Identification and feature analysis of Stephanoascus ciferrii isolated from 12 cases with chronic suppurative otitis media / 临床检验杂志

Objective To study the laboratory features of Stephanoascus ciferrii isolated from middle ear excretion of the patients with chronic suppurative otitis media for providing the basis of diagnosis for the rare fungus infection.Methods The study was performed for 12 patients who were suffered from chronic suppurative otitis media associated with Stephanoascus ciferrii.The secretions of middle ear were cultured and the macroscopic features of colony and cells of the fungi were observed.The species of the fungi was identified by Vitek 2 Compact system.Results Stephanoascus ciferrii may grow on common media such as blood plate,chocolate plate and sabourauad plate,at 28 to 35 ℃.The form of the colonies displayed cream-colored,rough,hard and wrinkled quality after 1 week incubation.On CHROMagar medium the colonies looked like small mushroom with blue center and white edge.Under microscopy,abundant branched septate mycelium with small ramified chains of oval blastoconidia and the conidia were arranged along the side of hyphae.The fungi strain may assimilate lots of carbohydrates and common amino acid.Vitek 2 Compact automated system should be able to identify Stephanoascus ciferrii.Conclusion Stephanoascus ciferrii is a rare opportunistic pathogenic fungus.In clinical laboratories special attentions should be paid to culture and identification of the fungus from middle ear excretion.

Effect of Xing Nao Kai Qiao Needling on Cognitive and Motor Function of Patients in Acute Stage of Cerebral Infarction / 上海针灸杂志

Objective To observe the effect ofXing Nao Kai Qiao (awakening brain and opening orifice) needling method on the cognitive and motor function of patients in acute stage of cerebral infarction.Method Sixty-four patients in acute stage of cerebra infarction were recruited and randomized into a trial group and a control group, 32 cases in each group. The control group was intervened by Edaravone 30 mg plus Fibrinogenase injection 200 U mixed in 500 mL 0.9% NaCl via intravenous drip, once a day. The trial group was byXing Nao Kai Qiao needling method in addition to the intervention given to the control group, once a day, 5 times a week. The two groups were both given 2-week successive treatment. At the end of the intervention, clinical efficacy, cognitive function, motor function, nervous function, serum C-reactive protein and NO levels, and adverse reactions of the two groups were compared.Result After the treatment, the Chinese Stroke Scale (CSS) score dropped significantly, Loewenstein Occupational Therapy Cognitive Assessment (LOTCA) score, Barthel Index (BI) score and motor function score of both upper and lower limbs increased significantly in both groups (P<0.05); the total effective rate of the trial group was significantly higher than that of the control group (P<0.05); the CSS score was lower, and LOTCA score, BI score, and motor function scores of both upper and lower limbs were higher in the trial group compared with those in the control group, and the differences were statistically significant (P<0.05); on the 1st, 3rd, 7th and 14th day after the intervention, the serum CRP and NO levels were significantly lower in the trial group compared with those in the control group (P<0.05). There was no significant difference in comparing the adverse reaction incidence between the two groups (P>0.05).Conclusion Xing Nao Kai Qiao needling method can produce a significant efficacy in treating patients in acute stage of cerebral infarction, and it can improve the cognitive, motor and nervous functions with high security.

Effect of Xing Nao Kai Qiao Needling on Cognitive and Motor Function of Patients in Acute Stage of Cerebral Infarction / 上海针灸杂志

Objective To observe the effect ofXing Nao Kai Qiao (awakening brain and opening orifice) needling method on the cognitive and motor function of patients in acute stage of cerebral infarction.Method Sixty-four patients in acute stage of cerebra infarction were recruited and randomized into a trial group and a control group, 32 cases in each group. The control group was intervened by Edaravone 30 mg plus Fibrinogenase injection 200 U mixed in 500 mL 0.9% NaCl via intravenous drip, once a day. The trial group was byXing Nao Kai Qiao needling method in addition to the intervention given to the control group, once a day, 5 times a week. The two groups were both given 2-week successive treatment. At the end of the intervention, clinical efficacy, cognitive function, motor function, nervous function, serum C-reactive protein and NO levels, and adverse reactions of the two groups were compared.Result After the treatment, the Chinese Stroke Scale (CSS) score dropped significantly, Loewenstein Occupational Therapy Cognitive Assessment (LOTCA) score, Barthel Index (BI) score and motor function score of both upper and lower limbs increased significantly in both groups (P<0.05); the total effective rate of the trial group was significantly higher than that of the control group (P<0.05); the CSS score was lower, and LOTCA score, BI score, and motor function scores of both upper and lower limbs were higher in the trial group compared with those in the control group, and the differences were statistically significant (P<0.05); on the 1st, 3rd, 7th and 14th day after the intervention, the serum CRP and NO levels were significantly lower in the trial group compared with those in the control group (P<0.05). There was no significant difference in comparing the adverse reaction incidence between the two groups (P>0.05).Conclusion Xing Nao Kai Qiao needling method can produce a significant efficacy in treating patients in acute stage of cerebral infarction, and it can improve the cognitive, motor and nervous functions with high security.

VEGF shRNA enhances the sensitivity of multidrug-resistant leukemia cells to anticancer agent / 中国实验血液学杂志

This study was aimed to explore the effect of vascular endothelial growth factor (VEGF) on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference, and to investigate its mechanism. The 3 shRNA targeting human vegf gene were synthesized, then transfected into K562/A02 cells by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF, MRP1, AKT, P-AKT at the protein level; MTT was used to determine the IC(50) value of transfected cells to doxorubicin (DOX); flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention. The results showed that after vegf shRNA were transfected into K562/A02 cells, the expression of vegf at the mRNA level decreased, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant (p < 0.05), the greatest decrease was observed in the cells transfected with vegf shRNA3; and the protein level of VEGF was also down-regulated. The IC(50) value of positively transfected group was lower than that of control groups, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant (p < 0.05). The retention of intracellular Rho123 was enhanced in three positively transfected groups (p < 0.05). Cell apoptosis increased in positively transfected groups, and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group (p < 0.05). The expression of mrp1 at the mRNA level were decreased, and there were statistical difference between vegf shRNA3 group and HK group (p < 0.05), and the protein level of mrp1 was also down-regulated; the expression of P-AKT at protein level decreased in positively transfected groups, and the greatest decrease was seen in vegf shRNA3 group. It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels, and enhance the sensitivity of K562/A02 cell to doxorubicin, the mechanism of which may be the inhibition of apoptosis and down-regulation of MRP1 by inactivating PI3K/AKT signaling pathway.

Influence of YB-1 protein on the biological behaviour in K562/A02 cells / 中国实验血液学杂志

The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.

CD44 variant increases the invasive ability of human breast cancer cell line MCF-7 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms.</p><p><b>METHODS</b>The full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction (RT-PCR) and subcloned into pMD19-T vector. The CD44v17 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant.</p><p><b>RESULTS</b>The new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid (HA) treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK.</p><p><b>CONCLUSION</b>A new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR cells. The expression vector pcDNA3.1-CD44v17 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras/MAPK signaling pathway.</p>

Effect of YB-1 gene knockdown on human leukemia cell line K562/A02 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry.</p><p><b>RESULTS</b>The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3.</p><p><b>CONCLUSION</b>Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.</p>