BACKGROUND:At present, many researches have shown that tacrolimus has protecting effect on peripheral nerves,however,whether tacrolimus has a similar effect on central neural system has not been known yet.OBJECTIVE:To investigate the effect of tacrolimus on neural behaviors and memory by observing the neural behaviors and detecting the expression of synaptophysin in cortex,hippocampus and basipodite.DESIGN:Parallel and before and post contrast study.SETTING:Department of neurosurgery, affiliated hospital of the two medical universities.MATERIALS:The experiment was carried out in the Laboratory of Department Neurosurgery,Shanghai Changzheng Hospital in November 2002.Twenty-four qualified Sprague-Dawley rats supplied by Shanghai Experimental Animal Center of medical institution of Fudan University,with body mass of 180-220 g,were randomly divided into three groups:control, injury and treatment,with 8 rats in each group.INTERVENTIONS:Anesthesia was induced by administration of sodium pentobarbital(with the concentration of 20 g/L,40 mg/kg) twenty-four hours before the operation.The coup energy was 151.95-172.21 kPa that was equal to medium injury. The control rats did not receive the coup. Tacrolimus was given to the treatment group in the dosage of 1 mg/kg body weight by intraperitoneal injection at 5 minutes post trauma. The drug was given once physiological saline. Before and post trauma (on the third and seventh day)all rats Accepted Beam Walking Test,Beam Balance Test and Memory Test,respectively. One week after trauma,all rats' heads was removed and the brain was taken out within seconds.The hippocampus,cortex and basipodite of the traumatic hemisphere were kept in methanal with the concentration of 4 percent. Immunohistochemistry and image analysis system were used to study the expression of synaptophysin in cortex,hippocampus and basipodite of the traumatic hemisphere.MAIN OUTCOME MEASURES: ①The performance records of neural behaviors and memory of the three groups. ②Results of the expression of synaptophysin in cortex, hippocampus and basipodite of the three groups.③Apoptotic cellscalculation of synaptophysin in cortex,hippocampus and basipodite.RESULTS:Totally 24 rats entered the final analysis. ①On the third day and the seventh day,the scores recorded in Walking Test and Balance Test of the treatment group were less than those in the injury group[Walking Test: (7.5±2.5) s,(5.5±2.1) s,(10.5±2.5) s,(8.2±2.5) s. Balance Test:(3.4±0.5)score,(2.5±0.2) score,(5.7±0.2) score,(5.0±0.5) score,P ＜ 0.05-0.01].But the scores recorded in memory test of the treatment group were still more than those in the control group,memory test[(4.9±1.7) s,(6.2±2.3) s,(4.0±1.5) s,(4.4±2.6) s,P ＜ 0.05-0.01]. ②The expression of synaptophysin in cortex,hippocampus and basipodite in the treatment group was still more than that in the injury group(140.36±3.87, 45.52±2.16,31.67±2.35,96.25±2.85,24.35±2.47,20.49±2.08,P ＜ 0.01);The expression of synaptophysin control group was especially more than that in treatment group (162.24±3.52,50.58±2.31,42.69±2.53, P ＜ 0.01).Apoptotic cells calculation of cortex,hippoeampus and basipodite in treatment group was far less than those in injury group,(10.37±2.12)%,(18.39±2.87)%, (12.78±2.45)%,(21.14±4.85)%,(38.57±3.78)%, (21.18±4.59)%,P ＜ 0.01].And apoptotic cells calculation in injury group was more than those in control group [(3.85±2.56)%,(4.96±2.15)%, (3.52±2.17)%,P ＜ 0.01].CONCLUSION:Tacrolimus can facilitate the expression of synaptophysin,refrain from calcineurin involving in the process of transcription-dependent and nontranscription-dependent apoptosis,and can also contribute to the recovery of memory and neural behaviors post trauma.

Objective To analyse the significance of proto oncogene protein in oral squamous cell carcinoma and oral leukoplakia.Methods 40 patients who were diagnosed with oral leukoplakia (OLK),40 patients who were diagnosed with oral squamous cell carcinoma(OSCC),and at the same time, 40 healthy volunteers as control group were collected.the lesions and normal oral mucosa were taken as samples, left supernatant after grinding, MDM2 protein, p53 protein and Ki-67 protein were detected by ELISA in three groups,and analyse the correlation between three groups.Results Among three groups,MDM2, p53 expression:OSCC group>OLK group>control group (P<0.05).Ki-67 expression level among three groups, OSCC group>OLK group>control group (P<0.05).MDM2, p53, Ki-67 expression had a positive correlation in OSCC group and OLK group (P<0.05), but there was no obvious correlation in control group.Conclusion When oral leukoplakia developes into oral squamous cell carcinoma, MDM2, p53, Ki-67 shows high expression, and there is a positive correlation between MDM2, p53, Ki-67.

