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Objective@#To investigate whether fusion protein SD-HA could regulate its downstream signaling molecule activity by competing with the phospho-BCR-ABL Y177 site, and its mechanisms to inhibit proliferation and induce apoptosis of K562 cells.@*Methods@#Co-immunoprecipitation interaction technology analysis of fusion protein SD-HA functioned by potently binding to the phospho-BCR-ABL Y177 site, Ras, MAPK and Akt activities were observed in the Ad5F35-SD-HA-treated cells. Western blot analyses of SD-HA fusion protein on cell membrane receptor pathway to death cascade caspase-8, caspase-3 and PRAP were performed.@*Results@#Exploration into the underlying mechanisms revealed that Ad5F35-SD-HA infection functioned by binding to the phospho-BCR-ABL Y177 site, which lead to a complex with Grb2. competitively disrupted the Grb2 SH2-phospho-BCR-ABL Y177 formation. The fusion protein SD-HA could reduce the activation of Ras and phosphorylation of MAPK (p-MAPK) and the expression level of p-ELK, inhibition of Ras-MAPK signaling pathway; SD-HA fusion protein could reduce p-Akt and Akt substrate p-GSK with inhibition of PI3K-Akt signaling pathway, thereby inhibiting the proliferation of K562 cells. Caspases-8-induced apoptosis signal could be activated by DED protein binding to DED domain of precursor caspases-8.@*Conclusions@#The strategy of fusion protein SD-HA inhibiting-Y177 BCR-ABL and Grb2 binding could be used as a novel entry point for the treatment of chronic myeloid leukemia.
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Biofilm formation is associated with invasive fungal infection, which is considered to be an important virulence factor.Candida albicans has become one of the important bacterial flora in hospital infection,and it is the most common clinical fungi strain, mainly isolated from respiratory tract.The formation of biofilm can promote the adhesion,proliferation and proliferation of Candida albicans, resulting in a significant increase in the minimum inhibitory concentration and drug resistance.This article reviews the current research advances of the mechanism of Candida albicans biofilm formation,structural characteristics and its prevention and treatment,to provide a reference for the development of clinical prevention and control strategies of Candida albicans biofilm formation.
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Objective To construct BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and investigate its effects on apoptosis of K562/G01 cells.Methods SH3-T79Y mutant was amplified by overlapping PCR with pMig210 as template and cloned into recombinant adenovirus vectors .After identifying , packaging and amplifying , the recombinant adenovirus vectors containing SH 3-T79Y mutant was collected .Recombinant adenovirus vectors were transferred into K562/G01 cells.Then transfection efficiency was determinated , changes of cell morphology were observed by Wright 's staining , cell apoptosis was evaluated by flow cytometry , BCR-ABL and CrkL phospho-rylation was detected by Western blot .Results The vectors were successfully constructed .Transfection efficiency was more than 80%after transferring into K562/G01 cells for 72 h;there was obvious apoptosis phenomenon , cell apoptosis significantly increased to 32.46% compared with the control groups ( P<0.05 ) , BCR-ABL and CrkL phosphorylation significantly decreased and so did the expression of BCR-ABL( P<0.05 ) .Conclusions Success-fully constructed the SH 3-T79 Y mutant recombinant adenovirus vectors and it may promote the apoptosis of K 562/G01 cells by inhibiting BCR-ABL and CrkL phosphorylation .
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Guided by the demands of society,we propose a new professional training objectives of medical laboratory technology,which is to cultivate comprehensive talents covering the entire industrial chain of medical laboratory technology with profound foundation,broad caliber,high quality and outstanding ability.According to the training objective,we build up 4321 talents training system and try to carry out preliminary practice and exploration on talent cultivation model from the following aspects,such as the construction of curriculum system,the reform of the teaching contents and methods,training of students' professional skills and entrepreneurial innovation spirit.
