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.

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.

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

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.

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.

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 Routine perioperative intravenous antimicrobial agents,was administered as surgical prophylaxis.However,whether balanced ultrafiltration during extracorporeal circulation can remove antimicrobial agent remains unclear.The concentrations of antimicrobial agent in plasma and ultrafiltrate samples were measured in this pseudo-extracorporeal circulation model.Methods Extracorporeal circulation consisted of cardiotomy reservoir (Ningbo Fly Medical Healthcare CO.,LTD.Ningbo,China),D902 Lilliput 2 membrane oxygenator (Sorin Group Asia Pte Ltd,Beijing,China) and Capiox (R) AF02 pediatric arterial line filter (Terumo Corporation,Beijing,China).HEMOCONCENTRATOR BC 20 plus (MAQUET Cardiopulmonary AG,Hirrlingen,Germany) was placed between arterial purge line and oxygenator venous reservoir.Fresh donor human whole blood was added into the circuit and mixed with Ringer's solution to obtain a final hematocrit of 24％-28 ％.After 30 minutes of extracorporeal circulation,zero-balanced ultrafiltration was initiated and arterial line pressure was maintained at approximately 100 mm Hg(1 mm Hg =0.133 kPa) with Hoffman clamp.The rate of ultrafiltration (12 ml/min) was controlled by ultrafiltrate outlet pressure.Identical volume of plasmaslyte A was dripped into the circuit to maintain stable hematocrit during 45 minutes of experiment.Plasma and ultrafiltrate samples were drawn every 5 minutes and concentrations of antimicrobial agent (including Cefmetasole and cefotiam) were measured with high performance liquid chromatography.Results All these two antimicrobial agents were detected in ultrafiltrate,demonstrating hemoconcentration may remove antimicrobial agent.The concentration of plasma antimicrobial agent decreased lineally with the increase of ultrafiltrate volume.At end of balanced ultrafiltration,the concentration of plasma cefotiam was (104.96 ± 44.36) μg/ml,which is about (44.38 ± 7.42) ％ of the initial concentration (238.95 ± 101.12) μg/ml; the concentration of plasma cefmetazole decreased linearly to (25.76 ± 14.78) μg/ml,which is about (49.69 ± 10.49) ％ of the initial concentration (51.49 ± 28.03) μg/ml.The total amount of cefotiam in ultrafiltrate is (27.16 ± 12.17)％ of the total dose administered,whereas cefmetasole in ultrafiltrate is (7.74 ±4.17)％.Conclusion Balanced ultrafiltration may remove antimicrobial agent from serum and has significant influence on plasma concentration of antimicrobial agent.The strategy of surgical prophylaxis should consider this unique technique during extracorporeal circulation.

Liver fibrosis is a common pathological process for chronic liver injury caused by multiple etiological factors and an inevitable phase leading to liver cirrhosis. According to the previous studies, hesperidin (HDN) shows a very good protective effect on CCl4-induced chemical hepatic fibrosis in rats. In this experiment, based on the findings of the previous studies, a platelet-derived growth factor (PDGF)-induced HSC-T6 model was established to observe the inhibitory effect of HDN on HSC-T6 proliferation. The ELISA method was adopted to detect the content of collagen I in HSC-T6 supernatant. Transforming growth factor (TGF)-beta1, Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) mRNA expressions were measured by RT-PCR; TGF-beta1 and CT-GF protein expressions in HSC-T6 were determined by Western blot, in order to study HDN's effect on TGF-beta1 signaling pathway in HSC and its potential action mechanism. The results demonstrated that HDN could notably improve HSC-T6 proliferation, Collagen I growth and TGF-beta1, Smad2, Smad3 and CTGF mRNA.expressions. After being intervened with HDN, it could notably inhibit HSC-T6 proliferation and Collagen I growth, reduce TGF-beta1, Smad2, Smad3 and CTGF mRNA and TGF-beta1, CTGF protein expressions and increase Smad7 mRNA expression. HDN's antihepatic fibrosis effect may be related to the inhibition of HSC proliferation and activation by modulating TGF-beta/Smad signaling pathway.
Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Connective Tissue Growth Factor ; physiology ; Hesperidin ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Rats ; Signal Transduction ; drug effects ; Smad Proteins ; physiology ; Transforming Growth Factor beta1 ; physiology

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.

Objective The purpose of this study is to construct eukaryotic gene vector of herpes simplex virus type 1 thymidine kinase(HSV1-tk)and to observe the expression of HSV1-tk in lung adenocarcinoma AGZY cell line.Methods The full length HSV1-tk gene was amplified by PCR from plasmid pHSV 106 and was inserted into pMD18-T.The recombinant plasmid was recombined with eukaryotic vector plRES 2-EGFP u-sing gene recombinant technique .HSV1 -tk was transfected into adenocarcinoma AGZY cell line with Lipo-fectamineTM 2 000.Fluorescence microscopy was used to detect the transfection and expression of HSV 1-tk.RT-PCR was used to detect the mRNA levels of HSV 1-tk.The cell proliferation was measured by MTT assay .Re-sults A length of 1 130 bp gene sequence was obtained by PCR .The expressions of HSV 1-tk at mRNA and protein levels were displayed by RT -PCR and Western blot .MTT analysis showed that there were no significant changes cell survival on after transfection .Conclusion The eukaryotic expression vector of HSV 1 -tk report gene is successfully constructed and HSV 1-tk is effectively expressed in transfected AGZY cells .