Progress in the structure and function of rotavirus non-structural protein 4 / 中华微生物学和免疫学杂志

Rotavirus (RV) is one of the main pathogens causing diarrhea in children under five years old, but the mechanism of RV-infected diarrhea is still unclear. The RV genome encodes six structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6), among which NSP4 can interact with other non-structural proteins or structural proteins of RV to produce corresponding biological functions, and is a key factor in the formation of RV morphology, the process of infection and the pathogenesis of diarrhea. In this paper, the current domestic and foreign studies on the structure and function of NSP4 are reviewed.

KLK6 Promotes Growth, Migration, and Invasion of Gastric Cancer Cells

PURPOSE: Kallikrein (KLK) proteases are hormone-like signaling molecules with critical functions in different cancers. This study investigated the expression of KLK6 in gastric cancer and its potential role in the growth, migration, and invasion of gastric cancer cells. MATERIALS AND METHODS: In this study, we compared protein levels of KLK6, vascular endothelial growth factor (VEGF), and matrix metallopeptidase (MMP) 9 in normal gastric epithelial and gastric cancer cell lines by western blot. Fluorescence-activated cell sorting was employed to sort 2 clones of SGC-7901 cells with distinct KLK6 expression, namely, KLK6-high (KLK6high) and KLK6-low (KLK6low), which were then expanded. Lastly, immunohistochemical analysis was performed to investigate KLK6 expression in gastric cancer patients. RESULTS: The expression levels of KLK6, VEGF, and MMP 9, were significantly higher in the gastric cancer cell lines SGC-7901, BGC-823, MKN-28, and MGC-803 than in the normal gastric epithelial cell line GES-1. Compared to KLK6low cells, KLK6high cells showed enhanced viability, colony-forming ability, migration, and invasion potential in vitro. Importantly, immunohistochemical analysis of a human gastric cancer tissue cohort revealed that the staining for KLK6, VEGF, and MMP9 was markedly stronger in the cancerous tissues than in the adjacent normal tissues. KLK6 expression also correlated with that of VEGF and MMP9 expression, as well as several key clinicopathological parameters. CONCLUSIONS: Together, these results suggest an important role for KLK6 in human gastric cancer progression.

Expression of MYH9 in human osteosarcoma tissues and its effects on epithelial-mesenchymal transition and invasion of osteosarcoma cells / 医学研究生学报

Objective Non-muscle myosin heavy chain 9 (MYH9) plays an important regulatory role in the development of tumor.This study aimed to explore the expression of MYH9 in osteosarcoma tissues and its effects on epithelial-mesenchymal transition and invasion of osteosarcoma cells.Methods We collected 52 cases of osteosarcoma tissues and para-carcinoma tissues at 5 cm form the edge of the tumor.RT-PCR and immunohistochemistry were used to analyze the expression level of MYH9 mRNA and protein in the osteosarcoma tissues and para-carcinoma tissues.MYH9 shRNA plasmid was transfected into U2-OS cells to silenced the expression of MYH9, after transfected, the cells were divided into three groups: the normal U2-OS cells were the control group, the U2-OS cells transfected with empty plasmid were the empty group and U2-OS cells transfected with MYH9 shRNA were interference group.RT-PCR was used to detect the changes of MYH9 mRNA levels in the U2-OS cells, the protein level of MYH9, EMT related protein E-cadherin and Vimentin were detected by Western blot, and the ability of cell invasion was evaluated by Transwell assay.Results The results of RT-PCR showed that the relative expression MYH9 mRNA in para-carcinoma tissues(1.526±0.148) was significantly lower than that in cancer tissues (3.547±0.195) (P<0.05).The results of immunohistochemistry showed that MYH9 protein was mainly expressed in cytoplasm, and the expression in cancer tissues was significantly higher than that in para-carcinoma tissues, the positive expression rate were 59.6%(31/52) and 26.9%(14/52) respectively, the difference was statistically significant(P<0.05).The results of Western blot showed that the relative expression of MYH9 mRNA in interference group was significantly lower than that in control group and empty group (P<0.05) after silenced MYH9 gene, and compared with the control group, the E-cadherin in U2-OS cells was significantly up-regulated but the Vimentin was down-regulated.After 48h, all of the groups had cells through the microfiltration membrane, the numbers of cells through the microfiltration membrane in interference group(41.2±15.1) was significantly lower than that in control group(117.3±12.4) and empty group(193.5±14.7) (P<0.05).Conclusion The expression of MYH9 protein in osteosarcoma tissues was significantly higher than that in para-carcinoma t tissues, silenced MYH9 gene can reduce the invasive ability of osteosarcoma by reducing the epithelial interstitial transition.