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<p><b>OBJECTIVE</b>To investigate the effect of indomethacin on the proliferation and Wnt/β-catenin pathway in K562 cells.</p><p><b>METHODS</b>The cell growth of K562 cells treated with different concentrations of indomethacin was assessed with MTT assay, and the colony-forming ability of the cells was evaluated by colony-forming assay. The mRNA expressions of BCR/ABL and β-catenin were detected by RT-PCR, and the protein expressions of pBCR/ABL, total BCR/ABL, β-catenin, pGSK-3β and c-myc were analyzed by Western blotting.</p><p><b>RESULTS</b>Indomethacin significantly suppressed the growth and colony-forming ability of K562 cells in a dose-dependent manner. Indomethacin treatment dose-dependently decreased the protein level of pBCR/ABL and total BCR/ABL without affecting bcr-abl mRNA expressions. Compared with the control groups, indomethacin-treated cells showed obviously decreased mRNA and protein expressions of β-catenin and decreased protein expressions of pGSK-3β and c-myc.</p><p><b>CONCLUSION</b>Indomethacin inhibits the proliferation of K562 cells by suppressing the activity of bcr-abl-Wnt/β-catenin pathway.</p>
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Humans , Cell Cycle , Cell Proliferation , Fusion Proteins, bcr-abl , Metabolism , Indomethacin , Pharmacology , K562 Cells , RNA, Messenger , Wnt Signaling Pathway , beta Catenin , MetabolismABSTRACT
Objective To explore the relationship between ERG4 gene overexpression and azole resistance in clinical Candida albicans strains. Methods The National Committee for Clinical Laboratory Standards (NCCLS)M27-A2 broth microdilution method was conducted to evaluate antifungal susceptibility of 34 clinical Candida albicans isolates in vitro. Total RNA was extracted from these Candida albicans strains and transcribed into cDNA. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of ERG4 gene. Statistical analysis was carried out by a two-sample t-test. Results The expression level of ERG4 mRNA was significantly higher in fluconazole-resistant than in -sensitive Candida albicans strains (4.20 ± 2.56 vs. 1.72 ± 1.33, t = 3.99, P < 0.05), higher in itraconazole-resistant than in -sensitive Candida albicans strains (3.60 ± 2.47 vs. 1.66 ± 1.61, t = 3.71, P < 0.05), and higher in voriconazole-resistant than in -sensitive Candida albicans strains (3.99 ± 2.72 vs. 2.07 ± 1.58, t = 2.91, P <0.05). Further more, increased ERG4 mRNA expression was also observed in isolates cross-resistant to all the three azole antifungal agents compared with those susceptible to all of them (4.49 ± 2.73 vs. 1.69 ± 1.82, t = 3.81, P < 0.05). Conclusions The overexpression of ERG4 gene may be associated with cross resistance to fluconazole, itraconazole and voriconazole in clinical Candida albicans strains, but its exact role is expected to be investigated through downregulation of the ERG4 gene.
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Objective To investigate the potential of chronic myeloid leukemia ( CML) cell line KCL22 in indu-cing leukemia in NOD-SCID mice for setting up a basis for constructing a CML mouse transplantation tumor model. Methods 2 ×107 KCL22 cells in logarithmic growth phase were injected via the tail vein into experimental NOD-SCID mice whereas PBS was injected to the mice of control group.General condition of the mice of both groups was observed.Wright staining was used to observe the changes of blood and bone marrow smears.PCR was conducted to detect the transcription level of BCR-ABL, and histology with HE staining was used to evaluate the tumor cell invasion in the liver and spleen. Results Four weeks after the injection of KCL22 cells, the mice in experimental group showed physical signs of decreased reactivity, depression, swollen hindlimb muscles and petechia on the hindlimb femur.Peripheral white blood cells ( WBC) began to increase after 5 weeks, with a significantly increased quantity compared with the control group (P90 days) (P<0.05).Conclusions A NOD-SCID mouse model of CML transplantation tumor is successfully established with leukemia KCL22 cells.
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Objective The aim of this study was to investigate the diagnostic impact of polymerase chain reaction (PCR) assays for fungal pathogens in fluid samples,and to evaluate the feasibility of fast PCR diagnostic method for the invasive fungal infection.Methods The sterility body fluid samples from 60 cases hospitalized immunocompromised patients with clinical underlying diseases and suspect of invasive infections with fungi between January 2008 and December 2011 were processed for microscopy and cultures.Applying fungal ITS4 and ITS86 universal primers to amplify pathogenic fungi genes from the sterility body fluid with method of rapid PCR.The results were compared between the standard and PCR methods.Results Humoral direct clinical specimens by PCR amplification of DNA fragments with the scan results were similar.Positive rate of PCR test with clinical body fluid samples and the traditional fungal cultivation was similar.There was no significant statistical difference [38.3％ (23/60) vs 33.3％ (20/60),P ＞ 0.05].Conclusions PCR test is feasibility with clinical fungal diagnosis from directly humoral specimens.To amplify the clinical sterility body fluid samples with ITS fungal universal primers and PCR method might provide an accurate and rapid approach to detect the pathogenic fungi.Its methods on early diagnosis and prognosis of invasive fungal infections are of guiding significance.
