AIM:To investigate apoptosis and the expression of death receptors of TRAIL, TNF and Fas on hepatic veno-endotheliocyte ED25 cell strain induced by dengue virus type 2(DV2).METHODS: Flow cytometric analysis was used to detect the number of apoptotic cells and the expression levels of TRAILR1-4 ,TNFR1-2,Fas on ED25 cells before/after DV2 infection. RESULTS: The numbers of apoptotic cells of ED25 increased after DV2 infection, there were only about 5.7%?1.2% of apoptotic cells before virus infection while there were approximately 27.3%?1.6% of apoptotic cells after virus infection. At the same time the expression level of Fas also increased, before virus infection about 44.3%?2.2% of ED25 cells expressed Fas while 63.0%?2.3% of ED25 cells expressed Fas after virus infection. CONCLUSION: DV2 infection can induce apoptosis of ED25 cells, and it suggests strongly that Fas/FasL may be involved in the apoptotic signal transduction.

Objective To study the expression and clinical significance of Notch3 and Notch intracellular domain (NICD) in ovarian carcinoma and the effects of N-[N-(3 ,5-difluorophenyl) acetyl-L-alanyl]-S-phenyl glycine t-butyl ester (DAPT), a γ-secretase inhibitor on the proliferation and apoptosis in OVCAR3, A2780 ovarian carcinoma cell lines. Methods Western blot was used to detect the expression of NICD in the tissues from 58 ovarian carcinomas patients and 21 normal ovarie, who were admitted in Peking University First Hospital from July 2006 to June 2009. Immunohistochemistry was also used to detect the expression of Notch3 in these tissues. The relationship with clinical features of ovarian carcinoma was also analyzed. Proliferation of OVCAR3 and A2780 ovarian cancer cells was determined by methyl thiazolyl tetrazolium (MTT) assay, cell cycles and apoptosis and index of proliferation were detected by flow cytometry method. The expression of NICD in OVCAR3 and A2780 cells incubated with DAPT was detected by western blot. Results (1)The expression level of NICD in ovarian carcinomas was significantly higher than that in normal ovarian tissues (1.64 ±0. 19 vs. 0.98 ±0.20;P <0.05). The NICD expression was higher in ovarian cancers with low grade or advanced stage than those in high-middle grade or early stage,respectively (1.90 ± 0. 22 vs. 1.25 ± 0. 21,1.80 ± 0. 21 vs. 1.21 ± 0. 15; all P < 0. 05). The Notch3 protein was stained positively in cytoplasm, nuclear and cell membrane. The expression of Notch3 was higher in ovarian carcinomas than that in normal ovaries [78% (45/58) vs. 24% (5/21); P < 0. 01]. While,there were no stasistical difference in different pathological types, stages, differentiation of ovarian carcinoma. There was no difference between the patients with adjuvant chemotherapy or not. (2)After OVCAR3 and A2780 cells incubated with DAPT 24, 48, 72 hours, NICD expression was significantly lower than that in control group (P < 0. 05). The effects of DAPT inhibited the proliferation and prompted the apoptosis of OVCAR3 and A2780 cells were depended on the concentrations and times. Conclusions Notch3 and NICD may play a key role in the occurrence and progress of ovarian carcinoma. The mechanism of DAPT inhibited the proliferation and prompted the apoptosis of OVCAR3 and A2780 cells may be due to decreased the formation of NICD.

