Objective To investigate the therapeutic effects of mesenchymal stem cells (MSCs) on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in C57BL mice.Methods Different doses of MNU (30 mg · kg-1,45 mg · kg-1,60 mg · kg-1,75 mg · kg-1 and 90 mg · kg-1) were injected to C57BL mice for 7 days.Then electroretinogram (ERG) detection and HE staining were performed to examine retinal electrophysiological function and morphological changes on day 1,day 3 and day 7 after MNU treatment,respectively.Then we could explore the optimum condition to establish stable animal model of retinitis pigmentosa.MSCs were transplanted to C57BL mice by intravitreal or tail intravenous injection.Then ERG detection and HE staining were performed to evaluate the effect of MSCs on retinitis pigmentosa induced by MNU.Results When compared with control group,30 mg · kg-1 and 45 mg · kg-1 MNU could cause mild retinal damage in morphology and function in mice;while 60 mg · kg-1 and above dose of MNU induced serious retinal damage,leading to decreased ERG amplitude of the retina (all P ＜ 0.001) and outer nuclear layer (ONL) thickness (all P ＜ 0.001).On day 1 and day 3 after single dose of 60 mg · kg-1 MNU injection,ERG amplitude of the retina was decreased,and outer nuclear layer thickness became thin;while the retinal damage was serious badly in morphological structure on day 7,with the ERG amplitude extinguished (all P ＜ 0.001),ONL thickness thin (all P ＜ 0.001) and internal and external nuclear layer fusion.When compared with MNU alone treatment group,following injection of 60 mg · kg-1 MNU for 1 day MSCs were transplanted to C57BL mice by intravitreal or tail intravenous injection,and the amplitude of ERG and retinal ONL thickness were increased on day 7 after MSCs transplantation (all P ＜ 0.001).Conclusion MSCs transplantation has a certain therapeutic effect on MNU-induced retinitis pigmentosa in C57BL mice.

ObjectiveTo investigate the effects of grape seed proanthocyanidin extracts (GSPE) on advanced glycation end product receptor (RAGE),NF-Κb and connective tissue growth factor (CTGF) in the myoeardium of diabetic rats.MethodsTotal 30 streptozotocin (STZ) induced diabetic rats were randomly divided into 2 groups:diabetic group (DM1,n-15) and GSPE (250 mg/ kg,i.g) treated diabetic group (DM2,n= 15).Another two control groups:normal rats(C1,n= 10)and normal rats treated with GSPE (250 mg/kg,i.g) (C2 group,n= 10) were also observed.After 24 weeks,blood was collected to measure fasting plasma glucose (FBG) and RAGE.The protein expression of NF-Κb was determined in myocardial tissue by immunohistochemical staining and Western blot.The protein expressions of RAGE and CTGF were measured by Western blot.Results The levels of FBG and RAGE were significantly higher in diabetic rats than in control rats (P＜0.05).After GSPE treatment,RAGE level significantly reduced (P＜0.05),but FBG had no change in diabetic rats.The protein expressions of RAGE,NF-Κb and CTGF in the myocardial tissue of diabetic rats had marked increase compared with control rats (P＜ 0.05),however,their levels significantly reduced after GSPE treatment (P＜0.05).ConclusionsGSPE may protect diabetic rats against cardiomyopathy,possibly by decreasing the protein expressions of RAGE,NF-Κb and CTGF.

Objective To analyze the protein expression changes of retina in diabetic mice using isobaric tags for relative and absolute quantitation (iTRAQ) approach and to study proteins of apoptosis.Methods 8 diabetic mice were chosen as the diabetic model group (DM group),8diabetic mice as the normal control group.The animals were housed in wire-bottomed cages and received normal pellet chow and tap water in a constant environment.After 10 weeks,all mice were killed,and their retina were dissected.After hematoxylin and eosin(H&E) staining,the sections of retina were examined using light microscopy.The changes of protein expression in retina were studied using iTRAQ approach.Expression of apoptosis associated proteins was analyzed using ingenuity pathway analysis (IPA).Results Compared with control group,mice retina in DM group developed looser structures,tissue edema and obvious telangiectasia under light microscopy.Using iTRAQ approach,a total of 348 differential proteins were identified.Among those proteins,16 proteins were related with apoptosis,including Ataxin-10,Protein NDRG1,mucin-4,Aquaporin-1 and annexin A4,etc.There were 8 apoptosis-related proteins in retina with up-regulation,and the other 8 proteins with down-regulation in the DM group.The relationship between these proteins were analyzed and charted by IPA.Conclusions Apoptosis may be involved in the development of diabetic retinopathy.The identification of the apoptosis-related proteins will be helpful for the further study.

