Background To establish the ideal animal model of retinitis pigmentosa (RP) is very important for onward relevant study.Previous research determined that N-methyl-N-nitrosourea (MNU) can selectively damage photoreceptors via intravenous injection in mammal.However,whether MNU can be used to create an RP model needs to be investigated.Objective This experiment was designed to evaluate the toxic effect of MNU on photoreceptor cells of cats.Methods MNU was injected into 20 2-year-old cats via femoral vein and randomized into 20mg/kg, 25mg/kg, 30mg/kg, 35mg/kg and 40mg/kg MNU groups,and equal amount of normal saline solution was used in the same way in 4 normal cats as the control group.The activity,pupil size and light reflex were observed after injection of MNU.The cats were sacrificed and eyeballs were enucleated for histological examination to evaluate the structural and morphological changes of photoreceptors at 24 hours,72 hours,7 days and 14 days after the administration of MNU.This experimental study complied with the Statement for the Use of Animals in Ophthalmic and Vision Research.Results Dilated pupil and inertia of light reaction were found in experimental cats on the 7th days in the various groups.In 24 hours after MNU injection,the damage of photoreceptors was primarily characterized by pyknosis and disorder.In 72 hours after MNU injection,attenuation of the outer nuclear layer and disruption of cells were seen.Loss of photoreceptors and disappearance of the outer nuclear layer were observed on the 7th and 14th day.The extent of retinal photoreceptor cell damage was dependent on the dose of MNU.Conclusion MNU can selectively induce serious damage of the photoreceptor cells in cats retina in a time- and dose-dependent manner.

Retinal vein occlusion ( RVO ) is a common vascular disease of the retina and is one of the main reasons for blindness. ln recent years, there have been some new understanding about the diagnosis and treatment of the disease, especially some new researches about treatment,for example ,in the therapy of the intravitreal injection of triamcinolone acetonide and anti-VEGFs as well as dexamethasone implant ( Ozurdex ) . This article will make a brief summarization of the progress about the diagnosis and treatment of RVO.

Objective:To investigate the existence of side population cells with the potency of stem cells in human gallbladder carcinoma cell line GBC-SD and the differences in ABCG2,Oct-4 and CD34 expression among SP cells,non-SP cells and GBC-SD cells.Methods:SP and non-SP cells were sorted from GBC-SD cells by fluorescence-activated cell sorting(FACS).The expression of ABCG2,Oct-4 and CD34 in SP cells,non-SP cells,and GBC-SD cells was detected by reverse transcription-polymerase chain reaction(RT-PCR),Western blot,flow cytometry(FCM)and immunofluorescence chemistry.Results:SP cells with stem cell potency were isolated from GBC-SD cells with a proportion of(0.64±0.08)%.The metastatic ability of SP cells was obviously higher than that of non-SP cells and GBC-SD cells(P<0.05).The expression of ABCG2 was significantly higher in SP cells than in non-SP cells and GBC-SD cells[(89.56±3.86)%vs.(1.32±0.49)%and(12.37±1.61)%,P=0.001].The expression of Oct-4 in these cells was(94.87±1.40)%,(88.16±2.34)%and(90.17±1.61)%,respectively(P>0.05).CD34 was neady absent in these cells on protein level[(1.78±0.51)%vs.(0.63±0.21)%and(0.96±0.381)%,P>0.05)],but it was highly expressed in non-SP cells and GBC-SD cells and absent in SP cells off mRNA leve;.Conclusion:SP calls which hava the potency of stem cells,exist in human gallbladder carcinoma GBC-SD cell line and have the phenotype of ABCG2+Oct-4+CD34-.

OBJECTIVETo investigate the role of CD40 ligand (CD40L) in dendritic cells (DC) of CEA transgenic mice and to evaluate the specific cellular immunity induced by activated DC.

METHODSBone marrow cells of the CEA transgenic mice were used to generate immature dendritic cells under the condition of GM-CSF and IL-4. CD40L was added to activate dendritic cells into mature phenotype. Dendritic cells cancer vaccine was pulsed with CEA526-533 peptide which made the vaccine specific for cancer immunity. The immunophenotype molecules were identified by flow cytometry. The cytokines produced by cells were determined by ELISA. T cells proliferation was measured by (3)H-thymidine essays.

