Objective To investigate the protective effects of different ATP-sensitive potassium (K_ ATP) channel openers. Pinacidil and Diazoxide, on myocardium injury in isolated rabbit hearts caused by ischemia/reperfusion and the possible changes after application of ATP-sensitve potassium channel blocker, 5-HD. Methods Observation was made on rabbit hearts perfused with a Langendorff apparatus. Forty rabbits were randomly divided into five respective groups: 1. Pinacidil (Group P), 2. Diazoxide (Group D), 3. 5-HD+Pinacidil (Group HP), 4. 5-HD+Diazoxide (Group HD) and 5. the control (Group C). All groups were subjected to 40 minutes of occlusion, followed by 20 minutes of reperfusion as cardiac arrested by cold cardioplegia. Any one of Pinacidil, Diazoxide , Pinacidil or Diazoxide mixed with 5-HD was infused 15 minutes before cardioplegic heart rested in the experimental group. Cardiac tissue ultrastructure, hemodynamics variables, levels of adenine nucleotides and lipid peroxide of the myocardium were measured. Results (1) In Group P, Group D and Group HP, the recovery of myocardial contractility and heart rate after reperfusion was faster but MDA level and the amount of albumen released were lower than Group C. Moreover levels of myocardial adenosine triphosphate (ATP) were much higher compared with Group C. (2) In Group HP the recovery of myocardial contractility and heart rate was not as good as Group P, but its myocardium MDA level and amount of albumen released was higher than group P. Conclusion ATP-sensitive Potassium Channel openes may enhance myocardial protection against ischemia /reperfusion injury. The above effect of myocardial protection was only partially closed down by ATP-sensitive potassium channel blocker: 5-HD. The mitoK_ ATP channels on myocardium may be an important pathway of protection during ischemia/reperfusion process.

Objective: To investigate perioperative changes of gastrointramucosal pH (pHi) in major operative patients. Method: Six patients underwent major abdominopelvie operation with radial artery and internal jugular vein catheters, and nasogastric tonometries in place. The phi and hemodynamic, oxygen, and metabolic variables were measured simutaneously before anesthesia, 30 minutes after anesthetic induction, during intraoperation, at the end of operation, and the first 24 hours postoperatively. Result: There was significant decrease in mean pHi(P

Objective To sudy the changes in mTOR signaling pathway in childhood aplastic anemia(AA) by detecting the expression levels of the molecules of mTOR signaling pathway in T cells,and to explore immunologoical pathogenesis of AA in children from T cell intracellular signal transduction pathway.Methods Peripheral blood samples were collected from 16 newly diagnosed severe AA(SAA) patients and 8 patiens treated with effective immunosuppressive therapy,and the findings were compared with those of 17 healthy children (normal controls) and CEM cells (positive controls).The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K in CD3 + T cells in peripheral blood were detected by flow cytometry(FCM).Results 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,pp70S6K of the newly diagnosed SAA group were higher than those of the normal control group (P ＜ 0.05),but were lower than the postive control group (CEM group) (P ＜ 0.05).The mean fluorescence intensity (MFI) of p-Akt of three groups was 8.04 ± 3.78,2.59 ± 1.01 and 20.23 ± 8.98 respectively ;p-TSC2 was 49.73 ± 19.49,16.10 ± 8.04 and 101.05 ± 29.78 respectively ; p-mTOR was 13.90 ± 9.32,2.92 ± 1.09 and 34.3 ± 19.03 ;p-4EBP1 was 142.69 ± 53.36,26.91 ± 13.70,256.01 ± 53.79 ; p-p70S6 K were 17.67 ± 10.48,3.69 ± 2.22,31.73 ± 12.85 respectively.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K of the effective treatment groups were lower than those of the newly diagnosed SAA group (P ＜ 0.05) ; the expressions of p-Akt,p-TSC2,p-mTORC1,p-p70S6K were similar to those of the normal control group(P ＞ 0.05),but the expressions of p-4EBP1 were higher(P ＜ 0.05).The MFI was followed by 3.28 ± 1.27,16.50 ± 10.91,3.54 ± 1.66,74.89 ± 49.69 and 4.21 ± 1.69.Conclusions 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K were increased in the newly diagnosed SAA patients,the mTOR signaling pathway was activated in SAA patients.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p4EBP1,p-p70S6K were lower than those of the newly diagnosed SAA patients.The degree of activation of mTOR signaling pathway was associated with disease status.The signaling pathways may be involved in the T cells of AA of the immune abnormalities.

