Inhibitory effect of luteolin on the growth, migration and vasculogenic mimicry formation of a melanoma cell line B16 / 中华皮肤科杂志

Objective To evaluate the effect of luteolin on the growth,migration and vasculogenic mimicry formation of a melanoma cell line B16.Methods In vitro cultured B16 melanoma cells were divided into 4 groups:low-,middle-and high-dose luteolin groups treated with 2.5,5,10 μmol/L luteolin respectively,and control group treated with 0.1％ dimethyl sulfoxide (DMSO).Scratch assay,Transwell invasion assay and vascular channel formation assay were performed to assess the migration,invasion of and vascular channel formation by melanoma cells.A model of subcutaneous transplanted B 16 melanoma was established in 12 C57 mice,which were randomly and equally divided into 4 groups:control group gavaged with ultrapure water,low-,middle-and high-dose luteolin groups gavaged with 10,20,40 mg/kg luteolin respectively every day.The above treatment for the tumor-bearing mice lasted till day 28,and then these mice were sacrificed.Meanwhile,the lung and tumor tissues of the mice were excised,and the growth,metastasis and vasculogenic mimicry of transplanted melanoma were observed.Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin (VE-cadherin),vascular endothelial growth factor receptor 1 (VEGFR1),VEGFR2,matrix metalloproteinase-2 (MMP-2) and MMP-9 in the transplanted melanoma.Means were compared among several groups by using one-way analysis of variation or rank sum test.Results In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group,low-,middle-and high-dose luteolin groups (0.47 ± 0.04,0.64 ± 0.04,0.73 ± 0.03,0.84 ± 0.04 respectively;F =34.51,P ＜ 0.001),and the migration ability of B16 cells was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.05).At 24 hours,there were significant differences in the number of cells crossing the Transwell membrane among the control group,low-,middle-and high-dose luteolin groups (281.00 ± 8.79,169.00 ± 15.35,92.00 ± 14.79 and 57.00 ± 13.72 respectively;F =275.30,P ＜ 0.001),and the invasive ability was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (P ＜ 0.01).Meanwhile,the number of formed vascular channels also differed among the above 4 groups (20.00 ± 2.77,11.00 ± 1.28,7.00 ± 1.86 and 2.00 ± 1.32 respectively;F =48.61,P ＜ 0.001),and the number of vascular channels was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (all P ＜ 0.01).In vivo study showed that the tumor size significantly differed among the control group,low-,middle-and high-dose luteolin groups (5.10 ± 1.72,4.02 ± 2.13,2.98 ± 0.92,1.49 ± 1.13 cm3 respectively;F =28.76,P ＜ 0.001),and was significantly lower in the low-,middle-and high-dose luteolin groups than in the control group (t =3.86,7.11 and 13.06 respectively,all P ＜ 0.01).CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-,middle-and high-dose luteolin groups (all P ＜ 0.01).In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group (P ＜ 0.05).Conclusion Luteolin can effectively inhibit the growth,metastasis and vasculogenic mimicry formation of melanoma.

The effects of Ulinastatin on the inflammatory response in cerebral cortex of Wistar rats after cardiopulmonary resuscitation / 中华急诊医学杂志