RESULTSImmunophenotype molecules expressions of CD40L-activated dendritic cells were significantly higher than those in control group. IL-12 secretion by CD40L-activated dendritic cells was (937.81+/-51.99) pg/10(6) DC, significantly higher than that in control group [(83.06+/-8.58) pg/10(6) DC, P<0.01]. CD8(+) T cells proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.05), and the secretion of IFN-gamma was(33.900+/-4.550) ng/L, significantly higher than that in control group [(5.226+/-0.460) ng/L, P<0.01]. Splenocytes proliferation induced by CD40 L-activated dendritic cells was stronger as compared to control group (P<0.01), and the secretion of IFN-gamma was (69.802+/-11.407) ng/L, significantly higher than that in control group [(2.912+/-0.562) ng/L, P<0.01].

CONCLUSIONThe method of using CD40L to stimulate bone marrow-delivered dendritic cells promotes the maturation and activation of dendritic cells, which enhances the cellular immunity in CEA transgenic mice.

Animals ; CD40 Ligand ; immunology ; physiology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Immunity, Cellular ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

Objective To explore the effects of intraventricular administration of insulin on the expressions of Bcl-2,Bax mRNA and neuronal hippocampus apoptosis in rats after cardiopulmonary resuscitation (CPR).Methods This experiment was implemented in the animal Laboratory center of Xuanwu Hospital of Capital Medical University.Thirty male SD rats were randomly (random number)divided into three groups:control group (n=6),CPR group (n=12),insulin treated group ( n =12).CPR was performed at 6 minutes after ventricular fibrillation induced by transesophageal overdrive pacing.Resuscitation procedures lasted until restoration of spontaneous circulation (ROSC).ROSC was defined as the recovery of the supraventricular heart rates and the increase of mean arterial pressure (MAP) ＞ 60mmHg for more than 10 minutes.Ten minutes after ROSC in rats,12.5 μL ( 1 U) regular insulin was injected into the left ventricle in the insulin group,and 12.5 μL isotonic saline was injected the control and CPR groups at least 10 minutes.Real-time PCR was used to observe the expressions of Bcl-2,Bax mRNA in hippocampus CAI after reperfusion 24 h and 72 h.TUNEL staining was used to observe the neuronal apoptosis in all groups after reperfusion 7 days.Blood glucose was monitored in rats before and after CPR.Results ① The Bcl-2mRNA in insulin groups were significantly higher than those in the CPR group after 24 h and 72 h (P ＜0.01 ).The expression of Bcl-2 mRNA in 24 h insulin group were significantly higher than those in 72 h insulin group ( P ＜ 0.01 ) ; There were no significantly different in the Bax mRNA between insulin groups and the CPR and the control group after 24 h and 72 h ( P ＞ 0.05 ) ; ②After CPR 7 d,the apoptotic neurons of hippocampal CA1 area in the CPR group ( 124.75 ± 17.35 ) were significantly higher than those in the control group (5.12 ± 3.26) ( P ＜ 0.01 ) and the insulin group (92.79 ± 7.35 )(P ＜0.01 ); the apoptotic neurons in the insulin group were higher than those in the control group (P ＜0.0l ),and the differences were statistically significant.③There were no significant difference in venous blood glucose in the CPR and insulin groups (P ＞ 0.05).Conclusions Insulin may regulate Bcl-2mRNA expression in hippocampus,inhibit neuronal apoptosis and protect neurons after CPR in rats.