Objective To observe the effect of mPFC neuron synaptic plasticity changes in the formation of morphine related reward memory.Methods 40 SD rats were administered morphine (10 mg/kg,ip) or saline (2 ml/kg,ip)and sacrificed 0,2,4 and 8 h after the treatment.The temporal profile of activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) expression in medial prefrontal cortex (mPFC) was analyzed.Another 40 rats receiving a single injection of morphine at different doses (0,5,10 or 20 mg/kg),and rats were sacrificed by decapitation 2 h later.In mPFC,changes of Arc/Arg 3.1 protein was analyzed by Western Blot,Arc/Arg 3.1 positive cells was detected by immunohistochemistry (IHC),and number of spines were analyzed by Golgi-cox method.In the second experiment,CPP model was established by 5 mg/kg morphine for 8 days.Arc/Arg 3.1 antisense oligodeoxynucleotide (AS) or the control (CS) was microinjected into mPFC 15 minutes before each morphine injection,then CPP score was evaluated.Results Compared with saline groups,Arc/Arg 3.1 protein,Arc/Arg 3.1 positive cells,number of spines ((1.01±0.04) vs (1.58±0.18),P＜0.01 ; (42.80±7.63) vs (74.47±8.02),P＜0.01 ;(17.27±5.64) vs (39.47±7.56),P＜0.01) were significantly increased 2 hours after morphine administration.All three doses of morphine (5,10 and 20 mg/kg) increased Arc/Arg 3.1 protein expression in the mPFC,and there were no dose-dependent effects.In CPP experiments,compared with microinjection of Arc/Arg 3.1 CS (0.74±0.02),Arc/Arg 3.1 AS microinjection significantly decreased the CPP score (0.51±0.01) in morphine group (P＜0.01).Conclusion It is enough to increase Arc/Arg 3.1 protein content and synaptic plasticity in mPFC by 10 mg/kg,and the changes implied in formation of morphine relative reward memory.

Objective To study the protective effect of intraaortic protamine injection on lung in infants undergwent opening heart operation by cardiopulmonary bypass surgery. Methods Sixty infants (age ≤ 1 year,weight ≤ 10 kg)who accepted opening heart operation by cardiopulmonary bypass surgery were randomly assigned into 2 groups ( n = 30 in each group) reciving intra-aortic and intra-venous protamine injection respectively. P-peak, P-plate, CL, Oxygenation Index, the number of WBC and neutrophil segregated in lungs were compared between two groups before injecting protamine and 10 minutes, 1 hour, 3 hours after injecting protamine. The time of mechanical ventilation were compared as well. Results P-peak, P-plate, the number of WBC and neutrophil segregated in lungs of intra-aortic injection group significantly decreased than intra-venous injection group at 1 hour, 3 hours after injecting protamine (t =2.743, 3.512; 3.218, 3.469; 3.716, 5.243; 3.853,4. 783 respectively, Ps ＜ 0. 05 ), while the CL and Oxygenation Index increased significantly ( t = 3. 976,4. 267; 4. 557,4. 265 respectively, P ＜ 0. 05 ). The duration of mechanical ventilation follow operation in intraaortic injection group ( [8. 03 ± 5. 14] h ) was shorter compared with intra-venous injection group ( [10. 56 ±6.95]h) (t =2.599,P＜0.05). Conclusion By intra-aortic protamine injection the lung injury decreased significantly. It shows good protective effect on lung in infants underwent opening heart operation by cardiopulmonary bypass surgery.

Objective:To obtain highly purified recombinant human IGF-Ⅰ(rhIGF-Ⅰ) and identify it.Methods:rhIGF-Ⅰ Was purified through ion-exchange chromatography and gel filtration chromatography after the inclusion bodies of rhIGF-Ⅰ were extracted from Escherichia coli. The recombinant protein was characterized through molecular weight assay, Western-blot, and fluorescent chromatography. The renaturation and biological assay of rhIGF-Ⅰ were investigated. Results and Conclusions: The purity of rhIGF-Ⅰ was higher than 99%. The analysis of molecular weight, Western-blot, fluorescent chromatography and sequences of NH2-terminal 15 amino acids were same as those anticipated. 3-10 mg/ml was the concentration of renatured rhIGF-Ⅰ to support half-maximal stimulation of cell proliferation with BALB/c 3T3 cells.

ABSTRACT:Objective To explore the protective effect of the antioxidant N‐acetylcysteine (NAC) on the retinal nerve tissue of early diabetic rats .Methods We randomly divided 60 healthy adult Sprague‐Dawley (SD) rats weighing between 180 g and 220 g into 2 groups:normal control (CON , n=20) and diabetic (DM , n=40) .By intraperitoneal injection of streptozotocin (60 mg/kg) ,the model of diabetic rats was established .The rats were considered diabetic only when they had hyperglycemia (set at ≥16 .7 mmol/L) (32) .The CON group was injected with the same amount of citric acid and sodium citrate buffer solution .After successful model establishment ,the diabetic rats were randomly divided into 1‐month diabetes group and 2‐month diabetes group ,with 16 rats in each group .The left eye of each experimental diabetic rat was set for diabetes control group (D) while the right eye was set as NAC treatment group (NAC) .At 2 weeks of diabetes ,4μL (1 .6μg/μL) of NAC was injected into the vitreous chamber of NAC group and 4μL (0 .01 mmol/L) of PBS was injected into the vitreous chamber of the other diabetic rats .The thickness changes of outer nuclear layer retina was observed by HE ,ultrastructural changes of retinal ganglion cells were observed under the transmission electron microscope ,and the number of retinal ganglion cells was detected by immunofluorescence method .Results At different time points ,retina outer nuclear layer in NAC group was thicker than in D group (P<0 .01) .However ,the NAC group and the CON group did not differ (P>0 .05) .Under the transmission electron microscope ,NAC group had more retinal ganglion cell organelles ,higher electron density of the cytoplasm ,and milder mitochondria swelling than D group .The NAC group did not differ from CON group in the ultrastructure of retinal ganglion cells . NAC group had an increased number of retinal ganglion cells at different time points compared with the D group (P<0 .01) ,but the NAC and CON groups did not differ in the number of retinal ganglion cells (P> 0 .05) .Conclusion The antioxidant N‐acetylcysteine has a protective effect on the retinal nerve tissue of early diabetic rats .