Establishment of an orthotopic transplantation model of osteosarcoma labeled with three reporter genes / 军事医学

Objective To establish an orthotopic osteosarcoma nude mice model that co-expresses green fluorescent protein( GFP) , red fluorescent protein ( RFP) and luciferase for the purpose of monitoring the growth of osteosarcoma and screening drug candidates against osteosarcoma .Methods Human osteosarcoma cells of U 2-OS were infected with lentivirus carrying reporter gene .The reporter gene expression was verified by fluorescent microscopy and bioluminescence imaging.The cells were transplanted into tibia of the nude mice and monitored by bioluminescence imaging .Results The reporter gene was stably expressed in U 2-OS cells.The growth and metastsis of osteosarcoma could be detected in nude mice.Conclusion The established orthotopic osteosarcoma nude mice model is an ideal model for investigating the mechanism of growth and metastasis of osteosarcoma and for screening drug candidates against osteosarcoma .

Inhibitory effect of ursolic acid on high oxygen-induced mouse retinal neovascularization by intravitreal injection / 中华实验眼科杂志

Background Retinal neovascular diseases affect visual function.Although many drugs have been used to manage the visual diseases,their effectiveness is less than satisfactory.Studies showed that ursolic acid has multiple biological effects including anti-vascularization.However,the effect of ursolic acid on retinal neovascular diseases is unclear now.Objective This study was to observe the inhibitory effect of ursolic acid on the high oxygen-induced mouse retinal neovascularization after intravitreal injection.Methods Sixty clean 7-day-old C57BL/6J mice were divided into the blank control group,PBS control group,positive control group (triamcinolone) and low,moderate and high dose (1.5,3.0 and 6.0 μg) ursolic acid groups randomly.The blank control group mice were raised in normal environment,and the mice from other groups were fed in the environment with O2 concentration at (75±2)％ for 5 days together with the maternal mice.The mice then were back to the normal air environment to induce retinal neovascularization.Then,the drugs were intravitreally immediately injected in the mice of the different groups.The mice were sacrificed at the 17-day old for the preparation of retinal sections.Retinal new blood vessel was examined by haematoxylin and eosin stain under the light microscope,and the number of vascular endothelial cell nucleus breaking the inner limiting membrane was counted.The gene expressions of vascular endothelial growth factor (VEGF),cyclooxygenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2) in the mouse retinas were quantitatively assayed using reverse transcription PCR.Results The number of endothelial nuclei newly-generated vessel breaking internal limiting membrane in the mice of PBS control group was (18.65±3.24)/field,which was more than (0.78±0.11)/field of the blank control group obviously (t =2.24,P＜0.05).The number of endothelial nuclei newly-generated breaking internal limiting membrane in the moderate-or high-dose ursolic acid group was less than that of moderate group obviously,it was statistically significant(P＜0.05).The number of vascular endothelial cell nuclei breaking internal limiting membrane in high high-dose group was (13.32 ± 1.87)/field and (8.93 ± 1.09) /field,showing significant decreases in comparison with the PBS control group and low-dose ursolic acid group (18.65±3.24)/field (15.44±2.02)/field (all at P＜0.05).However,no significant difference were seen in the number of new vascular endothelial cell nucleus between the high-dose ursolic acid group and the positive control group(9.14±1.13)/field (t=1.17,P＞0.05).The relative expressions of COX-2 mRNA,VEGF mRNA and MMP-2 mRNA in the mouse retinas were higher in the PBS control group than those in the blank control group (t =13.45,12.49,14.32,all at P＜0.05),and those in the moderate-dose or high-dose ursolic acid group were lowed in comparison with the PBS control group and the low-dose ursolic acid group (all at P＜0.05),but there were no significant differences between the high-dose ursolic acid group and the positive control group (all at P＞0.05).Conclusions Ursolic acid can suppress retinal neovascularization by down-regulating the expressions of VEGF,COX-2 and MMP-2 in oxygen-induced retinopathy of mouse in dose-dependent manner.

