Objective To explore the effect of hypoxia-ischemia(HI)on expression of second mitochondria-derived activator of caspase proteinc(Smac)in brain tissue of neonatal rats.Methods All neonatal rats were divided randomly into HI group,pseudo-operation group,and normal control group.The animal models of HIE were made.At 24,48,72,96 h after HI,Western blot was used to detect the expression of Smac protein in hippocampus.The relative activity of Caspase-3 in hippocampus tissue was determined by colorimetric assay.Results In HI group,at 24,48,72,96 h after HI,the expression levels of Smac protein in hippocampus significantly increased compared with that of pseudo-operation group and normal control group(Pa

Objective To explore the roles and possible mechanism of calcium-sensing receptor(CaSR) in cell cardiac hypertrophy model using angiotensin Ⅱ (Ang Ⅱ ).Methods The cultured neonatal rat ventricular myocytes were treated with Ang Ⅱ as cell cardiac hypertrophy model.Hypertrophic neonatal rat cardiomyocytes were treated with GdCl3(a specific agonist of CaSR) and/or with Ro318220(a specific inhibitor of PKC pathway).To evaluate the status of cardiac hypertrophy,cell diameter was observed by HE dyeing,and protein content was determined through coomassie brilliant blue protein kit.The intracellular calcium concentration( [ Ca2+]i) was determined by laser scanning confocal microscope.The protein expression of CaSR and PKC pathway were analyzed using Western blotting.Results ①Compared to the control group(0.1263 ± 0.0443),the protein expression of CaSR was increased in Ang Ⅱ group and in GdCl3 group(0.1963 ± 0.0375,0.2778 ± 0.0564,all P＜ 0.05).Moreover,compared with Ang Ⅱ alone,the increase was significant in GdCl3 group(P ＜ 0.05).②Compared to control group(222.70 ± 22.09),AngⅡ group(392.16 ± 36.85) remarkably increased [Ca2+]i(P＜ 0.05),and this increase of [Ca2+]i was further enhanced in GdCl3 group (502.60 ± 44.21) versus Ang Ⅱ group (P ＜ 0.05).③Compared to control group,Ang Ⅱ could induce cardiomyocyte hypertrophy,and GdCl3 enhanced the effect.Moreover,this enhancement was attenuated by Ro318220.④Compared to control group(0.27 ± 0.07,0.69 ± 0.06,0.87 ± 0.04),the protein expression of PKCα,PKCε and PKCδ was increased in Ang Ⅱ group(0.60 ± 0.16,1.02 ± 0.13,1.20 ± 0.18,all P＜ 0.05) and the protein expression of PKCα,PKCε was increased in GdCl3 group(0.82 ± 0.16,1.34 ± 0.12,all P ＜ 0.05).Moreover,compared with Ang Ⅱ group,the protein expression of PKCα,PKCε was obviously increased in GdCl3 group (all P ＜ 0.05);compared with GdCl3 group,the protein expression of PKCα,PKCε(0.41 ± 0.10,0.85 ± 0.14) was obviously decreased in Ro318220 group(all P ＜ 0.05).Conclusions CaSR is involved in cardiac hypertrophy induced by Ang Ⅱ through PKC pathway in cultured neonatal rat cardiomyocytes.

Objective: Changes in the ?2 integrin of adhesion molecules between cells are closely associated with the invasion and migration of tumor cells.This study aimed at the effect of anti-?2 integrin on the invasion and migration of osteosarcoma MG63 cells. Methods: The osteosarcoma MG63 cell line was cultured in the DMEM medium.The effect of the anti-?2 integrin monoclonal antibody on the migration and invasion of tumor cells were measured by scratch assay and Transwell assay.The migration and invasion cells were stained by Crystal violet staining and counted under the hundredfold microscope.Results: Compared with the control group,the migration and invasion abilities of the MG63 cells were significantly decreased in the anti-?2 treatment groups.Conclusion: Anti-?2 integrin may inhibit the migration and invasion of osteosarcoma cells.

OBJECTIVETo develop a method for determining epichlorohydrin in the workplace air by gas chromatograph-electron capture detector (GC-ECD).

METHODSEpichlorohydrin in the workplace air was collected by activated charcoal tubes, desorbed using acetone, and analyzed by GC-ECD.

RESULTSA good linearity was obtained in the range of 1.0-50 µg/mL (r=0.999 7). The detection limit was 0.012 µg/ml, while the recovery rate was 88.1% and relative standard deviation ranged from 1.11% to 3.57%. The samples could be stored for seven days at room temperature.

CONCLUSIONThis method effectively eliminates the interferences of alkanes on determination of epichlorohydrin and improves the sensitivity by 1 to 2 orders of magnitude, which can solve the problem of detection limit above standard in GBZ/T 160.58-2004.