Objective Multidrug resistance-associated protein 1 (MRP1) has been reported with a close correlation with tumor multi-drug resistance. Real-time quantitative PCR (QRT-PCR) was performed to detect the MRP1 gene expression in childhood acute lymphoblastic leukemia (ALL) and its clinical signiifcance was analyzed. Methods Sixty-seven denovo ALL patients and 10 healthier children as bone marrow donor were studied. The chemotherapy was given according to CCLG-2008 protocol. SPSS software was employed to analyze the data and p-value below 0.05 was regarded as statistic signiifcance. Results MRP1 expression level showed a close correlation with ALL risk, the median of MRP1 expression was 4.28 (2.75~6.12), 5.62 (4.99~8.60) and 7.56 (3.66~11.13) for standard-risk group (SR), intermediate-risk group (IR) and high-risk group (HR), respectively. MRP1 mRNA expression in T-ALL group was 7.71 (6.49~14.35), which is higher than that of B-ALL (5.18(3.89~8.46)) (P<0.01). The rate of leukemia cells’ sensitivity to prednisone on 7th day was 70.6%in high expression group (n=34), which was signiifcantly lower than that in low expression group (n=33, 90.9%, P=0.035). The complete remission rateon 33th day was 64.7%in high expression group, and 87.9%in low expression group, which showed a signiifcant difference between them (P=0.026). Conclusions In children ALL, the expression of MRP1 is closely related with immunophenotyping, treatment response, hazard level and disease relapse.

OBJECTIVETo study the direct effect of hawthorn leaf procyanidins on cardiomyocytes subjected to ischemia-reperfusion injury and elucidate their therapeutic mechanism on ischemic heart diseases.

METHODCultured cardiomyocytes of neonatal rats were subjected to anoxia-reoxia injury which simulated the ischemia-reperfusion injury in vivo, and hawthorn leaf procyanidins were applied. The therapeutic effect was valued by LDH leakage and MTT test. For a further mechanism study, contents of MDA and activities of SOD in cardiomyocytes were measured.

RESULTHawthorn leaf procyanidins in 24-60 mg x L(-1) significantly and dose-dependently inhibited LDH leakage (compared with the model group, all P<0.01 ) and cell viability decrease (compared with model group, 24-48 mg x L(-1) groups all P<0.05; 60 mg x L(-1) group, P<0.01) in cardiomyocytes induced by anoxia-reoxia injury. Furthermore, hawthorn leaf procyanidins in 24-60 mg x L(-1) significantly inhibited the increase of MDA content (compared with the model group, all P<0.01) and the decreased of SOD activity (compared with the model group, 24 mg x L(-1) group, P<0.05; other groups all P<0.01) in cardiomyocytes undergoing anoxia-reoxia injury.

CONCLUSIONHawthorn leaf procyanidins have a significant therapeutic effect on the simulated ischemia-reperfusion injury of cultured neonatal rat cardiomyocytes, which may relate to their anti-oxidation effects. And the direct protective effect of hawthorn leaf procyanidins on cardiomyocytes subjected to ischemia-reperfusion injury may be one of the key mechanisms of its therapeutic effect on ischemic heart diseases.

Animals ; Biflavonoids ; pharmacology ; Catechin ; pharmacology ; Cell Survival ; drug effects ; Crataegus ; chemistry ; Female ; Hypoxia ; metabolism ; pathology ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Plant Leaves ; chemistry ; Proanthocyanidins ; pharmacology ; Rats ; Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Superoxide Dismutase ; metabolism

Retroviral vectors are wildly used as vehicles for gene transfer into hematopoietic cells based on its potency for efficient gene delivery and integration of transgene in host genome. The development of better transduction protocols depends on gene markers that allow a rapid detection and effective selection of genetically transduced cells. In this study, the enhanced green fluorescent protein (EGFP), a gene that is optimized for detection and expression in mammalian cells, was firstly amplified and cloned by high-fidelity PCR. The vector LGSN carrying EGFP gene was then constructed and the retroviral producer cell lines that yield high titers of LGSN vector in supernatants were developed by liposome-mediated transfection in combination with cross infection. Both GP + envAm12 murine fibroblasts and K562 leukemic cells transduced with EGFP virus demonstrated a stable green fluorescence signal readily detectable by flow cytometry or fluorescence microscopy in up to 97% and 86% of examined cells, respectively. The integration of LGSN provirus in transduced cells was confirmed by PCR analysis. These results indicate EGFP is a suitable reporter molecule for gene transfer and expression in hematopoietic cells. Therefore, the bright and long-term expression of EGFP in living cells will advance the study of gene therapy in vitro and in vivo, particularly for human applications.