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ObjectiveTo investigate the genotype of a case with abnormal hemoglobin combined with hereditary persistence of fetal hemoglobin (HPFH).Methods Male patient,26 years old,were suspected abnormal hemoglobin combined with HPFH after receiving medical examination including hematology exmination,hemoglobin electrophoresis,erythrocyte osmotic fragility analysis in Guangzhou Kingmed Diagnostics in September 2010.Routine examination of anemia and hemoglobin electrophoresis at alkaine pH on agarose gels were applied to analyze the phenotype.Direct sequencing of the complete HBB gene was utilized to identify the hemoglobin variant.Multiplex ligation-dependent probe amplification (MLPA) assay was used to identify the presence of β-globin gene cluster deletion.Gap polymerase chain reaction (gap-PCR) was used to amplify the HBB gene fragment across the breakpoint,and the deletion breakpoint was characterized by direct sequencing the gap-PCR product and comparing the sequencing result with the reference sequence NC_000011.9.ResultsBy direct sequencing of the complete HBB gene,the patient in this study was found to carry a hemoglobin Ta-Li (HBB:c.250G ＞ T) mutation.By combining use of MLPA and gap-PCR with gene sequencing,we found that it had a gross deletion in the β-globin gene cluster,the deletion region was NC_000011.9:g.5222878_5250288del.Therefore,the genotype of this subject was SEA-HPFH combined with abnormal hemoglobin Ta-li.ConclusionCombining application of MLPA and gap-PCR with gene sequencing can help to make sure the genotype.
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Objective To explore the role of tumor inhibition of enhancer binding protein C/EBPα in the leukemic mice. Methods BALB/c nude mice were randomly divided into three groups. Three kinds of cells including pEGFP-C/EBPα-K562 cells, pEGFP-K562 cells and K562 cells as the control were injected into mice separately through the subcutaneous and tail vein, and subcutaneous tumors and leukemic models were formed. The changes of tumors were observed and the apoptosis of cells was detected by TUNEL; The capacity of proliferation of leukemia cells was observed in the bone marrow and the peripheral blood by Wright-Giemsa staining. The expression of genes of related to proliferation was detected by RT-PCR. Results The quality and the max diameter of tumors in the pEGFP-C/EBPα-K562 group were smaller than that of pEGFP-K562 group and K562 control group [(2.4±0.1) g vs (5.1±0.3) g and (5.7±0.4) g, both P ＜0.05; (11+2)mm vs (19+3) mm and (23+3) mm, both P ＜0.05]. More apoptosis cells were found in the pEGFP-C/EBPα-K562 group leukemic cells were found in the peripheral blood of leukemic models, and the proliferation of leukemic cells in the pEGFP-C/EBPo-K562 group were lower than that of other groups, accompany by the conspicuous cell differentiation. p53 was significantly elevated by RT-PCR, while down-regulated of c-myc.Conclusion Enhancer binding protein C/EBPα promote the apoptosis of cells and inhibit the proliferation of leukemia cells in leukemia mice, and further induce the cell differentiation. The inhibition of enhancer binding protein C/EBPα in the leukemia may have effect through the regulation of related genes.
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Objective To investigate the epidemiology and relevant risk factors of invasive fungal infection (IFI) in hospital old patients for non-respiratory tract. Methods Seventy-eight patients of IFI in non-respiratory tract were enrolled in this investigation. The incidence and risk factors of IFI were analyzed by prospective case-control study. Results In 78 old patients, 84 strains were isolated from different parts, and the most was Candida spp 82 strains (97.62%,82/84), followed by Candida albicans 55 strains (67.07%,55/82), Candida glabrata 13 strains ( 15.85%, 13/82), Candida krusei 6 strains (7.32%, 6/82), Candida tropicalis 4 strains (4.88% ,4/82), Candida parapsilosis 3 strains (3.66% ,3/82), Candida lusitaniae 1 strain ( 1.22%, 1/82). Aspergillus 2 strains (2.38%,2/84). Multivariate Logistic regression analysis showed that age, pathogen detection time, underlaying disease,glucocorticoids, immunosuppressants were the risk factors for IFI in non-respiratory tract. Conclusions Candida albicans is the main pathogens of Candida infections in old patients. To efficiently control the risk factors should be emphasized in old patients, including early diagnosis and treatment underlying diseases, appropriate use drugs, right to shorten hospital stay.
