Objective To establish the topotecan(TPT)-resistant cell line of human oophoroma (SKOV3/TPT), and to investigate its biological features. Methods To simulate the condition in clinical chemotherapy, drug resistant SKOV3/TPT cell line of human oophoroma was established by challenging SKOV3 with a large dose of TPT (2160ng/ml). Drug sensitivity was assessed by MTT assay. Light microscopy, electronic microscopy, trypan blue staining rejection, and cell counting were employed to determine the biological features of the cell line. Flow cytometry (FCM) was used to study the cell cycle distribution, the accumulation of intracellular drug, and the expression of P glucoprotein (Pgp). Results SKOV3/TPT cells were established successfully, and the resistance index (RI) was 10.67. They showed an obvious cross-resistance to CPT-11, MX and VCR, but still sensitive to taxol, cis-platin, VP16, MTX and 5-Fluorouracil. Comparing with the parent cells, their morphology was unchanged, but multiplication time was prolonged. The apoptosis rate and the proportion of G2+M phase cells of resistant cells were significantly higher than that of their parent cells (P

Objective To investigate the effect of high concentration hydrogen gas on neurons in the rat hippocampus CA1 region during global cerebral ischemic/reperfusion injury (GCIR) Methods Four-vessel occlusion was used to establish rat model with GCIR injury. One hundred and five healthy male Sprague-Dawley rats were randomly divided into sham operation group(SH group, n = 15), model group(4-VO group, n = 45) and treatment group(4-VO+H2 group,n = 45). After 72 h and 9 d reperfusion, hippocampal CA1 region pyramidal neurons in every group were detected with Nissle staining , immunohistochemical neuron-specific nuclear protein (NeuN), specific protein antibody microglial cells (Iba1) staining and the relationship of position between neurons and microglia was observed through fluorescence double staining. We used Morris water maze to test the space orientation ability and the learning and memory ability in rats after 9 d reperfusion. Results Compared with those of 4-VO group,the neurons of hippocampus CA1 region were closer to normal in 72 h and 9 d in 4-VO+H2 group and neuron form and the number of neuron survival were increased significantly (P < 0.05);immunohistochemical staining showed that the number of neuron survival in 4-VO+H 2 group was obviously higher than that in 4-VO group (P < 0.05) and the number of microglia in 4-VO group was obviously higher than that in 4-VO+H2 group (P < 0.05). Water maze experiment showed that the swimming time in quadrant Ⅳ in 4-VO+H2 group was longer than that in 4-VO group (P < 0.05). Conclusion Inhalation of high concentration hydrogen gas has prominent protective effect on neurons of rat hippocampal CA1 region during reperfusion. The mechanism may be related with inhibiting the microglia excitation and activation during GCIR.

Objective: To establish the method of percutaneous endoscopic gastrostomy(PEC) and percutaneous endoscopic jejunostomy (PEJ) for enteral nutrition. Methodes: PEG tubes were placed in 114 patients with Pull method. On the foundation of PEG, PEJ tubes were placed in 26 patients by pushing endoscopy to send tubes through Treitz ligment with usingthe the clip. Results: All PEG insertion was performed successfully. PEJ tubes were placed successfully with a new method in 26 patients. 15 patients had a little blooding and 8 patients had slight infection. 21 patients had respiratory tract infection and had been cured by using antibiotic. There was no severe complication. Conclusion: PEG is simple、safe、efficient. The new method of PEJ is feasible.

Objective To analyze the preoperative related factors of early weaning of infants with congenital heart disease after operation. Methods From January ,2014 to January ,2017 in Pediatric Cardiology CICU , infants with congenital heart disease were selected as research objects. The clinical data were retrospectively collect ed and the relationship between preoperative influencing factors and postoperative early weaning were analyzed. Early weaning meant mechanical ventilation time was shorter than 24 h and late weaning meant longer than 24 h. Results Single factor analysis showed that early weaning success rate was related to preoperative cardiac function grade 1,NNIS grade 0~1,ASA grade 1,no lung infection and no or mild pulmonary hypertension(average P<0.05). Non conditional logistic regression analysis showed that preoperative heart function grade 1 was an indepen-dent influencing factor,with OR value(95%CI)of 3.9(1.9~7.7). Conclusions In infants with congenital heart disease,preoperative heart function grade 1,NNIS grade 0 ~ 1 ,ASA grade 1,no lung infection and no or mild pulmonary hypertension benefit early withdrawing of ventilator and preoperative cardiac function grade 1 is an inde-pendent factor for early weaning.