Objective The aim of this study was to investigate the aquaporin-4(AQP4) expression in the ischemic penumbra tissues.Methods Thirty-six Wistar rats were divided into 7 groups randomly, including control group(n=6) and occluded groups(n=30). The occluded groups were studied after the right middle cerebral artery of the rats unilaterally occluded(MCAO) at an interval of 15 min, 30 min, 1 h, 3 h, 6 h and 24 h, respectively(n=5 for each group). The operation process of the control group was the same as the occluded group except occluded MCAO. Then all rats were imaged with T_1WI, T_2WI and diffusion weighted-imaging(DWI). The brain tissue, according to the method by LIU Meili reported, was regarded as the area of the graphic penumbra. The relative apparent diffusion coefficient of the graphic-penumbra (rADC_1) and the center infarction(rADC_2)(ratios between the values of the occluded side and the opposite side) were calculated. The animals were sacrificed and perfused with the mixture solution consisting of TTC at different time intervals. The graphic-penumbra of the biggest layer of the ischemic cerebral tissue which corresponded to the DWI was examined with immunohistochemistry and RT-PCR. Meanwhile, histologic examination was performed at same site of the lesion. Results There were no significant changes on MRI, the relative apparent diffusion coefficient and the expression of the AQP4. The abnormal high intensity was found on DWI at 15 min after MCAO. T_2WI detected the lesion at 1 h after MCAO. The value of the rADC_1 decreased within 24 h after MCAO in ischemic penumbra, especially, it descended quickly within 1 h after MCAO, from(70.4?6.9)% at 15 min to(53.5?10.9)% at 1 h. Whereas, in the infarct tissue, the changes of the rADC_2 had a rule of decrease from(71.5?6.6)% at 15 min to(45.7?10.5)% at 3 h at first time, and then follow an increasing up to(78.7?11.5)% at 24 h after MCAO. The expression of AQP4 increased gradually within 24 h after MCAO, from 0.42?0.05 at 15 min to 1.18?0.12 at 24 h, it showed negative relationship with the rADC_1 in the ischemic penumbra (r= -0.966,P

OBJECTIVETo revalidate the conversion factor（CF）for the conversion of BCR-ABL （P210）transcript levels to the international scale（BCR- ABLIS）in chronic myeloid leukemia（CML） which validated before.

METHODSPeking University People's Hospital（PKUPH）prepared the exchange samples for revalidation of CFs of 15 laboratories which validated nine or eighteen months ago. The fresh BCR-ABL（P210）（+）bone morrow or peripheral blood nucleated cells were diluted with BCR-ABL （P210）（-）cells to achieve different BCR- ABL levels, totally 16 sets and 24 samples per set were prepared. TRIzol reagent was added in each tube. Each laboratory tested BCR-ABL transcript levels of one set of samples. Agreement between BCR-ABLIS of each laboratory and PKUPH was assessed by the Bland- Altman method. For laboratories which did not meet the criteria of revalidation, linear regression equation was derived after the samples with maximum BCR-ABL deviation were removed until R²>0.98, then new CF was calculated.

RESULTS10 laboratories met the revalidation criteria with both bias within ±1.4 fold and 95% limits of agreement within ±6 folds, and their CFs still could be used for accurately conversion of BCR-ABLIS. New CFs were recalculated as of 1.8-6.3 folds of their previous CFs in 5 laboratories not met the criteria.

CONCLUSIONRevalidation of CF by sample exchange among laboratories was necessary for accurate and continuous application of BCR-ABLIS, which not only tested the validity of CF acquired before but also calculated new available CFs for those with invalid CFs.

Bone Marrow Cells ; Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.