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For the reform and development of clinical laboratory education, based on the previous course reform, Laboratory Medicine College of Chongqing Medical University has put the leading teachers'responsibility system of laboratory medicine course into practice. In the recent 3 years, the course is better organized.The students are more interested in the course and they communicate more with teachers than ever before. The effect of the course is obvious. The leading teachers' responsibility system in laboratory medicine course should be promoted.
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It is for estimating the laboratory statistics from the view of clinical medicine objectively,completely and appropriately that we establish ＜ Laboratory Sciences and Clinical Medicine ＞ which fits the requirement for the modern medical laboratory specialists. In this selective course, we use problem-based learning ( PBL ) teaching method which is different from the traditional one, stimulating students to participate in the study actively and passionately,training them to think in a scientific way and to analyse and solve the problem in a rational way. As a result, it works well which makes us believe it is worth popularizing.
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Objective: The BCR-ABL fusion gene induced by reciprocal translocation of t (9; 22) (q34; q11) plays an important role in the pathogenesis of chronic myeloid leukemia (CML). Using recombinant ade-noviruses carrying the N-terminal oligomerizaton domain (OD) of the BCR/ABL and chimeric ubiquitin ligase β-TrCP, this study was to investigate the effect of the targeted degradation of oncoprotein BCR-ABL by Ubiqui- tin-Proteasome System on the proliferation of leukemia call line K562. Methods: The recombinant adenovirus-es carrying wild-type β-TrCP gene (Ad5β-TrCP-OD-HA), mutational β-TrCP gene (Ad5 A F-TrCP-OD-HA)and green fluorescent protein gene (Ad5GFP)were amplified in 293 calls and co-infected into K562 cells respec- tively. The rates of infection were analyzed by flow cytometry (FCM). Recombinant protein and BCR-ABL ex-pression was detected by Western blot. Cell proliferation was determined by cell counting and methylcellu- cose clonal cell culture. Cell cycle was observed through FCM. Untreated K562 cells were used as blank con-trols. Result: The leukemia K562 cell lines with exogenous recombinant β-TrCP-OD-HA and F-TrCP-OD-HA gene were established. The infection rates in the three groups were over 66.4% and recombinant protein sus-tained to be expressed. Ad5β-TrCP-OD-HA down-regulated the expression of BCR-ABL and inhibited prolifer-ation of K562 cells. FCM showed that the percentage of cells at S phase was decreased to 10.88%±2.42%, while that of cells at G_0/G_1 was increased to 85.6%±5.61%, with a significant difference (P<0.05). No changes were found in the cell cycle in groups of Ad5 △ F-TrCP-OD-HA and Ad5GFP. Conclusion: There is sustained ex-pression of recombinant β-TrCP-OD-HA protein in K562 cells infected by recombinant adenovirus.β-TrCP-OD-HA could inhibit the proliferation and clonogenicity of K562 cells through targeted degradation of oncoprotein BCR-ABL and arresting the progression of call cycle.
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Objective:To study the influence of protein transduction domain (PTD)-oligomerization domain (OD)-HA fusion proteins on apoptosis of bcr/abl-positive cell lines. Methods:bcr/abl-positive cells were treated with PTD-OD-HA protein. The apoptoses of the cells were detected by flow cytometry (FCM), DNA ladder and transmission electron microscopy (TEM), and the levels of apoptosis-related genes bax and bcl-2 were detected by RT-PCR and Western blotting. Results:FCM examination demonstrated that PTD-OD-HA protein induced the apoptosis of bcr/abl-positive cells; DNA ladder showed that the classic DNA ladders appeared in BaF3-P210 and K562 cells after 48 h treatment with PTD-OD-HA proteins; the apoptoses of BaF3-P210 cells were observed by TEM; the levels of bax in mRNA and protein increased in BaF3-P210 and K562 cells, and bcl-2 decreased. Conclusion:PTD-OD-HA proteins specifically induced the apoptosis of bcr/abl positive cells.
