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BACKGROUND:Neuroblastoma is a common solid tumor in children. Pediatric tumors are little affected by environmental factors, but closely related to child development. The suspension method is an effective and reliable method to harvest neoplastic stem cel s. OBJECTIVE:To culture the cel clones of human neuroblastoma cel line SK-N-SH and to assess the biological properties of the cel clones. METHODS:Using the suspension method with no serum media, tumor cel clones were obtained. Immunofluorescence method was used to identify whether tumor cel clones exhibit stem cel properties. SK-N-SH neuroblastoma was labeled by luciferase, and tumor cel clones and tumor cel s were seeded onto the back of nude mice to monitor cel proliferative properties in nude mice using in vivo imaging. RESULTS AND CONCLUSION:Using the suspension culture method, SK-N-SH neuroblastoma cel s could successful y develop into cloning bal s. Under serum-free culture, cloning bal s were immunofluorescently used to detect molecular markers that showed strong positive expression. Cloning bal s subcutaneously implanted into nude mice showed the strong ability of self-renewal and differentiation as stem cel s. The cel clones cultured by the suspension method strongly expressed Nestin, but weakly expressed glial fibril ary acidic protein, neuron-specific tubulin, possessing stem cel characteristics and strong proliferation and metastasis in nude mice.
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Objective To detect the expression of FOXM 1 transcription factor in cervical cancer tissues and to evaluate its clini-cal significance .Methods Immunohistochemical staining was employed to detect the expression of both FOXM 1 and Ki-67 in a va-riety of cervical tissues respectively from 38 patients with cervical cancer ,22 with cervical intraepithelial neoplasia (ranging from CINⅠ to CIN Ⅲ) and 17 with normal cervical epithelium .Results Abnormal expression rate of FOXM 1 was respectively 5 .88% , 63 .6% and 92 .1% in normal cervix ,CIN and cervical cancer ;Deregulated FOXM1 expression in cervical cancer tissues was signifi-cantly correlated with patients’ pathological differentiation ,clinical stage ,post-operational recurrence and Ki-67 expression level . Conclusion FOXM1 expression may be correlated with the deregulated proliferation ,malignant progression ,staging and prognosis in cervical cancer .
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Objective To investigate the antimicrobial resistance of main clinical isolates from ICU during 2012 .Methods Auto-matic microbiology analysis system and the disk diffusion method were performed to determine the antimicrobial susceptibility .All the data were analyzed by WHONET5 .6 software according to the breakpoints of The American Association of Clinical Laboratory Standardization Institute (CLSI) 2012 .Results A total of non-repeated 1 374 clinical isolates were collected in ICU during 2012 , including 1 089 strains (79 .3% ) of Gram-negative bacilli and 285 strains (20 .7% )of Gram-positive cocci ;The top five pathogens were Acinetobacter baumannii ,Pseudomonas aeruginosa ,Escherichia coli ,Klebsiella pneumoniae and Staphylococcus aureus ;Some Enterobacteriaceae strains were resistant to imipenem or ertapenem .2 strains of Enterococcus faecium were found resistant to van-comycin .Conclusion Non-fermenting bacteria ,Enterobacteriaceae and Staphylococci remain the predominant pathogens isolated from the patients in ICU ,their drug resistance is serious ,it is important to use antibacterial agents rationally and strengthen the sur-veillance of bacterial drug resistance .
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Objective To investigate the changes of hepatocyte growth factor (HGF) and vascular endothelial growth factor(VEGF) after partial hepatectomy in patients with liver cirrhosis and their relationship with liver regeneration. Methods Thirty-five patients with partial hepatectomy between June 2007 and August 2009 were enrolled,according to whether cirrhosis were divided into cirrhosis group (16 cases) and non-cirrhosis group (19 cases). Liver size were measured with angiographic computed tomography at 7,30,90 d after operation. Regeneration rate of remnant liver were calculated. The serum concentrations of HGF and VEGF were meaaured. Postoperative hepatic function and complications incidence rate were comparatively analyzed. Results Compared with non-cirrhosis group, the postoperative hepatic function of cirrhosis group suffered serious damage. In non-cirrhosis group, the remnant liver regeneration rate reached (63.6± 15.9)%, (79.4 ± 17.2)%, (97.2 ± 18.3)% at 7,30,90 d after operation,in cirrhosis group,it reached (41.7 ± 10.7)%, (55.7 ± 13.2)%, (76.6 ± 12.5)%, liver in non-cirrhosis group regenerated rapidly (P <0.05). After hepatectomy,the HGF levels in cirrhosis group increased significantly at 1,3,7 d than those in non-cirrhosis group(P ＜ 0.05), but the VEGF levels were lower. Conclusions Liver in the patients with cirrhosis regenerate slowly and it may be due to in part through a decrease in VEGF. Whether it may,when given therapeutically represent a strategy to optimize liver regeneration in problematic patients needs to be clarified.