Objective:To investigate the anti-oxidative stress effects of microRNA 125b (miR-125b) on lens epithelial cells (LECs) and its possible mechanism.Methods:Twenty-four anterior capsule specimens were collected from 24 eyes of 24 age-related cataract patients during phacoemulsification and 20 normal anterior capsule specimens were obtained from 20 eyes of 20 donors in Henan Eye Hospital from July 2018 to March 2019 under the approval of a Medical Ethics Committee of Henan Eye Hospital (No.YKYY20193151).The reverse transcription PCR and Western blot assay were employed to detect and compare the relative expression levels of miR-125b and nuclear factor E2-related factor 2 (Nrf2) in different specimens.The human lens epithelial cell line HLEB-3 was divided into control group and oxidative stress model group.The oxidative stress models were established by coculture with different concentrations (100, 200, 400 μmol/L) of H 2O 2 for 24 hours, and the cells were cultured with normal medium without H 2O 2 in the control group.The reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe, and the activities of total-antioxidative capability (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as malondialdehyde (MDA) concentration were detected by ELISA, and compared among the groups.The expression levels of miR-125b and Nrf2 were detected by reverse transcription PCR and Western blot assay, respectively.The cells were transfected with miR-125b mimics, miR-125b control and miR-125b inhibitor for 24 hours, respectively, and ROS content was detected by DCFH-DA fluorescent probe and T-AOC, SOD and GSH-Px activities as well as MDA concentration were detected by ELISA and compared among different transfected groups.A dual luciferase reporter assay was used to assess an association between miR-125b and Nrf2.The expression level of Nrf2 protein was detected by Western blot assay and the expression levels of Nrf2 and Keap1 were assayed and located by immunofluorescence double staining. Results:The relative expression levels of miR-125b and Nrf2 in the normal lens anterior capsule specimens were 0.21±0.03 and 0.27±0.06, which were significantly lower than 0.89±0.05 and 0.84±0.12 in the cataract specimens, respectively ( t=15.355, P<0.05; t=18.647, P<0.05).The relative expression levels of miR-125b and Nrf2 were significantly increased in various H 2O 2 treated groups in comparison with the control group and were gradually elevated with the increase of H 2O 2 concentration (all at P<0.05).Compared with the control group, the T-AOC, SOD and GSH-Px activities were reduced, and ROS content and MDA concentration were significantly ascended (all at P<0.05).Compared with the miR-125b control group, the T-AOC, GSH-Px and SOD activities were increased, and ROS content and MDA concentration were decreased in the miR-125b mimics group (all at P<0.05).In addition, the T-AOC, GSH-Px and SOD activities were significantly weakened, and ROS content and MDA concentration were significantly increased in the miR-125b inhibitor group in comparison with the miR-125b control group (all at P<0.05).Dual luciferase reporter assay showed that miR-125b targeted to the expression of Nrf2 in the H 2O 2 model cells.The fluorescence of Nrf2 in the cytoplasm was the strongest with more nuclear transfer in the miR-125b mimics group, and the expression intensity of Keap1 in the cytoplasm was weaker.The expression of Nrf2 was the weakest with less nuclear transfer in the miR-125b inhibitor group, and the expression level of Keap1 in the cytoplasm was stronger. Conclusions:MiR-125b can enhance the anti-oxidative stress of LECs in age-related cataractous eyes probably by upregulating the expression of Nrf2 and activating the Keap1/Nrf2 signaling pathway.

The study of mammary gland bioreactor is in the ascendant. In order to generate transgenic goats of well-controlled expression of exogenic genes, we constructed a human lactoferrin (hLF) gene targeting vector containing promoter, exon 1, intron1 and some of exon 2 (about 6.1 kb fragment) and exon 6 approximately 9 (about 3.3 kb fragment) of the goat beta-casein gene as well as hLF minigene, neo gene inserted into them and tk gene ligated to the 3' end of the construct. The 9.4 kb goat genomic sequences as homologous arms were initially amplified by PCR with local goat tissue DNA. The expression vector was named pBC-tk-neo-hlf. Then the recombinant plasmid pBC-tk-neo-hlf containing hLF minigene was transfected into mice mammary tumor cell line C127 by liposome, cell clones were selected with G418. After proliferating, the transfected cells were induced with insulin, luteotropic hormone and hydrocortisone. The result of Western-blotting analysis showed that the transfected cells can secrete hLF protein, and the recombinant protein expressed in cultured cell supernatant has the similar molecular weight as the native protein. The expression level detected by ELISA was 0.21 microg/mL. This result indicated that the targeting vector could efficiently direct the expression of hLF in mammary cells,and it confirmed the validity of the constructed vector. At the same time, C127 cell line proved to be useful for evaluating the regulation of a foreign gene expression in mammary gland specific expression vector.
Animals ; Caseins ; genetics ; Cell Line, Tumor ; Goats ; Humans ; Lactoferrin ; genetics ; Mammary Glands, Animal ; cytology ; metabolism ; Mice ; Molecular Weight ; Transfection

