Animals ; Disease Models, Animal ; Facial Nerve Injuries ; etiology ; Female ; Male ; Rabbits

To generate recombinant adenovirus expression vector of human Sema4C gene and observe its expression in mouse myoblasts cell line C2C12 for ensuring easy access to investigate the role of Sema4C gene during myogenesis. The recombinant plasmid was packaged and amplified after being transfected in HEK293 cells through Lipofectamine. After infecting C2C12 myoblasts with recombinant adenovirus vector, the adenoviral infection efficiency was determined by confocal microscope which showed that the expression of green fluorescence could be detected at 12h and then reached peak at 24h after recombinant adenovirus infection. The infection efficiency was almost 100% confirmed by FACS examination. Detection of WB indicated that the expression of Sema4C in C2C12 of recombinant adenoviral infection group was significantly higher than that of the control group (P

The nucleic acid fragment of porcine circovirus type 2(PCV2)was amplified using PCR from the tissues of a domesticated wild boar with suspected postweaning multisystemic wasting syndrome(PMWS).The sample were then inoculated into the Dulac cells and passaged for 8 generation.The 4 nucleic acid fragments covered the complete genome for PCV2 were obtained by over-lapping PCR and sent to sequence.The sequence of genome was compared with other 9 strains originated from piglets in the same area.The result showed that these strains were in high homology.

Hide melting presents itself as one of the most critical processes in the production of donkey-hide gelatin. Here a NIR-based method was established for the rapid analysis of in-process hide melting solutions as well as for end-point determination of this process. Near infrared (NIR) spectra of hide melting solutions were collected in transflective mode. With the contents of total nitrogen determined by the Kjeldahl method as reference values, partial least squares regression (PLSR) was employed to build calibration models between NIR spectra and total nitrogen. Model parameters including wavelength range and PLS factors were optimized to achieve best model performance. Based on the contents of total nitrogen predicted by calibration model, end point of hide melting was determined. The constructed PLS model gave a high correlation coefficient (R2) of 0.991 3 and a root mean square error of prediction (RMSEP) of 0.807 g x L(-1). With the predicted total nitrogen and predefined limit, decisions concerning the proper times of melting were made. This research demonstrated that NIR transflectance spectroscopy could be used to expeditiously determine the contents of total nitrogen which was subsequently chosen as the indictor for determining the end-point of hide melting. The proposed procedure may help avoid unnecessary raw material or energy consumption.
Animals ; Calibration ; Endpoint Determination ; methods ; Equidae ; anatomy & histology ; Gelatin ; chemistry ; Least-Squares Analysis ; Nitrogen ; analysis ; chemistry ; Skin ; chemistry ; Spectrophotometry, Infrared ; Time Factors ; Transition Temperature

AIM: To evaluate cell proliferation, apoptosis and migration of human retinal pigment epithelial cells ( RPE) when co - cultured with human marrow mesenchymal stem cells ( hMSCs ) in condition of hypoxia and hyperglycemia so as to explore possible mechanisms of diabetes aggravating choroidal neovascularization ( CNV) preliminarily.
METHODS:Both hMSCs and RPE cells were co-cultured in a transwell system. The experiment was divided into four groups: 21% O2 with 5. 56mmol/L glucose ( control group, A ), 21% O2 with 30mmol/L glucose ( hyperglycemia and normoxia group, B ) , 5% O2 with 5.56mmol/L glucose ( normoglycemia and hypoxia group, C ) and 5% O2 with 30mmol/L glucose ( hyperglycemia and hypoxia group, D) . Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of RPE cells in each group at 12, 24 and 48h respectively. Flow cytometry was performed to observe apoptosis of RPE cells at 24h. Additionally, we assessed migration
capabilities of RPE via transwell assay under the condition of hyperglycemia and hypoxia by co-culturing of hMSCs.RESULTS:In this co-culturing system, at 12, 24 and 48h, group B (1. 61±0. 41, 1. 80±0. 34;1. 91±0. 35), C (1.34±0. 46, 1. 94±0. 40, 2. 14±0. 41) and D (1. 98±0. 47, 2.26±0.42, 2. 55±0. 40) showed significantly higher proliferation rate than group A (0. 92±0. 45, 1. 27±0. 32, 1.59±0. 41, P<0. 05). The migration capabilities of RPE in group B (149. 5±9. 19), C (140±9. 90) and D (170. 5±7. 78) increased dramatically compared with group A ( 114. 5±7.78, P<0.05) at 24h, whereas there was no significant difference of apoptosis ratio among four groups (P>0. 05).
CONCLUSION:By coexistence with hMSCs, the synergy of hyperglycemia and hypoxia can improve migration and proliferation of RPE cells, and have no effect on apoptosis of RPE cells within short period.