Aim To investigate the role of autophagy and its mechanism in Raji cell death induced by arse-nic trioxide. Methods Transmission electron micros-copy ( SEM) and MDC fluorescence staining were used to observe autophagy. MTT colorimetry was employed to assay the cellular proliferating activity. Cell apopto-sis and cell cycle analysis were performed using FITC-Annexin-V/PI double staining and flow cytometry ( FCM) . The expressions of LC3 and the conversion of LC3-I to LC3-II were measured by western bloting. The expression of bcl-2 mRNA and p53 mRNA were detected by reverse transcription-polymerase chain re-action ( RT-PCR ) . Results Arsenic trioxide could obviously inhibit the proliferation of Raji cells, arrest the cells at G2/M phase and induce apoptosis. Mean-while, arsenic trioxide markedly inhibited the expres-sion of bcl-2 mRNA and enhanced the expression of p53 mRNA in Raji cells. Arsenic trioxide also induced autophagy synchronously which paralleled with the in-duction of apoptosis in Raji cells, and 3-MA, an auto-phagy inhibitor, was able to reverse the arsenic triox-ide-activated autophagic activity, up-regulate bcl-2, down-regulated p53 expression and suppress the lethal effect of arsenic trioxide on Raji cells to reduce their sensitivity to arsenic trioxide. In contrast, the Rapamy-cin, an autophagy inducer, possessed the completely opposite effects on Raji cells compared with 3-MA. Conclusions The apoptosis and autophagic cell death are coexistent in arsenic trioxide-triggered death of Raji lymphoma cells, and Bcl-2 and p53 may play a key regulating role in this process.

Objective: To investigate the effect of cold self-blood cardioplegia with ulinastatin on immature myocardial cell apoptosis and protein expressions of Bcl-2, Bax in ventricular septal defect (VSD) infants.
Methods: A total of 60 infants received VSD repairing operation with cardiopulmonary bypass (CPB) in our hospital were summarized. The patients were randomly divided into 2 groups:Test group, the infants received cold self-blood cardioplegia with ulinastatin when aortic cross-clamp was closed. Control group, the infants received cold self-blood cardioplegia when aortic cross-clamp was closed. n=30 in each group. The right atrium tissue was collected before CPB and 10 min after releasing aortic cross-clamp. The index of myocardial cell apoptosis was observed by TUNEL method, and the protein expressions of Bcl-2, Bax were examined by immunohistological method.
Results: Both groups showed the higher index of myocardial cell apoptosis at 10 min after releasing aortic cross-clamp than 5 min before CPB, and the apoptosis index in Test group was lower than that in Control group, all P<0.05. The protein expressions of Bcl-2 and Bax were obviously increased at 10 min after releasing aortic cross-clamp than 5 min before CPB in both groups. Compared with Control group, Test group presented the higher Bcl-2 protein expression and lower Bax protein expression, all P<0.05.
Conclusion: Cold self-blood cardioplegia with ulinastatin could protect immature myocardum from ischemia-reperfusion injury in VSD infants during CPB operation in clinical practice.

Objective To investigate the effects of ulinastatin-containing autologous cold blood cardioplegic solution on the cardiac function of infants after cardiopulmonary bypass surgery.Methods Sixty infants younger than 10 months old,who underwent ventricular septal defect repair under cardiopulmonary bypass,were randomized into autologous cold blood cardioplegia group (30 patients,Group A)and ulinastatincontaining cold blood cardioplegia group (30 patients,Group B).CI,SI and LCWI were monitored 1 and 6 hours after opening the aorta.The time and rate of cardiac resuscitation,as well as the dependence on the inotropic drugs,were intraoperatively monitored.Results The automatic resuscitation rate in two groups was not siynificantly ( P ＞ 0.05).The time for automatic resuscitation were (34.2 ± 4.7) s and (52.1 ± 6.5 ) s for Group B and Group A,respectively ( P ＜ 0.05 ).The rate of dependence on inotropic drug were 40.0％ (12/30) and 66.7％ (20/30)for Group B and Gro~p A,respectively (P ＜ 0.05).Mter the operation,the CI,SI and LCWI of group B were higher than that of group A ( P ＜0.05 ).Conclusion Ulinastatin-containing autologous cold blood cardioplegic solution is beneficial to the functional cardiac recovery of the infants after heart bypass surgery by protecting the immature myocardium.