Objective To study the expression of Notch 1,Jagged1 and Notch intracellular domain (NICD) in epithelial ovarian carcinoma tissues and analyze the clinical significance.To explore the activity of γ-secretase in epithelial ovarian carcinoma cell line SKOV3 and the effect of N-[N-(3,5-dil uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT),a γ-secretase inhibitor on the activity of γ-secretase in SKOV3.Methods Immunohistochemistry staining method was performed in 43 patients with epithelial ovarian carcinoma and 11 patients with benign epithelial ovarian tumor to detect the expression of Notch1,Jagged1 and NICD.The differences of expressionof Notch1,Jagged1 and NICD between malignant and benign ovarian tumors was compared and alsoanalyzed the correlation with clinicopathological parameters of ovarian carcinoma.Human serous ovarian cancer cell line SKOV3 and immortalized nontumorigenic ovarian epithelial cell line T29 were incubated in vitro.The activities of γ-secretase in SKOV3 and T29 with dimethyl sulfoxide (DMSO) and DAPT were detected respectively by Gal4VP16/UAS and dual luciferase reporter assay system.Results (1) The immunohistochemical composite scores (ICS) of Notch1 in epithelial ovarian carcinoma (6.7±2.2) were not significantly different with those in benign epithelial ovarian tumor (5.4± 2.7,P=0.153),while the ICS of Jagged 1 and NICD in epithelial ovarian carcinoma (5.3± 2.4,5.3± 2.3) were higher than those in benign epithelial ovarian tumor (1.6± 1.4,3.1± 1.7; all P＜0.01).The expression of Notch 1,Jagged 1 and N ICD had no correlation with patients' aged,history of carcinoma,ascites,the level of serum CA125,maximum length of ovarian tumor,Federation International of Gynecology and Obstetrics (FIGO) stage,grade and pathology subtypes (all P＞0.05).The hazard ratio between the high expression of Notch1,Jagged1,or NICD and the moderate to low expression of Notch1,Jagged1,or NICD,and Jagged1 were 0.771,1.648 and 1.316,respectively (all P＞0.05).The 5-year survival rate and median survival time between the high expression of Notch,Jagged 1 or NICD in subgroup and moderate to low expression in subgroup were of no difference (all P＞0.05).The activity of γ-secretase in SKOV3 was significantly higher than that in T29 [(12.2± 1.4)％,P=0.019].(2)After DAPT treated,the relative activity of γ-secretase in SKOV3 (50 μmol/L) was declined from (100.0±5.3)％ to (6.6±0.8)％ (P=0.001).Conclusions Jagged1 and NICD in Notch1 pathway may play a key role in the occurrence of ovarian carcinoma.The activity of γ-secretase in epithelial ovarian carcinoma was higher than that in ovarian epithelial cell which suggest that DAPT,γ-secretase inhibitor,may become the target of ovarian carcinoma treatment.

Objective To study the expression and clinical significance of Notch intracellular domain (NICD) in cervical cancer and the effects of N-[N-(3,5-difluorophenyl)acetyl-L-alanyl]-S-phenyl glycine t-butyl ester (DAPT), a γ-secretase inhibitor on the proliferation and apoptosis of cervical cancer cell lines. Methods Western blot was used to detect the expression of NICD in the tissues of 40 cervical cancers and 21 normal cervix and its relationship with clinical features of cervical cancer was also analyzed. Proliferation of SiHa and HeLa cervical cells was determined by methyl thiazolyl tetrazolium (MTT) assay, cell cycles and apoptosis and index of proliferation were detected by flow cytometry method. The expression of NICD in SiHa and HeLa cells incubated with DAPT was detected by western blot. Results The expression level of NICD in cervical cancers was significantly higher than that of normal cervical tissues (1.237±0.353 vs 0.938±0.105, P<0.05). The NICD expression was higher in cervical cancers with high grade,lymph node involvement and parametrial invasion than that with low-middle grade (1.496±0.540 vs 1.150±0.216), without lymph node involvement (1.419±0.532 vs 1.159±0.210) and no parametrial invasion (1.718±0.710 vs 1.183±0.258), respectively (all P<0.05). The expression of NICD in cervical adenocarcinoma was higher than that of squamous cell cancer (1.463±0.395 vs 1.162±0.187, P<0.05). After SiHa and HeLa cells were incubated with DAPT, NICD expression was significantly lower than that in control (P<0.05). The effects of DAPT inhibited the proliferation and prompted the apoptosis of SiHa and HeLa cells was depended on its concentrations and times. Conclusions NICD may play a key role in the occurrence and progress of cervical cancer. The mechanism of DAPT inhibited the proliferation and prompted the apoptosis of SiHa and HeLa cells may be due to decreased the formation of NICD.

