Objective To set up a calibration system for automated hematology analyzers with fresh blood in clinical laboratory.Methods Fresh blood assigned by a traceable measurement system was used to calibrate nine hematology analyzers, and compared the bias before and after calibration.Results In the parameters to be calibrated for the hematology analyzers, there was about 55.6% (25/45) over allowable bias before calibration but 15.6% (7/45) after calibration with fresh blood. Among the results of bias over allowable upper limit were mostly existed in 3-part differential hematology analyzers, and mainly focused on WBC and PLT.Conclusion It is available to calibrate different hematology analyzers with fresh blood in a clinical laboratory.

OBJECTIVETo screen the lentiviral vector carrying siRNA and capable of significantly suppressing the ROCK2 gene expression in the corpus cavernosum smooth muscle cells of spontaneously hypertensive rats (SHR).

METHODSWe designed and synthesized 4 siRNA fragments targeting the ROCK2 gene and packaged them into lentiviral vectors. We collected corpus cavernosum smooth muscle cell samples from 5 male SHRs and randomly divided them into groups A (non-transfection control), B (GFP lentiviral transfection), C, D, E, and F (lentiviral transfection with siRNA fragments 1 -4 targeting the ROCK2 gene). Each group consisted of 5 samples and each sample 3 x 10(4) cells. At 48 hours after transfecting MOI = 80 into the SHR corpus cavernosum smooth muscle cells, we detected the expression of GFP under the fluorescent microscope and the mRNA expression of the ROCK2 gene by RT-PCR.

RESULTSThe transfection efficiency of the SHR corpus cavernosum smooth muscle cells was > 50%. Compared with group A, the expression of ROCK2 mRNA in the corpus cavernosum smooth muscle cells showed no remarkable change in group B (P > 0.05) but was inhibited very significantly in C ([43.91 +/- 8.19]%), D ([47.15 +/- 6.64]%), and F ([25.7 +/- 6.03]%) (P < 0.01), and significantly in E ([16.81 +/- 5.94]%, P < 0.05).

CONCLUSIONWe successfully constructed 4 lentiviral vectors carrying siRNA targeting the ROCK2 gene, all of which can significantly suppress the ROCK2 expression in the SHR corpus cavernosum smooth muscle cells, and one has a highly strong inhibitory effect.

Animals ; Gene Silencing ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Myocytes, Smooth Muscle ; Penis ; cytology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Random Allocation ; Rats ; Rats, Inbred SHR ; Transfection ; methods ; rho-Associated Kinases ; genetics

Objective To examine the impact of diabetic foot patients′ negative emotion on the caregiver′quality of life. Methods Totally 100 pairs of diabetic foot patients and their caregivers were investigated using convenience sampling method. Results The incidence of anxiety, depression of hospitalized patients with diabetic foot was 41.5% (23/200), 44.0% (88/200) respectively. Pearson correlation analysis showed that anxiety score were negatively correlated with caregivers′ quality of life except for mental health dimension, physical pain dimension and the total score of physical health, mental health and the MOS item Short from Health Survey (SF-36) (r=-0.471--0.117, P<0.05), and depression score were negatively correlated with caregivers′ quality of life except for physical pain dimension and the total score of physical health, mental health and SF-36(r=-0.519--0.220, P<0.05). Multiple regression analysis indicated that caregivers educational level, provided support, social support , relationship with patients, self-evaluation of health, live together time with patient, patient care burden, caregivers gender, depression score, patient age, diabetic foot Wagner grade were the influence factors of the caregiver′ quality of life. Conclusions Diabetic foot patients′ negative emotion has an important impact on caregiver′quality of life, we can improve the quality of life of patients and their caregivers by reducing the negative mood of patients with diabetic foot.

Objective:To explore the influence of RNAi mediated Slug silencing on the growth and metastasis of colon cancer in nude mice.Methods: HCT116 colon cancer cells use for 24 five-week-old nude mice implanted subcutaneously , established colon cancer xenograft model in nude mice ,all divided into blank control group ,negative control group and the experimental group ,each group had eight nude mices.All group were injected with saline , negative plasmid and lentivirus vectors respectively.Tumor growth was observed and draw tumor curved growth ,changes in tumor growth and lymph node metastasis between the groups were observed ,Slug gene and protein expression were detected by immunohistochemistry ,qRT-PCR and Western blot analysis.Results: Slug gene shRNA intervention group compared with the control group and negative control group ,tumor grew slower ,tumor mass was significantly reduced (3.08±0.31 vs 7.37±1.18,7.46±1.16,P＜0.01),experimental group of lymph node-positive rate was 36.3%( 4 /11 ) ,compared to the negative control group 77.8% ( 14/18 ) and the control group was 68.4% ( 13/19 ) ( P＜0.01 ).Conclusion: Targeted Slug RNA interference can significantly inhibit the growth of colon cancer in nude mice ,lymph node metastasis and the expression of the gene protein in cancer tissue ,Slug may be a potential molecular target for colon cancer gene therapy.