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Objective To investigate the etiology features and relevant risk factors of non-albicans candida infections in hospital. Methods 256 patients of non-albicans candida infections admitted in the second hospital of shanxi medical university from April 2006 to March 2008 were enrolled in this investigation, and a prospective case-control study was executed on 256 cases of non-albicans candida infections and 1220 cases of non-fungal infections. The incidence and risk factors of non-albicans candida infections were analyzed by statistical software SPSS13.0. Results Candida glabrata was the most common reason of non - albicans candida infections (38. 28% ) , followed by candida krusei (37. 11% ), candida parapsilosis ( 12. 50% ), candida tropicalis (9. 77% ), candida lusitaniae (2. 34% ). Univariate analysis and multivariate logistic regression analysis showed that aging, length of stay, underlying disease, losing albumin, using prophylaxis antifungal drugs, using broad spectrum antibiotics, invasive examination and treatment ( such as total parenteral nutrition ( TPN ), invasive procedures, central venous catheters, hemodialysis and mechanical ventilation,et al. ) were the independent risk factors for non-albicans candida infections. Conclusions Non-albicans candida was the main of fungal infections in patients. To efficiently control the disease, it will be helpful by early diagnosis and treatment underlying diseases and commodities and using appropriate tools of examine and treatment methods.
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ion sections were not significantly different between 2005 and 2006, the rest inter-annual comparisons were significantly different (P<0.01).
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Objeetive To construct the cell DNA damage models for the human CML K562 cell line before or after imarlnib mesylate treatment and observe the repairing process dynamically for investigating the iniluence of imatinib mesylate on the repair function of K562 cells after cell DNA danlage.Methods The MTT assay was used to estimate the optimal pretreatment concentration of imatinib mesylate in K562 cells and Western blot was employed to evaluate the phosphorylation status in K562 cells after imatinib mesylate treatment to estimate BCR/ABL tyrosine kinase inhibition by imatinib mesylate.The comet assay was used to detect the DNA damage induced by hydrogen peroxide at various concentrations in K562 cells with or without the pretreatment of imatinib mesylate.A dynamic observation on the repairing process after cell DNA damage was made by the comet assay.Results The pretreatment by imatinib mesylate for K562 cells was optimized to be at a final concentration of 1 μmol/L for 24 h as revealed by the MTT assay additionly imatinib mesylate treatment at this concentration could effectively inhibit the phosphorylation of the BCR-ABL fusion protein at Tyr177(Deusityrate 0.100±0.018).When compared with the control group(Deusityrate 0.425±0.039),the BCR/ABL phosphorylation at Tyr177 was significantly decreased by (77. 11±5.59) % (t=4. 57,P＜0. 05). The cell DNA damage models for both imatinib mesylate-nontreated and imatinib mesylate-pretreated K562 cell groups were constructed with hydrogen peroxide treatment at a final concentration of 10 μmol/L for 10 min at 4℃ as confirmed by the comet assay. When compared with the control imatinib mesylatenontreatod K562 cell group,the time duration required for the DNA repair in imatinib mesylate-pretreated K562 cell group was significantly prolonged (F= 97.79,P＜0. 05 ). Conclusions The cell DNA damage models for the leukemic K562 cell groups before or after imatinib mesylate treatment were successfully constructed and the tyrosine kinase inhibitor imatinib mesylate for BCR/ABL fusion protein was revealed to attenuate the DNA repair capacity of the K562 cells after DNA damage.
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@#ObjectiveTo observe the effect of reverse acupuncture by stages combined with facilitation techniques on motor function of stroke patients.Methods114 stroke patient were randomly divided into the treatment group (n=68) and control group (n=46). The patients of the treatment group were treated with reverse acupuncture by stages combined with facilitation techniques. Those of the control group were treated with general acupuncture combined with facilitation techniques. The period of treatment of two group was 30 days.ResultsAfter treatment, the scores of motor function and activities of daily living of the treatment group were significantly higher than that of the control group ( P<0.01).ConclusionThe reverse acupuncture by stages combined with facilitation techniques can obviously improve motor function of stroke patients.