BACKGROUND: Previous studies showed that the prevalence and extent of carotid artery stenosis increased with thedevelopment of coronary artery disease. There was a higher incidence of intracranial small-vessel disease, but lower of carotid artery disease in the Chinese stroke patients as compared with the white.OBJ ECTIVE: To observe the distribution of carotid and intracranial artery stenosis in patients with 3-vessel coronary artery disease.DESIGN: An observational study.SETTING: Department of Neurology; Heart Center, Beijing Chaoyang Hospital affiliated to Capital Medical University.PARTICIPANTS: From August 2003 to August 2004, The coronary angiography was performed in the outpatients and inpatients suspected to be coronary arteriosclerotic cardiopathy in the Department of Neurology, Beijing Chaoyang Hospital affiliated to Capital Medical University, and 126 patients of them with 3-vessel diseases were examined with carotid arteriography, including 56 males and 70 females, 47-76 years of age. Informed contents were obtained from all the participants.METHODS: Digital substraction angiography (DSA) was performed immediately after coronary angiography in the 126patients. All catheterizations were performed through a transfemoral approach using the Seldinger technique, and thenan appropriate amount of nonionic Ominipaque was injected. The angiography of bilateral carotid arteries, subclavian artery, or vertebral artery was taken from different angles. The percentage of stenosis was calculated directly from DSAmachine. Evaluative standards: Based on the stenosis degree from carotid angiography results, the patients were divided into 5 categories as normal, mild stenosis, moderate stenosis, severe stenosis and complete occlusion.MAIN OUTCOME MEASURES: Severity of carotid stenosis.RESULTS: All the 126 patients were involved in the final analysis of results. There were 13 (10.32%), 18 (14.29%), 12(9.5%), or 10 (7.9%) patients found to have mild, moderate, severe carotid stenosis, or complete occlusion, and the incidences of these changes were fairly similar. However, the incidence of angiographic carotid stenosis coupled with 3-vessel carotid artery disease was 42.06%.CONCLUSION: The prevalence of carotid stenosis in patients with 3-vessel carotid artery disease was as high in the Chinese population as that in Westem countries. In patients with 3-vessel disease, the prevalence of carotid stenosis was higher than that of intracranial artery stenosis, thus they may require both coronary and carotid interventions.

Objective To evaluate the possible molecular pathogenesis of intractable temporal lobe epilepsy. The potassium ion channel gene KCNJ4 encodes one of the subfamilies of Kir channels, Kir2.3 subunit, which may play an important role in modulating neuronal excitation. Interference in the function or expression of this gene would cause disturbance of ionic concentrations, thus leading to seizure activity. Methods Reverse transcription polymerase chain reaction (RT-PCR) and Western-blot analysis were used to measure the expression alterations of KCNJ4 mRNA as well as its protein product Kir2.3 channel in temporal cortex samples from patients who had undergone temporal lobectomy for intractable epilepsy (n=12). Tissue from 10 subjects who did not have epilepsy served as controls. Results The expression of KCNJ4 mRNA (0.438?0.178) and its protein Kir2.3 (M 50=0.063) were significantly decreased in epileptic brain compared with the controls (P

Apoptosis of PK-15 cells induced by Foot-and-Mouth Disease Virus (FMDV) in vitro was reported in this paper. Typical cell apoptosis was detected by use of Hoechst 33258 fluorescence probe, agarose gel electrophoresis and in situ end-labeling (TUNEL). After PK-15 cells were infected by titration of 4.8 lg TCID50/mL FMDV for 32 h, apoptosis characteristics of nuclear condensation, fragmentation, accompanied by apoptotic bodies formation (Hoechst 33258 staining), 180-200 integer-fold sized pieces DNA Ladders (agarose gel electrophoresis) and strong green fluorescence dots (TUNEL) were all exhibited, and cell apoptosis was approximately 20%. In addition, the quantitative analysis of apoptosis in PK-15 cells induced by FMDV showed that apoptosis was correlated with infection of virus, and it was also time-dependent. Results indicate that FMDV can induce apoptosis of host cells and apoptosis plays an important role in the cytopathogencity effect of FMDV.

Objective To investigate a method of repairing complex tissue defects one stage in one finger or several fingers for more fine recipient site repairing and less donor area traum. Methods From August 2007 to August 2010,eight cases of 20 fingers were reconstructed according to the state of complex tissue defects in a hand,second toes (right or left side) were split into two or three parts as complex tissue flaps that may including joints,tendons,skin,nail beds,et al.These flaps then were translated to hand to repair complex tissue defects by anastomosing vessels.Results Twenty fingers in 8 cases were successfully reconstructed.Follow-up ranged from 3 months to 36 months,external appearance were fine.According to the Trial Evaluation Standard of Reconstructed Finger Function of Handsurgery Society of China,sixteen fingers were excellent,three fingers were good,one finger was fine.And there was no effect on foot.Conclusion Spliting single second toe is a good method for repairing more complex tissue defects in a hand.