AIMTo explore the effects of interleukin-1beta (IL-1beta) on inducible nitric oxide synthase (iNOS)-nitric oxide (NO) system activity in arginine vasopressin (AVP)-induced rat cardiac fibroblasts (CFs).

METHODSCFs were isolated by trypsin digestion method. Nitric acid reductase method, spectrophotometry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect NO contents, NOS activity and iNOS mRNA expression.

RESULTSAVP significantly increased iNOS mRNA expressions, NOS activity and NO contents (P < 0.05) in CFs. IL-1beta enhanced the effects of AVP on iNOS-NO system activity in a concentration-dependent manner, moreover the iNOS mRNA expressions, NOS activity and NO contents of AVP + 3 ng/ml, AVP + 5 ng/ml IL-1beta group were both significantly higher than those of AVP group (P < 0.05). But when IL-1beta concentration increased to 5 ng/ml, the iNOS mRNA expressions, NOS activity and NO contents did not increase accordingly, slightly decreased instead.

CONCLUSIONWithin certain range of concentrations IL-1beta cooperates with AVP to increase iNOS-NO system activity in CFs.

Animals ; Arginine Vasopressin ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Interleukin-1beta ; pharmacology ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Rats ; Rats, Sprague-Dawley

Background Chemical crosslinking agent can be used to strengthen the intensity of sclera tissue,but the intensity of the sclera may be influenced by different crosslinking methods.Objective The aim of this study was to compare the effectiveness of collagen crosslinking on porcine sclera between whole-eye crosslinking method and scleral strip crosslinking method.Methods Whole-eye crosslinking or sclera strip crosslinking was performed on 70 fresh porcine eyeballs in five groups using 1％ genipin,1％ glutaraldehyde or PBS respectively for 40 minutes.After crosslinking,10 sclera strips with l0 mm×4 mm from the temporal lateral were prepared in every group for the stress-strain measurement using a Instron5848 microtester,and the other 4 scleral strips in each group were extracted for the thermal shrinkage temperature test.Results Biomechanical property test reveled that the elastic modulus value of sclera strips reduced by 70.0％-82.8％ in the whole-eye crosslinking method group compared with scleral stip crosslinking method group after treated with 1％ genipin ((8.98 ± 1.81) MPa vs.(10.85 ± 1.83) MPa,t =3.375,P =0.003)) and 1％ glutaraldehyde((12.78 ±2.91) MPa vs.(18.25 ±5.16) MPa,t =4.007,P =0.001)) ;The tensile stress of whole-eye crosslinking method group was 54.9％-90.1％ of scleral stips method group,showing significant decline after corsslinked of whole-eye in 5％,10％,15％ and 20％ strain conditions (all P ＜ 0.05).Thermomechanics test showed that the thermal shrinkage temperature was lower in the whole-eye crosslinking group compared with scleral stip crosslinking group after treated with both 1％ genipin ((68.8 ±0.9)℃ vs.(74.8± 1.3)℃,t=11.129,P=0.000)) and 1％ glutaraldehyde((73.3±0.9)℃ vs.(79.3±1.3)℃,t=11.112,P=0.000)).Conclusions Different crosslinking methods have an influence on the efficacy of collagen crosslinking on porcine sclera.Sclera strip crosslinking offers a better crosslinking intensity for selera.