Build of lentiviral mediated RNA interference of mammalian target of rapamycin / 重庆医学

Objective To build of lentiviral mediated RNA interference of mammalian target of rapamcin (mTOR) ,because the mammalian target of rapamycin plays a important role in tumor development and the signal path .Methods According to RNA in-terference (RNAi) design rules Completely ,in view of the gene called mTOR was desiged four interference targets and Negative control (FAM ) sequence ,first of all ,synthetic oligonucleotides nucleotide fragments with artificial ,and can obtain siRNA fragments effectively by the method of PCR joining together ,then undertake transfection on lung adenocarcinoma A 549 cell by Lipofectamine transfection reagent 2000 .To begin to observe the enhanced green fluorescent protein expression in lung adenocarcinoma A 549 cells by fluorescence microscopy at high magnification after 1 days .We can use semi-quantitative RT-PCR method ,and detect of mTOR gene expression of mRNA level after 1 days ,meanwhile ,testing the expression of protein levels by Western Blot after 2 days ,in or-der to select the most efficient interference target sequence ,,afterwards ,synthetic double-stranded DNA ,and it can be make up vec-tor system with plasmid pHelper 1 .0 and pHelper 2 .0 by the pGCL-GFP carrier ,further transfect 293 T cells ,at last produce lenti-viral after packaging ,then detection GFP protein expression levels by Western-Blot method ,and consequently detect the virus drops degree of 293T cells ,at the same time ,identify the activity .Results The high efficiently target of mTOR gene has been successfully selected ;mTOR siRNA infecte of human lung adenocarcinoma A549 cells in vitro culture ,and the gene is obviously silence no mat-ter from the mRNA level or protein level ;3 mTOR gene lentivirus siRNA carrier was successfully build .geting virus supernatant al-so ,and virus drops to 1 × 108 UT/mL .Conclusion MTOR siRNA infected of human lung adenocarcinoma A549 cells in vitro cul-ture ,and could lead to mTOR gene obviously silence ;The construction was successfully gene mTOR siRNA lentivirus vectors .

Effect of lutein on oxidative stress of rats with high homocysteine and its relevent mechanism / 中华急诊医学杂志

Objective To reveal the effect of lutein on the status of oxidative stress in rats with high homocysteine levels (HHcy) and relevent molecular mechanisms.Methods The wistar rat HHcy model was established by intra-gastric administration with L-methionine suspension and treated with lutein.The oxidative stress status and the gene expression changes of transcription Nf-E2-related factor2 (Nrf2),a regulation factor of down stream antioxidant protein gene expression,were detected in HHcy rats and lutein intervention rats.Results Compared with the rat serum SOD activities (134.32 ± 12.65) U/mL in the control group,the serum SOD activities in the HHcy model group (95.6 ± 10.92) U/mL were significantly lower (P ＜ 0.05).The serum glutathione peroxidase (GPx) activities in the HHcy model group (121.66 ± 18.64) U/mL were also significantly lower as compared with the the control group (183.17 ± 21.29) U/mL,P ＜ 0.05.However,the serum SOD (126.75 ± 11.26) U/mL and GPx activities (167.18 ± 19.66) U/mL in the lutein intervention group were significantly higher as compared with the HHcy model group.As compared with the rat serum malondialdehyde (MDA) content (5.11 ± 0.68) μmol/L as well as the hydroxyl free radical levels (0.53 ± 0.05) U/L in the control group,the serum MDA content (7.65 ± 0.87) μmol/L and the hydroxyl free radical levels (0.92 ± 0.09) U/L in the HHcy model group were significantly higher.The serum MDA content (6.44 ±0.91) μmol/L and the hydroxyl free radical levels (0.74 ± 0.06) U/L in the lutein intervention group were significantly lower as compared with the HHcy model group.RT-PCR and Western blot results also showed decreased expression of SOD2 and GPxl mRNA in aorta epithelial tissues of HHcy model rats.With lutein intervention,the expressions ofSOD2 and GPx1mRNA were significantly increased and the gene expression of Nrf2 also up-regulated.Conclusions The Hhcy model rats were under the status of augmented oxidative stress,and carotenoid lutein could attenuate the Hcy-mediated oxidative stress,and its mechanism might be potentially associated with up-regulating the expression of Nrf2,thereby inducing the expression of its downstream antioxidant proteins.