This study developed a novel approach of targeting malignant glioma with pMAGE-A1(278-286)-specific cytotoxic T lymphocytes (CTLs) induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen (HLA)-A2-restricted pMAGE-A1(278-286) peptide-pulsed dentritic cells. Cytotoxic assays were performed by the colorimetric CytoTox 96 assay to analyze cytotoxic activity of the induced CTLs against various target cells. The induced CTLs showed approximately 45% specific lysis against T2pMAGE-A1(278-286) (pMAGE-A1(278-286) peptide pulsed T2 cells) and U251 (HLA-A2(+), MAGE-A1(+)) at an effector:target ratio of 40:1, and approximately 5% cytolysis against T2pHIV, A172 (HLA-A2(-), MAGE-A1(+)), K562 and T2 cells without being pulsed with peptide at any effector:target ratio. The specific killing activity of the induced CTLs against T2pMAGE-A1(278-286) and U251 was much more obvious than in any other control group (P<0.05). The cytotoxic activity against the T2pMAGE-A1(278-286) and U251 was significantly eliminated by anti-HLA class I mAb W6/32. These results suggest that pMAGE-A1(278-286) epitope may serve as a surrogate tumor antigen target of specific immunotherapy for treating HLA-A2 patients with malignant glioma.

Modern pharmacological studies have shown that Cistanche deserticola (C. deserticola) has a protective effect on the liver, but its active fraction and mechanism are not clear. In order to identify the effective fraction of C. deserticola Y. C. Ma, an acute alcoholic liver injury model in mice was established with 56-proof Erguotou and different fractional extracts of C. deserticola Y. C. Ma (total glycosides, polysaccharides, and oligosaccharides) were administered. After 14 days of oral administration, liver pathology and lipid deposition were measured and the expression of nuclear factor E2-related factor (Nrf-2), kelch-like ECH-associated protein-1 (Keap-1), and plasmalemma vesicle-associated protein-1 (PV1) were measured by immunofluorescence. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), endotoxin (ET), diamine oxidase (DAO), and D-lactic acid (D-LA) in serum, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and malondialdehyde (MDA) in liver were measured by ELISA. All animal experiments were carried out with approval of the Experimental Animal Welfare Ethics Committee of the Peking University Health Science Center. The results show that the total glycosides of C. deserticola Y. C. Ma (400 mg·kg^-1) could decrease liver pathology, decrease serum endotoxin, diamine oxidase, and D-lactic acid, and reduce hepatic lipid deposition. Total glycosides also promoted Nrf-2 transfer into the nucleus and decreased the expression of Keap-1 and PV1. In summary, the total glycosides of C. deserticola Y. C. Ma had a protective effect in acute alcoholic liver injury and the mechanism may be related to the activation of the Nrf-2/Keap-1 pathway, improvement of intestinal wall integrity, and inhibition of the transport of harmful substances into the liver.

Objective To assess community health service ability of general practitioners (GPs) with the Standardized General Practitioner Training(SGPT) in Shanghai.Methods One hundred and four GPs receiving SGPT and matched 102 GPs without SGPT were enrolled in this retrospective cohort study from September to November in 2014.Community health service ability,including abilities in basic medical service and public health service were assessed and compared between two groups.Written examination was applied in the public health services ability assessment.Objective structured clinical examination (OSCE) was applied for evaluation of basic medical service abilities.Results The scores of written examination in GPs with SGPT were higher than those without SGPT[(66 ±4) vs.(62 ±7),P ＜0.01].The OSCE scores in GP with SGPT were higher than those without SGPT [(494 ± 78) vs.(448 ± 80),P ＜ 0.01].GPs with SGPT also abtained the higher scores in consultation competence and clinical skills assessment [(245 ± 44) vs.(225 ±40),P＜0.01;(252 ±59) vs.(225 ±61),P ＜0.01].In OSCE,GPs with SGPT got higher score than those without SGPT in 5 stations (medical history collection,doctor-patient communication,basic operation,ECG interpretation,and CPR) (all P ＜ 0.01);however,no significant differences were observed in 3 stations (physical examination,case analysis and X-ray interpretation) between two groups (all P ＞ 0.05).Conclusions GPs with SGPT have better basic medical service and public health service ability.But some aspects of basic medical service ability need to be strengthened in the training and practice.

