movement. The movement circuit in the brain displays an unusual state. The attack may be caused by reducing of inhibition in brain areas.

Adult ; Chorea ; physiopathology ; Humans ; Magnetic Resonance Imaging ; Male ; Motor Cortex ; physiopathology ; Movement ; physiology

OBJECTIVETo establish an semi-automated effective method for large-scale purification of islet cells from human pancreas.

METHODSHuman pancreas tissue was digested with collagenase P using a semi-automated pancreas-digestion system followed by purification in a HCA-Ficoll continuous gradient using Cobe2991 cell separator. After isolation, the islet cell yield and purity was evaluated with light microscope with DTZ staining, and the islet function assessed by insulin release assay in vitro.

RESULTSThe number of the islets collected from each pancreas averaged 38 6201-/+78 219 islet equivalents (IEQ) before purification, and 231 420-/+28 054 IEQ after the purification with discontinuous gradient centrifugation. From each gram of the pancreatic tissue, 3148-/+317 IEQ were obtained with an average purity of (62.81-/+2.68) %. The purified islets responded well to high-concentration (16.7 mmol/L) glucose stimulation with a 2.53-fold increase of insulin secretion over the basal level (3.3 mmol/l, P<0.001).

CONCLUSIONThe established semi-automated method can be applicable for large-scale purification of fully functional islet cells from human pancreas.

Cell Count ; Cell Separation ; instrumentation ; methods ; Cell Survival ; Glucose ; pharmacology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Reproducibility of Results

OBJECTIVETo study the effect of combined continued estrogen and progestin replacement therapy on knee osteoarthritis (OA) symptoms of postmenopausal women.

METHODSSixty-four postmenopausal women with radiological knee OA and symptoms aged 45-75 were divided into treatment group and control group. They were given estradiol velerate (E2V) 1.0 mg/d and medroxyprogestetone acetate (MPA) 2 mg/d (treatment group) or placebo (control group) for 6 months. Calcium 400 mg/d were given to all cases. Then 0-100 mm visual analon scale (VAS) was used to evaluate the severity of knee pain at baseline and after 1, 3, 6 month of treatment.

RESULTSSignificant differences on pain at night and tenderness around knee were seen in the treatment group compared with the control group after 1 months of treatment (P = 0.036 and 0.035, respectively). The improvement of pain at night, during walk and morning stiffness between the two groups showed significant difference after 6 months (P = 0.026, 0.027, and 0.011, respectively).

CONCLUSIONCombined estrogen and progestin replacement therapy can relieve the knee OA symptoms of postmenopausal women.

Aged ; Double-Blind Method ; Estradiol ; therapeutic use ; Estrogen Replacement Therapy ; Female ; Humans ; Medroxyprogesterone Acetate ; therapeutic use ; Middle Aged ; Osteoarthritis, Knee ; drug therapy ; Postmenopause ; Prospective Studies

Objective To evaluate the clinical value of neonatal peripheral blood smear spherical erythrocyte count in the diagnosis of ABO-HDN.Methods 165 cases clinically diagnosed with ABO-HDN in Zhongshan Boai Hospital from 2009 to 2015 were listed as the experimental group by retrospective analysis,68 cases of non-ABO-HDN were listed as control group.Besides,relevant clinical data and experimental examination were investigated,and the results of their hemolysis test were analysed.Results Peripheral blood smear spherical erythrocyte count were positive in 110 cases of 165 patients with ABO-HDN,the positive rate of spherical erythrocytes was 66.7 ％ (x2 =58.069,P＜ 0.05).The spherical erythrocyte positive rates were 68.8 ％,60.5 ％ and 66.7 ％ in patients aged ≤2d,3 ～4d,≥5d respectively.The diagnostic sensitivity of spherical erythrocytes to ABO-HDN was 66.7 ％,the specificity was 88.2 ％,the positive predictive value was 93.2 ％,and negative predictive value was 52.2 ％.When spherical erythrocyte count positive point was set as ≥5 ％ spherical erythrocytes,the diagnostic sensitivity of spherical erythrocytes to ABO-HDN was 66.7％ and the specificity was 88.2％.If the positive point was set as ≥10％ spherical erythrocytes,the sensitivity of ABO-HDN decreases to 9.3％,and the specificity reaches 98.5 ％.In ABO-HDN group,the levels of nucleated red blood cell,RDW and Ret were higher,along with the lower level of hemoglobin compared with non-ABO-HDN group (all P＜0.05).Conclusion The peripheral blood smear spherical erythrocyte count had a high sensitivity and specificity for the diagnosis of ABOHDN.Combined with jaundice,anemia and RDW,peripheral blood smear spherical erythrocyte count can provide guidance for the early diagnosis,prevention and treatment of ABO-HDN.