The effects of constraint-induced movement therapy on expression of tyrosine hydroxylase and glial cell derived neurotrophic factor in Parkinson's disease model rats / 中华物理医学与康复杂志

Objective To explore the effects of constraint-induced movement therapy (CIMT) on the expression of tyrosine hydroxylase (TH) and glial cell derived neurotrophic factor (GDNF) in Parkinson's disease (PD) model rats. MethodsPD models were established by microinjection of 6-hydroxydopamine (6-OHDA) solution into substantia nigra of rats' right cerebral hemisphere.Forty-two model rats were divided randomly into an exercise group and a control group 1 week after microinjection.The exercise group rats were forced to use their impaired limbs by placing their nonimpaired fore-limbs in casts.The control group rats were housed in the same environment without any special treatment.Two weeks after 6-OHDA infusion and exercise training,the behavioral changes of rats were examined after intraperitoneal injection apomorphine ( APO).The content of dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) was measured by high performance liquid chromatography with electrochemistry ( HPLAEC) ; the expressions of TH and GDNF in striatum were detected by immunohistochemical methods and TH,GDNF mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results After 2 weeks of training,the rotating laps of the rats in exercise group within 30 min after APO induction,reduced to a significantly greater extent when compared to the control group (P ＜ 0.05).The content of DA and it's metabolites DOPAC in striatum homogenate was significantly higher in exercise group than that in the control group ( P ＜ 0.05 ),and the expression levels,of TH and GDNF protein/ mRNA were also significantly higher in the exercise group than those in control group ( P ＜ 0.05 ).Conclusions CIMT can improve the behavioral performance of PD rats,probably through promoting the expressions of TH and GDNF protein/mRNA in striatum,and increasing DA and it's metabolites DOPAC level.

Influence of acidic peptide on learning and memory of rats with Alzheimer disease / 中国组织工程研究

BACKGROUND: Acidic peptide is the tripeptide composed of 3 glutamic acids, which cannot bring excitatory nerve signal transmission into playlike single glutamic acid through presynaptic release and integration withpostsynaptic NMDA receptor directly as excitable neurotransmitter. It is quite possible that acidic peptide plays its actions by integrating with multiple metabolic glutamic acidic receptors so as to promote neuron proliferation or release nerve growth factor (NGF). OBJECTIVE: To probe into whether acidic peptide induces changes in learning and memory of model rats with Alzheimer disease (AD).DESIGN: Randomized controlled single experiment was designed.SETTING: Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University.MATERIALS: The experiment was performed in 2nd Research Room and Experimental Animal Room of Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University.Totally 100 SD male rats were selected and some of them were excluded due to retarded response in step down test. Totally 84 rats were included in the experiment and randomized into 7 groups, named normal control,model group, physiological saline group (PS group), piracetam group, acidic peptide groups of 60, 30 and 15 mg/kg, 12 rats in each group. Acidic peptide is a new small molecular peptide separated from bovine brain in this research team and is tripeptide composed of three glutamic acids.METHODS: Except normal control, in the rest groups, after 1 week routine breeding, cerebral stereotactic microinjection was used to inject 5 μg ibotenic acid in hippocampus of rats to destroy bilateral Meynert's basal ganglia to establish AD model. In normal control and model group, no medication was applied. In PS group, physiological saline was used for gastric perfusion. In piracetam group, piracetam of 0.3 g/kg was used for gastric perfusion and in acidic peptide groups of 15, 30 and 60 mg/kg,acidic peptide of 60, 30 and 15 mg/kg was applied for gastric perfusion successively, continuously for 20 days, once per day, 2 mL/time. On the expiration of gastric perfusion, learning and memory of rats were examined with step down test in every group. The animal was placed on the safe table on step down platform to adapt to the environment for 3 minutes, afterwards, 36 V electric current was given. Error response was recorded if the animal jumped to the copper railings after electric shock and correct response was recorded if the animal jumped back the safe area. Step-up latent phase and frequency of correct response were recorded in 3 minutes.MAIN OUTCOME MEASURES: Comparison of learning and memory of rats in every group. RESULTS: Totally 84 rats were all included in the result analysis. ①Comparison of learning in every group: Compared with model group, stepup latent phase was shortened remarkably in every acidic peptide group[(102.03±5.33), (71.77±4.38), (68.28±9.53), (69.13±8.79) s, P ＜ 0.01] and the frequency of correct response was improved remarkably [(12.92±2.91),(16.17±2.79), (15.83±3.27), (16.33±2.53) times, P ＜ 0.01]. ② Comparison of memory in every group: Compared with model group, step-up latent phase was shortened remarkably in every acidic peptide group [(43.17±4.66),(29.78±4.48), (26.20±3.28), (22.09±4.43) s, P ＜ 0.01] and the frequency of correct response was improved remarkably [(15.67±2.15), (20.92±2.68),(20.83±2.29), (20.25±2.05) times, P ＜ 0.01].CONCLUSION: Acidic peptide can shorten remarkably the step-up latent phase of AD rats in step down test and improve the frequency of correct response. It is indicated that acidic peptide provides good intervention on learning and memory of rat model of Alzheimer disease.