Objective To explore the efficacy of calmodulin antagonist berbamine(BBM) in down-regulating survivin mRNA. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study. The cells were cultured with different concentration of BBM for 72 hours. The mRNA expression level of survivin gene in both MCF and MCF7/ADR cells was detected by semi-quantitative RT-PCR. Results After treating MCF7 and MCF7/ADR cells by 20?mol/L BBM, the mRNA expression level of survivin gene decreased from 0.43?0.02 to 0.21?0.04 in MCF7 cells, and from 0.57?0.05 to 0.45?0.04 in MCF/ADR cells(P

Objective To investigate clinical effect of mild hypothermia therapy assisted intracranial hematoma evacuation in treatment of cerebral hemorrhage.Methods One hundred and ten patients with cerebral hemorrhage were selected in Affiliated Hospital of North China University of Science and Technology from December 2011 to December 2013,and were randomly divided into two groups.Fifty-five patients treated intracranial hematoma evacuation as control group.Another 55 patients treated mild hypothermia therapy assisted intracranial hematoma evacuation as observation group.Treatment effect was compared between two groups.Results Serum S100β,neuron specific enolization (NSE) enzyme,tumor necrosis factor α (TNF-α),creactive protein(CRP),cognitive function score,daily life ability score,neurological function defect score before and after treatment in control group were (0.82±0.12) μg/L and (0.53±0.09) μg/L,(19.42±2.30) μg/L and (10.36±1.07) μg/L,(3.62±0.57) mg/L and (1.54±0.30) mg/L,(29.43±4.36) g/L and (10.25± 1.07) g/L,(13.42± 1.58) points and (25.03± 1.19) points,(21.45± 3.27) points and (37.92 ± 5.83)points,(13.27± 1.35) points and (4.84 ± 1.08) points,the differences were significant (t =8.471,11.834,17.026,22.539,12.230,10.619,25.531,P ＜ 0.05).Serum S100β,NSE,TNF-α,CRP,cognitive function score,daily life ability score,neurological function defect score before and after treatment in observation group were (0.84±0.13)μg/L and (0.41±0.10) μg/L,(19.48±1.76) μg/L and (8.75±0.84) μg/L,(3.64± ±0.61) mg/Land (1.17±0.29) mg/L,(29.58±3.62) g/L and (6.02±1.18) g/L,(13.29±1.34) points and (27.58± 1.27) points,(21.68±4.02) points and (48.26±7.14) points,(13.46± 1.21) points and (3.57±0.85) points,the differences were significant(t=13.498,16.739,25.728,41.836,13.769,15.857,36.352,P＜0.05).Compared with serum S100β,NSE,TNF-α,CRP,cognitive function score,daily life ability score,neurological function defect score before treatment,there were no difference between two groups (P ＞0.05).Serum S100β,NSE,TNF-α,CRP,neurological function defect score after treatment in observation group were lower than control group(t =5.926,4.839,6.162,10.054,6.714,P＜0.05).Cognitive function score,daily life ability score after treatment in observation group were higher than control group (t =4.008,5.973,P ＜0.05).Postoperative Glasgow prognosis classification in observation group (14 cases of grade Ⅰ,27 cases of grade Ⅱ,11 cases of grade Ⅲ,2 cases of grade Ⅳ,1 case of grade Ⅴ) was better than control group(8 cases of grade Ⅰ,12 cases of grade Ⅱ,23 cases of grade Ⅲ,7 cases of grade Ⅳ,5 cases of grade Ⅴ),the differences were significant between the two groups (Z=17.085,P =0.002).Total effective rate in observation group 94.5％ (52/55) was higher than control group 78.2％ (43/55),the differences were significant between the two groups (Z =6.253,P=0.012).Conclusion Mild hypothermia therapy assisted intracranial hematoma evacuation in treatment of cerebral hemorrhage,can significantly reduce inflammatory factor and S100βlevel,improve neurological function,has significant effect and good prognosis.It is worthy of clinical use.

OBJECTIVE:To evaluate the utilization of traditional Chinese medicine(TCM) in our hospital and to provide reference for TCM pharmacy and rational use of TCM in the clinic.METHODS:1 052 outpatient prescriptions of TCM in our hospital from May to Oct.in 2009 were analyzed statistically by Microsoft Excel program.RESULTS:Prescriptions of respiratory system diseases in our hospital were mainly TCM prescription.TCM in middle-aged and aged group was a universal phenomenon.The ingredients in every prescription were excessive with high dosage.CONCLUSION:Clinicians and pharmacists should attach great importance on the application of TCM prescriptions to ensure safety and effectiveness of drug use.

