[Effects of tourism disturbance on the habitat and water quality for Andrias davidianus in Zhangjiajie, Hunan, China]. / æ— æ¸¸å¹²æ‰°å¯¹å¼ å®¶ç•Œå¤§é²µç”Ÿå¢ƒåŠæ°´è´¨çš„å½±å“.

To understand the effects of tourism disturbance on the habitat and water quality for Chinese giant salamander (Andrias davidianus, CGS), the interference and characteristics of habitat for CGS under different tourism intensity (high, medium, and low) were investigated, and the physicochemical and microbial index of water were analyzed in Zhangjiajie City, Hunan Province, China from 2014 to 2016. The results showed that high tourism disturbance (>0.5 million per year) significantly increased noise and reduced the number of dens, decreased dissolved oxygen levels, increased total nitrogen, total phosphorus, and microbial abundance in water, especially that of Escherichiacoli. Under high disturbance, the characteristics of habitat and water quality still could meet the requirements of the growth of CGS. According to the National ground water class 2 standard based on the demands of E. coli in the surface water (2000 ind·L-1), the theoretical threshold of tourist numbers was 26.0471 million per year. The intensity of tourism interference should be effectively reduced by declining tourist numbers and tourism facilities, for the protection of the habitat quality of the CGS and the promotion of tourism development.

Role of histone methylation enzyme EZH2 and VEGF165 in momymoya disease / 国际生物医学工程杂志

Objective To study on the role of histone methylation enzyme enhancer of zeste homolog 2 (EHZ2) and vascular endothelial growth factor 165 (VEGF165) in momymoya disease. Methods The animal model of moyamoya disease was established by ear vein injection of horse serum in New Zealand rabbits. VEGF165 was over-expressed in situ by packaging lentivirus. Real-time quantitative PCR and Western Blot were used to detect the expression of VEGF165, EZH2 and H3K27me3 in the brain tissues of the animal models. Results Compared with the normal control group, the expression levels of mRNA and protein of EZH2 in the moyamoya disease model group were increased (EZH2 mRNA:P<0.01), and the level of histone H3K27me3 was increased. After overexpression of VEGF165 in the moyamoya disease model group, the expression levels of mRNA and protein of EZH2 was further increased (EZH2 mRNA: P<0.01), and the level of histone H3K27me3 was also increased. Conclusions EZH2 plays a certain role in the pathogenesis of moyamoya disease, and the expression of EZH2 is regulated by VEGF 165, which provides a theoretical basis for the study of the pathogenesis of moyamoya disease.

CRISPR-Cas9-based site-directed knock-in of VEGF165 gene in a HEK293T cell / 国际生物医学工程杂志

Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.

Expression and identification of VEGF165 in bone marrow mesenchymal stem cells of rhesus / 天津医药

Objective To detect the transferred vascular endothelial growth factor (VEGF)165 gene expression in rhesus autologous bone marrow mesenchymal stem cells (MSCs), and to explore the functional viability of transgenic MSCs. Methods MSCs from rhesus bone were isolated by Ficoll, which were used to detect the phenotype. After the culturing, the expression vector pcDNA-eGFP-VEGF165 was transfected into bone marrow MSCs. Fluorescence microscope and flow cytometry were used to detect the enhanced green fluorescent protein (eGFP) expression. At the same time, the phenotype in transfected MSCs was also indentified. The VEGF165 expression level was detected by RT-PCR. Results The highly purified MSCs were collected successfully. The transfected MSCs and daughter cells showed expressions of eGFP and VEGF165, which also remained the characteristics of MSCs. Conclusion The VEGF165 gene that is transfected into MSCs can maintain characteristics of MSCs, and stably express foreign genes.

Study on the autophagy of prostate cancer PC-3 cells induced by oridonin.

To investigate the mechanism of oridonin (ORI)-induced autophagy in prostate cancer PC-3 cells, PC-3 cells cultured in vitro were treated with ORI, and the inhibitory ratio of ORI on PC-3 cells was assayed by 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. The ultrastructural changes of the cells were observed under light microscope, scanning electron microscope (SEM), and transmission electron microscope (TEM). Acridine orange (AO) staining was used to observe the acidic vesicular organelles (AVOs). The level of autophagy-related proteins, MAP1-LC3, was detected by Western Blot, and RT-PCR was used to detect the level of mRNA of beclin 1. After ORI treatment, the proliferation of PC-3 cells was inhibited significantly in a concentration and time-dependent manner. SEM examination revealed cellular shrinkage and disappearance of surface microvilli in ORI-treated cells. Under TEM examination, the nuclei exhibited chromatin condensation and the appearance of a large number of autophagosomes with double-membrane structure in cytoplasm. AO staining showed the existence of AVOs. The expression of LC3 and the mRNA level of beclin 1 was increased by ORI. Furthermore, autophagy inhibitor 3-methyladenine reversed the increase of beclin 1 mRNA. The growth of PC-3 cells was inhibited, and autophagy was induced by ORI, indicating ORI may have a potential antitumor effect.

Primary extraskeletal osteosarcoma of omentum majus.

Extraskeletal osteosarcoma is a rare malignant soft tissue tumor. Here we present a case of a primary extraskeletal osteosarcoma arising from omentum majus in a 40-year-old Chinese woman. Ultrasonography of the pelvic cavity showed a large soft tissue mass with marked calcification. Complete surgical resection of the primary tumor was performed and the histopathological diagnosis was extraskeletal osteosarcoma of omentum majus. She was followed up without adjuvant radiotherapy and chemotherapy, and died from widespread intra-abdominal, lung and liver metastases 7 months postoperatively.

[Autophagy of prostate cancer PC-3 cells under the induction of oridonin].

OBJECTIVE: To investigate the mechanism of oridonin (ORI)-induced autophagy in prostate cancer PC-3 cells. METHODS: The PC-3 cells cultured in vitro were treated with ORI. The inhibitory ratio of oridonin In PC-3 cells was assayed by MTT. The ultrastructural cellular changes were observed under light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). AO staining was used to observe the acidic vesicular organelles (AVOs). The level of MAP1-LC3 was detected by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR) used to detect the level of mRNA of beclin 1. RESULTS: After oridonin treatment, the proliferation of PC-3 cells was inhibited significantly in a concentration and time-dependent manner. The SEM examination revealed cellular shrinkage and the disappearance of surface microvilli after ORI treatment. And on the TEM examination, the nuclei exhibited chromatin condensation and the appearance of a large number of autophagosome with double-membrane structure in cytoplasm. AO staining showed the existence of AVOs. Simultaneously, the expressions of autophagy-related proteins, MAP-LC3 and the mRNA level of beclin 1 were elevated by ORI. The autophagy inhibitor 3-MA reversed the elevation of beclin 1 mRNA. CONCLUSION: The growth of PC-3 cells is inhibited. And cellular is induced by oridonin with a potential anti-tumor effect.