Objective To study the effects of hypothermia therapy on coagulopathy in neonates with hypoxic-ischemic encephalopathy(HIE) of different degrees.Methods Twenty-five newborns with HIE were divided into 2 groups (moderate HIE group and severe HIE group) by amplitude integrated electroencephalogram(aEEG).The platelet (PLT) count,activated partial thromboplastin time (APTT),prothrombin time(PT),thrombin time(TT),fibrinogen (Fbg) and international normalized ratio (INR) were measured before hypothermia therapy and those after 24 hours,48 hours,72 hours after hypothermia therapy were collected.The hematological parameters of 2 groups were analyzed by repeated measurement analysis of variance.Results The APTT(t=2.239,P<0.05)and PT(t=2.453,P<0.05) of severe HIE group were significantly longer than those of moderate HIE group and significantly different.PLT,PT,APTT and Fbg(all P>0.05)showed no statistically significant difference between 2 groups after hypothermia therapy.The differences in INR(F=12.470,P<0.05),TT(F=8.275,P<0.01)between 2 groups were significant.Fbg at diffe-rent time points showed no significant difference (P>0.05) between 2 groups.The differences in PLT(F=32.420,P<0.01),PT(F=9.377,P<0.01),APTT(F=15.420,P<0.01),TT(F=7.326,P<0.01)and INR(F=12.699,P<0.01)at different time points were significant between 2 groups.The degree of brain injury and time had no interactive effects in PLT,INR and Fbg(all P>0.05).But the degree of brain injury and time had interactive effects in PT(F=3.374,P<0.05),APTT(F=4.227,P<0.05)and TT(F=3.585,P<0.05).It indicated that the change trend in PT,APTT and TT were different between 2 groups.The increase range of PT,APTT and TT of severe HIE group was higher than moderate HIE group after hypothermia therapy for 24 hours.The percentages of severe HIE group who received transfusion with blood product were higher than those of moderate HIE group(P>0.05).Conclusion The coagulopathy function is worse in neonates with severe HIE in hypothermia therapy which may aggravate the hemorrhage risk.And the effect of hypothermia on neonates with moderate HIE is less evident.So hierarchical intervention on coagulopathy in neonates with HIE during hypothermia should be adopted to relieve the hemorrhage risk.

Objective To investigate the effects of postoperative adjuvant chemotherapy (CT) and chemoradiotherapy (CRT) or radiotherapy(RT) for Ⅰ b-Ⅱ a cervical cancer with risk factors.Methods From March 1995 to June 2010,there were 137 patients underwent radical hysterectomy and systematic pelvic lymphadenectomy for stage Ⅰ b-Ⅱ a cervical cancer admitted at Peking University First Hospital.These patients had risk factors,intermediate risk factors including bulky tumor (＞4 cm),lymph vascular space invasion,deep stromal invasion; high risk factors including positive surgical margin,parametrial invasion,lymph node involvement.Of the all patients,79 cases of them were treated with CT,58 of them were treated with RT or CRT.The 5-year survival and prognosis factors were analyzed retrospectively,the prognosis was compared between two adjuvant therapy groups.Results The univariate analysis shown that types of pathology,different grade of risk factors,stroma invasion and lymph node involvement were prognostic factors of 5-year overall survival Patients with squamous cell carcinoma,intermediate risk factors,no parametrial invasion,and no lymph node involvement had better prognosis (P ＜ 0.05).Whether patients with high-risk factors or intermediate-risk factors,the 5-year overall survival and 3-year disease-free survival had no difference between CT and RCT or RT groups respectively.Cox regression multivariate analysis of survival indicated that clinical stages,types of histology,different grade of risk factors were independent prognostic indicator.Patients with early stage,squamous cell carcinoma,intermediate risk factors had better prognosis.Univariate and multivariate analysis indicated that different postoperative adjuvant therapies had no effects on the prognosis.The 5-year overall survival was 88.6％ in patients treated with CT,and 89.7％ in patients treated with RT or CRT (P =0.455).Conclusion There are equivalent therapeutic results between CT and RT or CRT for patients with risk factors after radical surgery,CT may be as one choice of postoperative adjuvant therapy for stage Ⅰ b-Ⅱ a cervical carcinoma with risk factors.

Objective To study the predictive value of continuous amplitude-integrated electroencephalogram (aEEG) monitoring for the neurodevelopment outcome in infants with hypoxic-ischemic encephalopathy (HIE) receiving hypothermia treatment.Method From April 2014 to May 2016,neonates admitted to our NICU with HIE receiving hypothermia treatment were continuously monitored using aEEG for 96 h,and assigned into moderately and severely abnormal groups according to aEEG results.The aEEG results before hypothermia treatment,within 24 h,48 h,72 h and 96 h after hypothermia treatment were recorded.The Bayley Scales of Infant Development Ⅱ examination was performed at 6 months of age.The sensitivity,specificity,positive and negative predictive values and Youden's index of aEEG for poor outcome at these timepoints was compared.Result A total of 30 neonates were enrolled.Among them 13 were moderately abnormal and 17 were severely abnormal.The gender,gestational age,birth weight and delivery method between two groups were similar (P ＞ 0.05).The 1 min Apgar score,arterial pH,base excess (BE) were significantly lower in the severely abnormal group (P ＜ 0.05).The neurodevelopment assessment at 6 months of age showed unfavorable outcomes in 16 cases,while the remaining 14 cases had generally good outcomes.The sensitivity and specificity of aEEG before hypothermia treatment for the prediction of poor outcome was 81.3％ and 71.4％ respectively.The sensitivity and negative predictive values of aEEG within 24 and 48 after hypothermia treatment for poor outcome was 100％.The Youden's index of aEEG within 72 h after hypothermia treatment for abnormal outcome was the highest 0.661.Conclusion The aEEG before hypothermia treatment alone is not a reliable indicator of poor outcomes in HIE neonates.The aEEG within 72 h after hypothermia is better.Continuous aEEG monitoring during hypothermia in HIE infants is very important because it provides reliable prediction of outcome.