Objective To establish an effective DNA isolation method for neonatal disease screening,so as to explore its application to the methylation detection.Methods The 20 dried blood spots samples were randomly divided into 2 groups according to the gender:the traditional method group (n =10) and the improved kit method group(n =10).The DNA quality was evaluated based on its concentration,integrity and whether it could be used in polymerase chain reaction (PCR).These DNA samples with or without bisulfite treatment were used as template in the methylation-specific polymerase chain reaction (MSP).The methylation levels of Leptin and tumor necrosis factor-α (TNF-α) gene promoter region were detected.Results DNA concentration of the improved kit method [(5.70 ± 0.81) mg/L] was significantly higher than that of the traditional method [(3.50 ± 0.45) mg/L] (t =2.79,P ＜ 0.05),and biochemical analyzer analysis showed a better DNA integrity.Agarose gel electrophoresis revealed that 18S gene fragment could be successfully amplified by PCR method,suggesting its potential application to PCR study.MSP results showed different DNA methylation levels of Leptin and TNF-α genes promoter regions from various samples.Conclusions The improved kit method can effectively extract DNA from dried blood spots samples,and these DNA can be used in methylation research.The study can provide a new research direction and technical method to reveal the pathogenesis of disease from the perspective of DNA methylation.

Objective To observe neural stem cells proliferation,migration and differentiation in hippocampus in developing rats with status epileptictus.Methods 320 healthy SD rats at age 7,14,21,28 d (P7,P14,P21,P28) were randomly divided into status epilepticus (SE) and normal control group.In each group those rats at the same age were further randomly divided into 1,7,14,21,28 d five time points after PTZ-induced SE (n =8).New cell proliferation and migration were observed by immunohistochemistry studies in the dentate gyms.Double labeling with Brdu/NeuN and Brdu/GFAP was performed in the P14 rats.Results Nestin positive cells appeared in the dentate gyms on 1 d after SE in P7,P14,P21,P28 rats.The number of nestin positive cells gradually increased on 7 d and reached a peak on 14 d,then gradually reduced on 21 d,finally fell to a minimum on 28 d after SE.The numbers of nestin positive cells on 7 d(177.00 ± 3.22,t =16.033) and 14 d (195.00 ± 3.41,t =28.840) were significantly higher in the SE group than the NS group (147.50 ± 2.08,136.50 ± 2.65,both P ＜ 0.05).The smaller age of rats with SE onset,the greater the nestin intensity.But the number of nestin positive cells in the dentate gyrus of normal rats were gradually decreased with increasing age.Nestin positive cells were distributed in subgranular zone of dentate gyrus on 1 d and 7 d after SE,then gradually migrated to the granule cell layer on 14 d with morphological changes.Small part of nestin positive cells were ectopically migrated to the hilus of dentate gyrus in P14,P21,P28 age rats,and were also seen in the CA1,CA3 of hippocampus and cortex with various cell morphology.For differentiation of newly generated cells,most of Brdu positive cells coexpressed NeuN and about 4％-5％ cells co-expressed GFAP.Conclusions SE could induce neurogenesis in the hippocampal dentate gyrus area in developing rats which has age-related characteristics.Most new cells migrate from the subgranular zone to the granule cell layer of the dentate gyrus,and a small number of newly generated cells ectopically migrated to the hilus of dentate.The majority of newly generated cells differentiate into neurons,and the others differentiate into glial.

Objective To study the distribution and expression of Semaphorins-3A protein in brain of postnatal rats.Methods Semaphorins-3A positive cells were observed by immunohistochemistry in the cerebral cortex,hippocampus,dentate gyms and entorhinal cortex in postnatal 0d,7d,14d,21 d and 28d of Sprague Dawley rats.Results Semaphorins-3A positive cells widely distributed in the granule cell layer ( Ⅱ ),external pyramidal cell layer ( Ⅲ ),internal granular cell layer ( Ⅳ ),pyramidal cell layer ( Ⅴ ) and layer of polymorphous cells ( Ⅵ ),in addition to the molecular layer ( Ⅰ ) of the parietal,occipital,frontal,temporal,insular,cingulate cortex,piriform cortex and entorhinal cortex with postnatal 0d,7d,14d,21d and 28d rats.The amount of semaphorins-3A positive cells(IOD) in the entorhinal cortex was 84916.23 ± 3266.34 in P0d,77711.41 ± 2634.26 in P7d,74124.25 ± 3989.09 in P14d,65887.63 ± 3406.57 in P21d and 57705.96 ± 3136.35 in P28d,meanwhile the region of semaphorins-3A positive cells narrowed in the part level with Ⅱ -Ⅵ levels of cortex.Similarly semaphorins-3A positive cells distributed mainly in granule cell layer of dentate gyrus,CA1,CA3 region and only a few of semaphorins-3A positive cells scattered in the multi-line layer in hippocampus.The expression level of semaphorins-3Awas significant difference among postnatal 0d,7d,14d,21d and 28d rats (P＜0.01).Conclusion Semaphorins-3A positive cells widely distribute in the various cortex and hippocampus in developing rat brain,and the region of semaphorins-3A is reduced with age growth of rats.