Wound secretion from 20 patients with gaseous gangrene was collected for Gram staining,bacterial culture and drug sensitivity tests.The results indicated that gaseous gangrene was caused by the co-infection of both aerobic and anaerobic bacteria.Gram-negative bacilli were slishtly more common than other aerobic bacteria in gageous gangrene wound,which was different from the findings of ordinary gaseous gangrene.

Objective To establish a method of competitive polymerase chain reaction combined with restrictive endonucleases to measure the methylation quantitatively in MDR1 promoter region.Methods One sense primer and two anti-sense primers were designed to amplify a fragment of MDR1 gene promoter region, which was located between -102 and +186 bp containing a methylation site. The internal reference DNA fragment, witch was less 30bp than target fragment was made by three times of polymerase chain reaction(PCR)using different anti-sense primer and then subcloned into the Pbluescriptsk+ plasmid. The genomic DNA digested by Hpa, a methylation-sensitive restrictive endonuclease and competitive internal reference DNA were competitively amplified for MDR1 promoter in the same tube by PCR. The PCR products were electrophoresed on agarose gel, stained by ethidium bromide and subjected to image analysis scanner. The amount of target fragment was calculated as following, the optical density ratio of target fragment to competitive internal reference fragment multiplied the amount of competitive internal reference DNA. The ratio of PCR products amplified from HpaⅡ digested DNA and undigested DNA was named the methylation rate.Results The genomic DNA serially diluted with optimal amount of competitive internal reference DNA were co-amplified for MDR1 promoter. The significant positive correlation between the ratio of two products and the amount of genomic DNA was demonstrated. The correlation coefficient was 0.992, P

Objective To express and purify recombinant human platelet glycoprotein Ⅵ extracellular domain and prepare the polyclonal antibody against it.Methods Human platelet glycoprotein Ⅵ extracellular domain fragment (123~268 residues) was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-3x.The recombinant plasmid was constructed and expressed in E.coli after induction with isopropyl-?-D-thiogalactopyranoside (IPTG).The fusion protein was identified by Western blot analysis after purification by affinity chromatography.Rabbits were immunized with the purified fusion protein, and the collected rabbit antiserum was evaluated by sandwich ELISA, Western blot and flow cytometry.Results The coding sequence of GPVI extracellular domain was successfully inserted into pGEX-3x.Sequencing result showed that the cloned gene was identical as reported.After induction, a Mr 42kd fusion protein was expressed and confirmed by Western blot, which was identical to that expected.The titers of the antisera were up to 1∶[KG-*2]128. Sandwich ELISA result demonstrated that the prepared antibody recognized GPVI in human platelet. Western blot and flow cytometry revealed that the prepared antibody reacted with GPVI of platelet lysate and the native GPVI on human platelet surface.Conclusions Using purified prokaryotic expressed the fusion protein as antigen, the specific antibody was elicited in the immunized animals. The prepared polyclonal antibodies react specially with GPVI on human platelet surface and can be used for further studies of GPVI.

Objective To explore the efficacy of calmodulin antagonist berbamine(BBM)on multidrug resistance(MDR)reversal and its mechanism. Methods Human breast cancer cell line MCF7 and its adriamycin-resistant counterpart MCF7/ADR were used in the study.The cells were cultured with ADR and different concentration of BBM. MTT assay was used to analyze the effect of BBM on cell growth inhibition.According to the MTT assay,the 50% inhibitory concentration(IC 50 ),the multiples of drug resistance and increased sensitivity of ADR were calculated.The concentration of intracellular ADR and expression level of P-glycoprotein(P-gp)were detected by flowcytometry(FCM).The mRNA expression level of mdr1 gene was detected by semi-quantitative reverse transcriptase polymerase chain reaction(RT-PCR)with ?-actin as internal reference. Results The IC 50 of ADR in MCF7 and MCF7/ADR cells were(0.98?0.06)?mol/L and(101.20?5.72)?mol/L,respectively.The resistant multiple of MCF/ADR cells to ADR was 103 folds higher than that of MCF7 cells.BBM increased the chemo-sensitivity of ADR in MCF7/ADR cells with dose-dependent relationship,i.e.when 5*!?mol/L ,10*!?mol/L and 20*!?mol/L BBM was added into the culture the chemo-sensitivity of ADR was increased to 2.76,5.88,and 28.26 folds(P

Objective To deepen the understanding of chronic eosinophilic leukemia (CEL).Methods The course of diagnosis and treatment in a case of FIP1L1/PDGFRα fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRα fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. Result A sixteen-year-old girl had severe anemia, fever, splenomegaly,thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRα was negative. An abnormal clone of T cells expressing CD3-,CD4-,CD8- was found in peripheral blood and pleural fluid, in which the cional T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. Conclusion The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRα(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD3-,CD4-,CD8- T-cell abnormality is related to the pathogenesis of CEL.