Background Monosodium L-glutamate (MSG) is a food flavour enhancer and its potential harmfulness to the heart remains controversial.We investigated whether MSG could induce cardiac arrhythmias and apoptosis via the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor.Methods Myocardial infarction (MI) was created by ligating the coronary artery and ventricular arrhythmias were monitored by electrocardiogram in the rat in vivo.Neonatal rat cardiomyocytes were isolated and cultured.Cell viability was estimated by 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay.Calcium mobilization was monitored by confocal microscopy.Cardiomyocyte apoptosis was evaluated by acridine orange staining,flow cytometry,DNA laddering,reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Results MSG (i.v.) decreased the heart rate at 0.5 g/kg and serious bradycardia at 1.5 g/kg,but could not induce ventricular tachyarrhythmias in normal rats in vivo.In rats with acute MI in vivo,however,MSG (1.5 g/kg,i.v.) induced ventricular tachyarrhythmias and these arrhythmias could be prevented by blocking the AMPA and N-methyl-d-aspartate (NMDA) receptors.Selectively activating the AMPA or NMDA receptor induced ventricular tachyarrhythmias in MI rats.At the cellular level,AMPA induced calcium mobilization,oxidative stress,mitochondrial dysfunction and apoptosis in cultured cardiomyocytes,especially when the AMPA receptor desensitization were blocked by cyclothiazide.The above toxic cellular effects of AMPA were abolished by AMPA receptor blockade or by H2O2 scavengers.Conclusions MSG induces bradycardia in normal rats,but triggers lethal tachyarrhythmias in myocardial infarcted rats probably by hindering AMPA receptors.AMPA receptor overstimulation also induces cardiomyocyte apoptosis,which may facilitate arrhythmia.

OBJECTIVETo establish a simple but effective method of laser scanning confocal microscopic imaging for Ca2+ oscillations of pancreatic acinar cells in adult mice.

METHODSPancreatic acinar cells from adult Kunming mice were isolated acutely with collagenase, and then loaded with fluo-4-AM, a Ca2+ indicator. A laser scanning confocal microscope armed with 488 nm laser was employed to record the dynamic fluorescent signals in-time and synchronously while acetylcholine (ACh) was added in the pancreatic acinar cells.

RESULTS(1) The classic pancreatic acinar cell Ca2+ oscillations were induced by a certain concentration of ACh (100 nmol/L) successfully and steadily, which could be blocked by atropine completely. (2) Plasmic Ca2+ oscillations from different parts of one acinar cell were usually with different amplitudes and almost the same frequencies. But both of amplitudes and frequencies were different among different cells. (3) The acinar cell Ca2+ oscillations were induced by ACh in a concentration-dependent manner.

CONCLUSIONThe laser scanning confocal microscopic imaging for adult mouse pancreatic acinar cell Ca2+ oscillations was established successfully. The features of being easy to use, direct to see lively, high efficiency and good flexibility make it a popular tool for researchers to choose.

Acinar Cells ; chemistry ; Animals ; Calcium ; analysis ; Calcium Signaling ; Cells, Cultured ; Mice ; Microscopy, Confocal ; methods ; Pancreas ; cytology

Little is known about the non-structural protein 6(NSP6)of rotavirus.This report describes expression of the NSP6 of a group A human rotavirus strain TB-Chen in bacteria,and its immunological properties and cellular distribution.The results showed that the recombinant NSP6(rNSP6)was expressed in high efficiency without any other proteins fused(possesses about 34.2% of total bacterial proteins).rNSP6 elicited mono-specific antibodies in immunized guinea pigs and the antibodies could react with the rNSP6 itself and the viral NSP6 proteins synthesized in SA11-or Wa-infected MA104 cells in Western blot and immunofluorescence assay.The NSP6 distributed evenly in the cytoplasm mainly around the nucleus of virus-infected cells,no viroplasm-like gathering observed;The top amount of NSP6 synthesized in SA11-infected cells or Wa-infected cells could be detected at 12h after infection.This is the first report about the high expression of entire NSP6(without any other proteins fused)in prokaryotic expression system and detection of NSP6 synthesis in virus infected cells by immunofluorescence assay.The results are important to understand the structure,biological properties and further application of the NSP6.