ObjectiveTo observe Semaphorins-3A and synaptophysin P38 expression in hippocampus of developing rat induced by status epilepticus.Methods 320 SD rats were divided into four groups (P7,P14,P21 and P28) according to day -old after birth (7d,14d,21d and 28d).Rats in each group were randomly divided into model group (SE group) and saline control group (NS group).SE was induced by Pentylenetetrazol (PTZ).Semaphorins-3A and synaptophysin P38 expression were determined by immunohistochemical staining on 1d,7d,14d,21d and 28d after SE in hippocampal CA1 of rats.ResultsSemaphorins-3A-positive cells could be seen in the hippocampal granule cell layer in all rats.Semaphorins-3A expression tended to decrease with the increasing of day-age,especially in P7 group(91 552.68 ± 4664.69 ).No matter how day-age,Semaphorins-3A expression was similar to that in NS group and was obvious reduced in 7d after SE(56 938.84 ± 5688.47 ).Meanwhile P38 expression in P7-day-age rats had had been gradually increasing between 1 day and 14d (5413.18 ±48.77,6223.40±29.19,6902.94 ±78.51 ) and then stabilized gradually on 21d(7523.42 ± 62.94) after rats were tested.P38 expression in other day-age rat was relatively stable on the same level in physiological state.On the other hand P38 expression in the hippocampal CA1 region of P7,P14,P21 and P28 rats was significantly higher than that in normal rats between 1day and 28day after SE episode(P＜ 0.05 ),and reached a peak on 14 day(8408.35 ± 55.73 ).ConclusionSemaphorins-3A and synaptophysin P38 involved in hippocampal synaptic plasticity of rat in developing stage and epilepsy.

Objective To identify the epidemiological changes in invasive fungal infection (IFI) in a neonatal intensive care unit (NICU) to provide information for prevention and treatment of IFI.Methods A total of 102 cases who were diagnosed with IFI among 42 187 neonates hospitalized in the NICU of Affiliated BaYi Children's Hospital,Chinese People's Liberation Army General Hospital from January 1,2009 to December 31,2014 were enrolled in this study.Since January 1,2012,the divisions of our NICU were more specific and intravenous fluconazole was administered as a routine preventive measure for high-risk infants.Clinical information of the IFI cases including general features,incidence,distribution of pathogens and drug (Amphotericin B,Fluconazole,Flucytosin,Itraconazole and Voriconazole) sensitivity were analyzed between former period (January 1,2009 to December 31,2011) and latter period (January 1,2012 to December 31,2014) by Chi-square test.Results The total incidence of IFI was 2.42‰ (102/42 187),and among the 102 IFI cases,73.5％ (75/102) were preterm infants and 75.5％ (77/102) were low birth weight infants.The incidence ofIFI in the latter period was lower than that in the former period [1.8‰ (48/26 046) vs 3.3‰ (54/16 141),x2=9.329,P＜0.01].The incidences of IFI in neonates with gestation age ＜28,≥ 28-＜32 and ≥ 32-＜37 weeks in latter period were decreased as compared with those in former period [10.6 ‰ (3/284) vs 76.9 ‰ (9/117),x2=12.569;6.1‰ (13/2 134) vs 21.9‰ (28/1 277),x2=16.868;1.4‰ (12/8 706) vs 1.9‰ (10/5 256),x2=7.165] (all P＜0.01).Altogether 103 pathogen strains were identified from 102 IFI cases as one Candida parapsilosis strain and one Laurent cryptococcus strain were both isolated from one patient.The most prevalent three pathogens were Candida albicans [51.5％ (53/103)],Candidaparapsilosis [24.3％ (25/103)] and Candida glabrata [8.7％ (9/103)].The isolated rates of Candida albicans and Candida glabrata strains in the latter period were higher than those in the former period [63.3％ (31/49) vs 40.7％ (22/54),x2=5.218;18.4％ (9/49) vs 0.0％ (0/54),x2=10.868],while the isolated rate of Candida parapsilosis strain was lower in the latter period than that in the former period [12.2％(6/49) vs 35.2％(19/54),x2=7.355] (all P＜0.05).All pathogen strains were sensitive strains except one Candida krusei strain which was isolated in the former period and was resistant to Fluconazole.Conclusions Premature infants born at lower gestational ages or with low birth weights are still at high-risk of IFI,but the incidence of IFI has declined in recent years.Routine administration of fluconazole in high-risk infants in NICU could prevente IFI without increasing drug resistance.Candida albicans is the predominant pathogen ofIFI.