Objective To evaluate the clinical capability of young general practitioners (GPs) in communities in Shanghai.Methods A cross-sectional survey on clinical ability of young GPs was adopted from October to December 2014.Total 211 GPs aged (31 ± 2) years (25-35) from 17 districts of Shanghai participated in the study.Written examination and objective structured clinical examination (OSCE) was applied in the evaluation.Results All 211 GPs (63 male and 148 females) completed the assessment.The overall score of the assessment was (602 ± 83) and the pass rate was 62.6％ (132/211).In written examination the average score was (64 ±7) and the pass rate was 72.1％ (152/211).In OSCE,the highest score (80 ± 15) and pass rate (92.9％,196/211) was in CPR skill,followed by communication skill [average score:(76 ± 15) and pass rate:85.8％ (181/211)].The lowest average score was physical examination (47 ± 15) with a pass rate of 26.1％ (55/211),followed by electrocardiogram reading [average score:(84 ±31) and pass rate:39.8％ (84/211)].In basic operation station,the lowest score was using funduscope and gynecologial examination (29.8％ and 45.4％,respectively).Conclusion Young GPs in Shanghai communities basically master clinical skills,but also have some deficiencies,the training in certain skills need to be strengthened.

Objective To investigate the effects of ozone (O3) on Wnt/β-catenin signaling pathway in the articular cartilage of rats with osteoarthritis (OA).Methods Eighteen male SPF Wistar rats,aged 3 months,weighing 200-250 g,were randomly divided into 3 groups (n =6 each) using a random number table:control group (group C),group OA,and O3 group (group O).OA was induced by injection of monosodium iodoacetate 3 mg (50 μl) into the right knee joint cavity.On 7th day after the model was established successfully,25 μg/ml O3 1 ml were injected into the knee joint cavity,once a week for 3 consecutive weeks in group O.Behavioral changes were observed after establishment of the model.At 1 day before establishment of the model,and 1,4,7,14,21 and 28 days after establishment of the model,the mechanical paw withdrawal threshold (MWT) was measured.At 28 days after establishment of the model,the total knee joint was removed and stained with haematoxylin and eosin for examination of the pathological changes of the cartilage (under light microscope) and for determination of the expression of β-catenin and matrix metalloproteinase-13 (MMP-13) in the cartilage (by immunohistochemistry).Results The signs of OA such as hind-limb motor dysfunction,knee joint swelling,or decreased joint motion,and signs of hyperalgesia such as lickings were observed after establishment of the model in rats.Compared with group C,the MWT was significantly decreased at each time point after establishment of the model,and the ex pression of β-catenin and MMP-13 in the cartilage was significantly up-regulated in the other two groups(P＜0.05).Compared with group OA,the MWT was significantly increased at 7-28 days after establishment of the model,and the expression of β-catenin and MMP-13 in the cartilage was significantly down-regulated in group O (P＜0.05).The pathological changes of the cartilage were significantly reduced in group O as compared with group OA.Conclusion The mechanism by which O3 mitigates OA is probably related to inhibition of Wnt/β-catenin signaling pathway activation in the articular cartilage in rats.

BACKGROUND:Cytoskeleton plays an important role in the transduction of mechanical signal, and intermittent tensile stress can promote osteogenic differentiation. However, there is no relevant study about the change of cytoskeleton in osteoporosis rat bone marrow mesenchymal stem cells under intermittent tensile stress. OBJECTIVE:To investigate the effects of intermittent tensile stress on the cytoskeleton of osteoporosis rat bone marrow mesenchymal stem cells during osteogenic differentiation. METHODS:Bone marrow mesenchymal stem cells were obtained from osteoporosis rats and cultured in vitro. The 5%, 10%and 15%tensile stress were strained on the bone marrow mesenchymal stem cells through FX-4000T Flexcell. No stress was in the control group. Osteogenic differentiation of bone marrow mesenchymal stem cells was observed through alkaline phosphatase staining, while the change of cytoskeleton was observed by confocal laser scanning microscopy with figures col ected for analysis by Image-ProPlus 6.0 software. The area of cells, ratio of length to width and integrated fluorescence intensity of cytoskeleton protein F-actin were measured. RESULTS AND CONCLUSION:Under tensile stress, bone marrow mesenchymal stem cells from osteoporosis rats arranged in the direction vertical to mechanical stimulation. cells under different tensile stress differentiated towards osteoblasts. The result of alkaline phosphatase staining showed the most significant difference in 10%group, and quite an amount of cells lining lost succession in the 15%group. Under stress, the F-actin filaments were rearranged in paral el accordingly, which showed a reconstruction of cytoskeleton. Imaging analysis indicated that the area of bone marrow mesenchymal stem cells was decreased in 10%and 15%groups (P<0.05) with the increased ratio of length to width (P<0.05), and expression of F-actin increased in5%, 10%, 15%groups (P<0.05) after tensile stress. Under mechanical stimulation, the cytoskeleton of bone marrow mesenchymal stem cells from osteoporosis rats is shown to have corresponding alterations during osteogenic differentiation.