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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 The present study was initiated to determine the expression profiles of the Fox genes in normal Balb/c mouse liver and their dynamic expression changes during fibrogenesis induced by experimental bile duct ligation(BDL).Methods RT-PCR was employed to detect 18 Fox family members including Foxo1,Foxo3,Foxm1,and Foxl1 in normal mouse liver.After mice were bile-duct-ligated,real time fluorescence quantitative PCR(FQ-PCR)was performed to ananlyze the dynamic mRNA expression changes of 9 inflammation-or proliferation-related Fox family genes.Results All the 18 Fox genes were found to be exDressed in the normal mouse liver and bile duct ligation profoundly influenced the expression of Fox transcriptional factor family genes.The expression of Foxol was significantly reduced by BDL,while FoxoL1 and Foxom1 expression were enhanced in this roodel.Moreover,the expression of Foxo1 and Foxo3 were the highest among the Fox members.Conclusion The Fox family genes-related to inflammation and proliferation were dynamically changed during BDL-induced liver injury,it indicates these genes were involved in the pathogenesis of liver fibrosis.
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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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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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Objective To construct the transformed mouse BaF3-P210 cell line stably expressing BCR/ABL and to initially investigate the influence of BCR/ABL on the cell biological characteristics of BaF3 cell line. Methods The retroviral vector with bcr/abl gene was transfected into the packaging cell line. The BaF3 cells were infected with the collected viral supernatant. The transgenic BaF3-P210 cell line stably expressing BCR/ABL were screened and subcloned. The integration of the bcr/abl gene in the genome of the target cell was determined by PCR and DNA sequencing,trypan blue staining assay,flow cytometry and MTT assay. Results The bcr/abl gene was integrated into the BaF3 cell genome; RT-PCR and Western blot verified the stable expression of the bcr/abl gene and protein in the screened subclone cell line BaF3-P210. Compared with the control group,the cell proliferation rate was promoted (P
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Objective To construct a recombinant eukaryotic expression plasmid of glycosylphosphatidyl inositol(GPI)-anchored bcr/abl and explore its expression at mRNA and protein level.Methods The gene fragment encoding bcr/abl was amplified by PCR using the plasmid containing the cDNA sequence of P210 as template and then inserted into a eukaryotic expression vector pBudCE4.1.The constructed recombinant plasmid pBudCE4.1-bcr/abl was identified by restriction analysis and DNA sequencing.Lymphocytes were isolated from human peripheral blood and their total RNA was extracted.The gene fragment encoding GPI was amplified by RT-PCR using the obtained RNA as template and was inserted into the constructed recombinant plasmid pBudCE4.1-bcr/abl in order to anchor GPI and bcr/abl.The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI was transfected into COS-7 cells,and the expressions of objective fragment were detected by RT-PCR and Western blotting.Results The results of restriction analysis,PCR and DNA sequencing proved that GPI-anchored bcr/abl fusion fragment was correctly inserted into vector pBudCE4.1.The expression of bcr/abl fusion gene and fusion protein were identified in transfected COS-7 cells and on their membrane.Conclusion The recombinant plasmid pBudCE4.1-bcr/abl-GPI was successfully constructed and expressed on the membrane of COS-7 cells,which found a basis of cell immunity with GPI-anchored bcr/abl fusion gene.
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Objective To investigate the effects of circadian clock gene Period2(Per2)on the proliferation,differentiation and apoptosis of K562 cells and its probable molecular mechanism.Methods The Per2 expression plasmid pcDNA3.1-Per2 and empty control plasmid were respectively transfected into K562 cells with cationic liposome,and the resistant cells stably expressing Per2 gene were obtained by G418 selection.Their morphological changes were observed under light microscope following Wright-Giemsa staining.Trypan blue excluding staining and MTT assay were employed to evaluate cell proliferation.Flow cytometry was performed to analyze cell cycle distribution and cell apoptosis,and electron microscopy was used to detect cell apoptosis.Meanwhile,the expressions of proliferation and apoptosis associated proteins,such as P53,Cyclin B1 and C-Myc,were respectively detected by RT-PCR and Western blot analysis at mRNA and protein level.Results The K562/Per2 cell line stably expressing Per2 gene was screened out.As compared with either the empty plasmid transfected group(K562/empty)or the untreated group(K562/untreated),K562/Per2 cells was smaller in volume and showed no obvious cellular differentiation.Circadian clock gene Per2 could significantly inhibit both growth and proliferation of K562 cells.The percentage of K562 cells in G2/M phase increased [K562/Per2 group(36.1?5.5)%,K562/empty group(12.5?2.9)%,untreated group(9.7?2.3)%,P