OBJECTIVE:To analyze and identify the metabolites of tuberostemonine in rats plasma,urine and feces,and to clarify metabolic pathway of tuberostemonine. METHODS:Rats were randomly divided into blank group(0.3% carboxymethyl cellulose)and medication group(tuberostemonine 50 mg/kg,i.g.),with 6 rats in each group.The plasma of rats was collected 0.25, 0.5,1,2,4,6,12 h after intragastric administration to prepare samples. Urine and feces were collected 0-12 h and 12-24 h after administration to prepare samples. UPLC-Q-TOF/MS and software of Triple TOFTMhigh resolution mass spectrometry were used to analyze and identifiy chemical structure of metabolites in samples. RESULTS:A total of 4 compounds were detected in rat plasma, including one prototype compound and 3 metabolites. 4 metabolites were detected in urine and 2 metabolites in feces. Phase Ⅰmetabolites of tuberostemonine mainly included hydration and hydroxylation. Phase Ⅱ metabolites were not found. CONCLUSIONS:Hydration and hydroxylation are the major metabolic transformation forms of tuberostemonine in rats in vivo.

Objective:To assess the validity and reliability of the Chinese version of the eleven-item Kutcher Adolescent Depression Scale (KADS-11)in Chinese adolescents,calculate its optimal cut-off value and the sensi-tivity and specificity,and explore the possibility of providing a useful tool to assess the severity of adolescent de-pressive symptoms.Methods:Totally 3180 students aged 11 -17 years were selected from schools in 6 provinces and Shanghai.All of them were asked to complete the KADS-11 and Children Depression Inventory (CDI). Students whose CDI scores were above 19 (including 19)were diagnosed with the DSM-IV criteria of depressive disorder,73 students from Shanghai sample were assessed with KADS-11 and CDI to analyze the test-retest reliabil-ity 1 month later.Results:Exploratory factor analysis showed that KADS-11 had 2 factors,and confirmatory factor analysis tested proved the 2-factor model fit better than the one-factor model.The KADS-11 total scores were posi-tively correlated with CDI total scores (r =0.74,P <0.01 ),and the KADS-11 scores were higher in depressive group than those in non-depressive group.The mean area under the curve (AUC)of KADS-11was 0.94,the mean area under the curve of each item ranged from 0.7 to 0.9.The optimal cut-off point of KADS-11 was total score≥9,sensitivity and specificity were 89% and 90% respectively.The Cronbach's alpha coefficient of the KADS-11 was 0.84,the spilt-half reliability coefficient was 0.71 (P <0.01),and the test-retest coefficient was 0.77 (P <0.01).Conclusion:The KADS-11 is appropriate for Chinese adolescents because of its good validity,reliability and diagnosis accuracy,it could be used to assess depressive symptoms for adolescents.

Objective:To investigate the clinical and gene variant characteristics of benign familial infantile epilepsy in generations of three families.Methods:The clinical data of the three benign familial infantile epilepsy patients with PRRT2 gene variant who were diagnosed and their family members were collected from Children′s Hospital Affiliated to Zhengzhou University between 2018 and 2019. All coding exons from the patients and their parents were screened by targeted next-generation sequencing, and detected variants were verified by Sanger sequencing.Results:In all the patients, a cluster of seizures was observed before one year old,but interictal clinical conditions were normal. The electroencephalograms were all normal in interictal stage. The father of proband 1 presented with convulsion onset at the age of eight months and showed remission before one year old. The grandpa, mother and uncle of proband 2 also presented with convulsion onset in their babyhood of life and showed remission before one year old. The mother of proband 3 presented with convulsion onset in their babyhood of life and showed remission before three years old. Proband 1 carried heterozygous c.937G>C variant in the PRRT2 gene which is inherited from his father. Proband 2 carried c.1075_c.1076insC variant inherited from his mother. A deletion of PRRT2 gene exon 2 was detected in both of proband 3 and her mother. The three variants had not been reported in the Human Gene Mutation Database.Conclusions:Benign familial infantile epilepsy is a kind of inherited epilepsy characterized by early onset of seizure in babyhood with better prognosis, a cluster of focal seizures with or without secondary generalization, and cessation of seizure mostly before two or three years of age. The variants c.937G>C, c.1075_c.1076insC and the deletion of exon 2 in the PRRT2 gene have enriched the gene variant spectrum of benign familial infantile epilepsy.