OBJECTIVETo detect the methylation pattern of the p15(INK4B) gene and to explore its significance in the pathogenesis of acute leukemia (AL) and leukemic transformation of myelodysplastic syndromes (MDS).

METHODSA total of 49 AL cases and 22 MDS cases were analyzed by methylation specific polymerase chain reaction (MSP) for methylation patterns in CpG islands of the p15(INK4B) gene.

RESULTSHypermethylation of the p15(INK4B) gene was found in 90% (26/29) of newly diagnosed AL, including 46% with complete methylation and 54% with partial methylation. All 3 evolved AL from MDS and 9 relapsed AL showed a methylated p15(INK4B) gene and the proportion of complete methylation was 67% and 56% respectively. Only 5 of 11 (45%) AL in remission, including 2 in complete remission (CR) and 3 in partial remission (PR), were partially methylated. The frequency of p15(INK4B) gene methylation in newly diagnosed or relapsed AL was significantly higher than that in AL in the remission stage (P = 0.002) p15(INK4B) gene methylation was found in 5 of 13 (38%) low-risk MDS (RA/RAS) patients and 80% of them showed only partial methylation. However, p15(INK4B) gene methylation was found in all 9 cases in the high-risk group (RAEB/RAEB-T), including complete methylation in 56%, significantly different from the low-risk MDS group (P = 0.002).

CONCLUSIONSHypermethylation of the p15(INK4B) gene occurs frequently in leukemia and high-risk MDS. It is possible that hypermethylation of this gene is related to the pathogenesis and development of AL and MDS. It may be used as a gene marker to detect minimal residual disease, relapse of AL and leukemic transformation in MDS.

Cell Cycle Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Myelodysplastic Syndromes ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Tumor Suppressor Proteins

A 14-week human fetal kidney was studied by the method of freeze-fracture, the results were as follows:1. All the nuclei of various cells at this stage of development possess distinct nuclear pores with the same diameter and they are distributed randomly on the nuclear membrane. The intramembraneous particles on the PF of both the inner and outer nuclear membranes are more numerous than that of the EF. The morphological features of the nuclear pores vary according to the plane of their fracture face; they appear as dimples on the EF and as valcano mouth on the PF. The interior of the nucleus usually contains homogeneously dispersed particles, but no such structures were seen in thin sections. In some nuclei a round vesicle was revealed.2. The cell membrane of various cells, at this stage, shows special structures to manifest their degree of differentiation. In the less differentiated epithelial cells of the renal tubules, the cell membrane is straight and the intramembraneous particles are randomly distributed, the intromembraneous particles on PF are more than on EF, comparetively well developed tight junctions are located at the latexal surface near the apical portion of the cell while in the differentiated proximal tubules, there are many microvilli on their luminal surface, but their lateral cell membrane is still straight without interdigitations. In some tubules small processes near the basal part may be seen, however, basal fold is still absent. The basal membrane of the renal tubules is very prominent. As to the epithelial cells of the viseral layer of the renal corpuscle, gap and tight junctions are revealed while they are columnar in shape, but as they differentiated into podocyte with primary and secondary processes, the structure of tight junctions become simple, less prominent and discontinuous.3. Cytoplasm: In the process of cell differentiation the amount of cytoplasm increases and the ratio of nuclear end cytoplasmic volume decreases. The orgauelles are less in the undifferentiated cells, so the structure of the cytoplasm of them is much simpler than that of the more differentiated ones. The compartmentation phenomenon of the cytoplasm of differentiated cells become distince. In the freeze-fracture micrographs the membraneous structures, such as mitochondria, Golgi complex and secretion granules etc. are more stereoscopic than those in thin sections, but the opportunity to reveal the fine structures of various cells is minimized due to only one fracture face for each specimen and what structure will be fractured is beyond the control of the operator.