Objective To develop a nylon membrane chip for rapid and systematic detection of the diabetes-associated 45 mutant loci in mitochondrial DNA(mtDNA).Methods The mutant-and wild-type probes were designed for detection of 45 mutant loci in mtDNA with Primer Premier 5.0 and NCBI BLAST softwares and the 90 probes with 8 poly T were immobilized on the Hybond N~+ nylon membranes which were treated with 5?SSC Buffer by UV-crosslinking;Then asymmetric PCR was employed to obtain the target single strand DNA(ssDNA).The PCR products were labeled with biotin after purification.NBT/BCIP was used as substrate that yields a very intense purple signal followed by AP-avidin,and the signals were observed in 24 samples with known sequences to evaluate the chips,each sample was repeatedly measured three times.Results The specific target fragments of 45 loci can be amplified under the same condition with nine sets of primers.The annealing temperatures of the wild-type [(59.01?1.42)℃] and mutant-type [(59.34?1.29)℃ ] probes are so close(t=1.046,P =0.301)that hybridization can be performed at the same temperature.The spots on the membrane chip are distinct,regular and well-distributed.The results of positive-and negative-control are perfect.The signals of negative probes and the background are similar.The results of chip were nearly concordant with that of DNA sequences(?~2=113.132,Kappa value =0.888,P = 0.000)except for T16189C mutant.Conclusions We have successfully developed a nylon membrane chip for rapid and systematic detection of the diabetes-associated 44 mutant loci in mtDNA.It could be used for screening for diabetic patients and high-risk people.

Objective To develop a real-time polymerase chain reaction(PCR)system to determine transcriptional level of MexAB-OprM multidrug efflux pump gene and to investigate the impact of androgra- pholide on MexAB-OprM gene transcription in Pseudomonas aeruginosa.Methods The fragments of mexB gene of mexAB-oprM operon and 30S rRNA gene rpsL were amplified and cloned into two plas- mids respectively.These plasmids were used as external standards for real-time PCR.Real-time PCR was applied to measure the mRNA transcripition of mexB and rpsL gene in Pseudomonas aeruginosa growing in medium with different concentrations of andrographolide.Results The plasmids for standard curve were constructed successfully.The relative mexB mRNA expressions in 50,100,150 and 200?g/mL andrographolide were 0.04?0.03,0.06?0.07,0.09?0.03 and 0.04?0.03 respectively, which were significantly lower than that in the control(0.24?0.04,P0.05).Conclusion Andrographolide can reduce the transcriptional level of MexAB-OprM,which may he one mechanism for its anti-infection effect.

Objective To study the gait of children with spastic cerebral palsy (SCP) using plantar pressure measurement (PPM).Methods Twenty SCP children and 84 healthy children were recruited,and PPM was used to compare their gait cycle time,cadence,and standardized gait cycle parameters.Results Compared with the control group,gait cycle times in the SCP group were obviously prolonged,and their cadence was significantly slower.The side support phase and swing time in the SCP group were shorter,while the double support phase was longer than that of children in the control group.Conclusion PPM can be used to assess the gait of SCP children efficiently.

Objective To observe the clinical effects of hyaluronidase iontophoresis in the treatment of in- fants with congenital muscular torticollis(CMT).Methods Fifty infants with congenital muscular torticollis were divided randomly into a treatment group and a control group.Manual stretching was performed with both groups,while hyaluronidase iontophoresis was also administered to those in the treatment group.The range of side-flexion and rota- tion of the neck was examined at the beginning of and after 1 and 2 months of treatment.At the end of the treatment, the overall outcome was assessed according to a scoring system.Results There was no significant difference be- tween the two groups at admission.Compared with the control group,the range of side-flexion and rotation of the neck,and the overall treatment outcome were all significantly better in the treatment group after treatment.Conclu- sion Hyaluronidase iontophoresis can effectively improve function in infants with CMT and alleviate their symptoms.

Objective　To investigate whether matrix metalloproteinase-9 (MMP-9) was synthesized in pregnant cervix during parturition and its source and distribution. Methods　Cervical species (n=10, each weighing about 0.3 g) were taken from pregnant women immediately after delivery. Other cervical species (n=7) were served as negative control from those non-pregnant women but undergoing uterotomy due to other benign diseases. Immunohistochemical method (ABC) was carried out to detect the expression of MMP-9, with a monoclonal antibody against MMP-9. Results　Positive staining of MMP-9 was found in the cytoplasm of polymorphonuclear leukocytes (PMN) that had infiltrated into cervix or located in blood vessels of cervix. Scattered light positive staining were found in some interstitial cells of the cervix. No other cells including fibrocytes and lymphocytes were positive to MMP-9. No positive staining was found in control tissues. Conclusion　There are strong expressions of MMP-9 in pregnant cervix in term labor, derived mainly from infiltrated PMN. MMP-9 may be an important regulator in the process of cervical ripening.