BACKGROUND: Despite the progress in perinatal-neonatal medicine, complications of extremely preterm infants continue to constitute the major adverse outcomes in neonatal intensive care unit. Human umbilical cord Wharton’s Jellyderived mesenchymal stem cells (HUMSCs) may offer new hope for the treatment of intractable neonatal disorders. This study will explore the functional differences of HUMSCs between extremely preterm and term infants. METHODS: UMSCs from 5 extremely preterm infants(weeks of gestation: 22+5 w,24+4 w,25+3 w,26 w,28 w) and 2 term infants(39 w,39+2 w) were isolated, and mesenchymal markers, pluripotent genes, proliferation rate were analyzed.HUVECs were injured by treated with LPS and repaired by co-cultured with HUMSCs of different gestational ages. RESULTS: All HUMSCs showed fibroblast-like adherence to plastic and positively expressed surface marker of CD105,CD73 and CD90, but did not expressed CD45,CD34,CD14,CD79a and HLA-DR; HUMSCs in extremely preterm exhibited significant increase in proliferation as evidenced by CCK8, pluripotency markers OCT-4 tested by RT-PCR also showed increase. Above all, in LPS induced co-cultured inflame systerm, HUMSCs in extremely preterm were more capable to promote wound healing and tube formation in HUVEC cultures, they promoted TGFb1 expression and inhibited IL6 expression. CONCLUSIONS Our results suggest that HUMSCs from extremely preterm infants may be more suitable as candidates in cell therapy for the preterm infants.

Objective:To analyze the brain functional fluctuation of benign epilepsy in children with central-temporal spikes(BECTS) by using ReHo algorithm based on the resting-state brain functional imaging, and to explore the connection of the brain function and changes of the connection pattern, so as to find the damage of the cognitive function of BECTS children in the early stage.Method:s Perspectiveness and simple random selection of 20 BECTS children and 20 healthy control children admitted to Shenzhen Children′s Hospital from January 2015 to December 2017 were conducted for basic information collection and functional magnetic resonance imaging (fMRI) testing in a resting-state.Result:s Significantly lower ReHo value appeared in the default mode network (DMN) area, and the precuneus (voxel=422, t=-5.085 6), cuneus (voxel=85, t=-4.240 3), angular gyrus (voxel=191, t=-4.681 2), cingulate cortex (voxel=313, t=-5.238 2), anterior central gyrus (voxel=12, t=-3.482 7), and supplementary motor area (voxel=1 356, t-6.596 2). The significantly increased ReHo was found in the bilateral cerebellum (voxel=71, t=5.658 2), right superior temporal gyrus (voxel=24, t= 5.184 0), operculum insulae (voxel=337, t=6.814 9), left parietal lobe (voxel=12, t=4.378 7), and inferior parietal lobule (voxel=11, t=3.433 7). Conclusions:Significant impairment of DMN, Wernicke and angular gyrus functions in BECTS children may be one of the mechanisms of cognitive dysfunction.Enhanced sensorimotor area and cortical brain functions near the operculum insulae and central sulcus lead to seizures with typical clinical symptoms.fMRI has a high specificity and sensitivity for evaluating the brain function of children with BECTS, and it can detect the impairment of cognitive function in children with this type of epilepsy at an early stage.

At present,China has not formed a complete ethical review agency certification system,actively participating in international certification and seeking external quality assessment is of great significance for the improvement of standard management and review capacity of ethics committee.Combined with the experience of Peking University Cancer Hospital applying for SIDCER international certification,this paper expounded the responsibilities of ethical secretary in international certification from the overview of international certification system and the work practice during the certification process,to promote the progress ethics committee applying for certification and achieve satisfactory quality assessment results.