Objective To evaluate the effects of Ulinastatin (UTI) on the expressions of TNF-α,IL-6 and neurons apoptosis in cerebral cortex of rats after cardiopulmonary resuscitation (CPR).Methods Thirty-six healthy male adult Wistar rats were induced ventricular fibrillation untreated for 7 min and then received CPR.The animals were infused UTI 100 000 U/kg or phosphate-buffered solution (PBS) at once after ROSC.At 2,4 and 8 h after ROSC,cerebral cortex were removed to determine the mRNA expressions and levels of TNF-α protein and IL-6 protein,the translocation ratio of NF-κB p65 from cytoplasm to nucleus and the apoptotic neurons.Results The plasma levels of TNF-α (ng/mL) in animals of UTI group were (17.7 ± 1.4),(21.9 ± 2.1) and (17.1 ± 0.6) at 2,4 and 8 h after ROSC respectively,and significantly lower than those in PBS group at the given intervals.Mean while,the levels of IL-6 (ng/mL) were (208.9 ± 14.1),(281.5 ±25.9) and (251.8 ± 15.3) at 2,4 and 8 h after ROSC respectivèly in animals of UTI group,and lower than those in PBS group.The expressions of TNF-α mRNA and IL-6 mRNA and protein levels of TNF-α and IL-6 in UTI group were both lower than those in PBS group at given intervals,respectively.The translocation ratio of NF-κB p65 from plasma to nucleus in PBS group at each given interval after ROSC was significantly higher than that in UTI group.The number of viable neurons in cerebral cortex in UTI group was higher than that in PBS group,while the number apoptosis neurons was fewer in UTI group.Conclusions UTI attenuated the general inflammatory response after ROSC in rat,decreased the activation of NF-κB pathway,and subsequently attenuated the expression of TNF-α and IL-6,and finally decreased the neurons apoptosis.

Drug susceptibility and UL97 gene mutation analysis of cytomegalovirus in recipients of hematopoietic stem cell transplantation / 南方医科大学学报

<p><b>OBJECTIVE</b>To monitor human cytomegalovirus (HCMV) drug resistance in recipients of hematopoietic stem cell transplantation by phenotypic and genotypic methods.</p><p><b>METHODS</b>HCMV clinical isolates was isolated from the urine of hematopoietic stem cell transplantation recipients treated with GCV. Tissue cell infection median dose (TCID50) of the isolates was calculated using Reed-Muench method, and their drug susceptibility was determined by plaque reduction assay. We amplified the UL97 DNA fragment of the virus by nested PCR followed by automated DNA sequencing.</p><p><b>RESULTS</b>HCMV clinical strain isolated from the urine samples of the recipients using a human fibroblast cell line showed a TCID50 value of 10(-4.618)/0.1 ml and a 50% inhibitory concentration (IC50) to GCV of 5.847 µmol/L, suggesting its sensitivity to GCV. Alignment with the AD169 DNA reference sequence identified 4 point mutations of the virus at 1509 (T-C), 1575 (C-T), 1794 (T-C), and 1815 (C-G), and only the last mutation resulted in one amino acid mutation to D605E. No gene mutation was found in relation to GCV resistance.</p><p><b>CONCLUSIONS</b>Phenotypic and genotypic assays were established to examine antiviral drug resistance of HCMV in recipients of hematopoietic stem cell transplantation. We did not find any drug resistance of the clinical HCMV isolate.</p>

The detection of antiviral susceptibilities of HCMV clinical isolates to ganciclovir from the patients after hematopoietic stem cell transplantation / 中国基层医药

Objective To investigate the susceptibilities of HCMV clinical strains isolated to ganciclovir from the patients after hematopoietic stem cell transplantation.Methods Eight HCMV clinical isolates were isolated from the blood or the urine of hematopoietic stem cell transplantation recipients who had been treated with GCV.Tissue cell infection median dose(TCID50) were calculated by Reed-Muench method.Drug susceptibility was determined by MTT assay.Results TCID50 values of eight HCMV clinical strains were 10-4.12/0.1ml,10-4.29/0.1ml,10-4.3/0.1ml,10-4.4/0.1ml 10-4.42/0.1ml,10-4.5/0.1ml,10-4.52/0.1ml and 10-4.62/0.1ml respectively.50％ inhibitory concentration(IC50) to GCV of eight HCMV clinical strains were 0.638,1.438,0.965,0.698,0.482,1.167,1.519,1.511 mg/L respectively.Conclusion Our results suggest that resistant HCMV strains are not prevalent in Guangzhou.Continuous monitoring of HCMV is needed to understand the antiviral resistance status of the virus in patients after hematopoietic stem cell transplantation and guide its clinical management.