BACKGROUND AND PURPOSE: It is essential to develop a reliable predictive serum biomarker for Parkinson's disease (PD). The accumulation of alpha-synuclein (αSyn) and up-regulated expression of Rab35 participate in the etiology of PD. The purpose of this investigation was to determine whether the combined assessment of serum αSyn and Rab35 is a useful predictive biomarker for PD. METHODS: Serum levels of αSyn or Rab35 were determined in serum samples from 59 sporadic PD patients, 19 progressive supranuclear palsy (PSP) patients, 20 multiple system atrophy (MSA) patients, and 60 normal controls (NC). Receiver operating characteristics (ROC) curves were calculated to determine the diagnostic accuracy of αSyn or/and Rab35 in discriminating PD patients from NC or atypical parkinsonian patients. RESULTS: The levels of αSyn and Rab35 were increased in PD patients. The serum level of Rab35 was positively correlated with that of αSyn in PD patients. Compared to analyzing αSyn or Rab35 alone, the combined analysis of αSyn and Rab35 produced a larger area under the ROC curve and performed better in discriminating PD patients from NC, MSA patients, or PSP patients. When age was dichotomized at 55, 60, 65, or 70 years, the combined assessment of αSyn and Rab35 for classifying PD was better in the group below the cutoff age than in the group above the cutoff age. CONCLUSIONS: Combined assessment of serum αSyn and Rab35 is a better biomarker for discriminating PD patients from NC or atypical parkinsonian patients, and is a useful predictive biomarker for younger sporadic PD patients.
alpha-Synuclein ; Humans ; Multiple System Atrophy ; Parkinson Disease ; ROC Curve ; Supranuclear Palsy, Progressive

OBJECTIVETo compare the difference of effects on SiO(2)-induced alveolitis and early fibrosis between bone marrow-derived mesenchymal-like stem cells (BM-MSCs) and BM-MSCs transfected by pcDNA3.1-HGF and to explore the mechanism of this effects.

METHODSThe Primary BM-MSCs from Wistar male young rats were cultured and labeled by 4, 6-diamidino-2-phenylindole (DAPI). Fifty Wistar rats were randomly divided into 3 groups:model group (10 rats),which was administered with SiO(2) by the trache, the next day,injected PBS via the tail vein; BM-MSCs group (20 rats),which was administered with SiO(2) by the trache, the next day,injected with 1 ml suspension of BM-MSCs via the tail vein; pcDNA3.1-HGF plus BM-MSC group (20 rats),which was administered with SiO(2) by the trache, the next day,injected with 1 ml suspension of BM-MSCs transfected by pcDNA3.1-HGF via the tail vein. On the 14th and 28th days after treatment, half of the animals were sacrificed, respectively, and the lungs were harvested for frozen section to observe the cell marked by DAPI. HE staining under a fluorescent microscope, and to observe the pulmonary alveolitis and fibrosis by HE and Masson staining under a light microscope. Western blot assay was used to detect the expression of HGF in rat lungs. The expression levels of tumor necrosis factor-α (TNF-α) in pulmonary tissues were analyzed quantitatively by ELISA. The contents of HYP in pulmonary tissues were analyzed quantitatively by sample hydrolysis method.

RESULTSOn the 14th and 28th days after treatment, the scores of pulmonary alveolitis and early fibrosis in pcDNA3.1-HGF plus BM-MSCs group were 2.36 ± 0.17, 2.8 ± 0.14 and 0.1 ± 0.11, 1.16 ± 0.13, which were significantly lower than those (1.68 ± 0.17, 1.58 ± 0.31 and 0.54 ± 0.15, 1.36 ± 0.13) in BM-MSCs group, also which were significantly lower those (2.36 ± 0.17, 2.80 ± 0.14 and 0.64 ± 0.09, 1.84 ± 0.17) in model group (P < 0.05); On the 14th and 28th days after treatment, the TNF-α contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 280.4 ± 23.11 and 249.78 ± 22.33 pg/mg, which were significantly lower than those (341.58 ± 35.34, 442.29 ± 36.76 pg/mg and 319.51 ± 17.84, 348.53 ± 33.95 pg/mg) in BM-MSCs and model groups (P < 0.05); On the 14th and 28th days after treatment, the HYP contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 0.46 ± 0.04 and 0.65 ± 0.05 µg/mg, which were significantly lower than those (0.63 ± 0.04, 1.04 ± 0.07 µg/mg and 0.72 ± 0.60, 1.39 ± 0.60 µg/mg) in BM-MSCs and model groups (P < 0.05).

CONCLUSIONThe effects of BM-MSCs transfected by pcDNA3.1-HGF on suppressing pulmonary alveolitis and early fibrosis induced by SiO2 were better than those of BM-MSCs. The mechanism may be associated with the reduced pulmonary inflammation.

Animals ; Bone Marrow Cells ; cytology ; Hepatocyte Growth Factor ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; metabolism ; Pulmonary Fibrosis ; chemically induced ; prevention & control ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Silicosis ; prevention & control ; Transfection