The pathogenesis of brain injury in fetal growth restriction (FGR) fetuses is likely associated with oxidative stress and neuroinflammation, although the exact mechanisms are not fully understood. This article mainly reviews the anatomical alterations, potential pathophysiological processes, and the specific molecular mechanisms involving various types of brain cells in FGR.

Objective To explore the protective effect of in-taking high concentration hydrogen gas on neurons and dendritic spines in hippocampus CA1 region of rats after globe cerebral ischemia/reperfusion (I/R) injury and its mechanism.Methods Four-vessel occlusion (4VO) was used to establish the models of global cerebral I/R injury in rats.One hundred and twenty healthy male Sprague-Dawley rats were randomly divided into 3 groups using a random number table:sham-operated group (inhaled 67％ N2 and 67％ O2,n=40),model group (inhaled 67％ N2 and 67％ O2 during reperfusion,n=40),and treatment group (inhaled 67％ H2 and 67％ O2 during reperfusion,n=40).After 72 h,5 and 9 d reperfusion,neuron-specific nuclear (NeuN) protein expression in the pyramidal neurons of the hippocampal CA1 region was detected with immumohistochemical staining and the positive cells were counted.And the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were tested with colorimetry.Water maze test was used to measure the spatial orientation and memory function and Golgi staining to detect the number of dendritic spines in neurons 9 d after reperfusion.Results (1) Immunohistochemical staining of NeuN results showed that as compared with those in the model group,the neurons ofhippocampus CA1 region were significantly closer to normal with relatively intact structure,and the number of positive neurons was significantly increased in the treatment group 72 h,5 d,and 9 d after reperfusion (P＜0.05).With the reperfusion time being prolonged,the number of NeuN stained positive neurons at different time points of reperfusion in model group was gradually decreased (P＜0.05),and the numeric of the NeuN stained positive neurons at different time points of reperfusion in treatment group was slightly declined without significant difference (P＞0.05).(2) The serum SOD activity in the treatment group was significantly higher than that in the model group and sham-operated group (P＜0.05),while the MDA content in the treatment group was significantly lower than that in model group 72 h,5 d,and 9 d after reperfusion (P＜0.05).And with the reperfusion time being prolonged,the SOD activity at different time points of reperfusion showed no significant difference among the difference groups (P＞0.05).But the MDA content at different time points ofreperfusion between model group and treatment group was significantly different (P＜0.05);with the reperfusion time being prolonged,the MDA content was gradually decreased in both groups.(3) Nine d after reperfusion,water maze test found that the incubation period of treatment group was significantly shorter than that of model group (P＜0.05);the IV quadrant swimming time of space exploration in the treatment group was significantly longer than that in the model group (P＜0.05).(4) Golgi staining showed that the complexity of the neuronal dendrites branch and the number of dendritic spines of neurons in the hippocampal CA1 region of treatment group were increased than those in the model group;high-power oil microscopy indicated that the density of dendritic spines in the treatment group was significantly higher than that in the model group (P＜0.05).Conclusion In-taking of high concentrations hydrogen gas during reperfusion can definitely protect neurons in hippocampal CA1 region after globe cerebral I/R injury,and improve learning and memory function,whose mechanism may be related to hydrogen protecting the structure and function of neurons and dendritic spines,and inhibiting oxidative stress to reduce oxidative damage.

Objective: To investigate the relationship between the eosin-5′-maleimide (EMA) binding test and the clinical severity of hereditary spherocytosis (HS). Methods: A total of 258 un-splenectomize HS patients were consecutively enrolled. Correlation of hemoglobin concentration, hemolytic parameters, compensating erythropoiesis and the EMA binding test were evaluated. Results: 258 (128 male and 130 female) patients were included in this study, including 91 compensatory hemolysis patients, 53 patients with mild anemia, 78 patients with moderate anemia and 36 patients with severe anemia. The median age at diagnosis was 23 (2-70) years. The median decreased fluorescence intensity of EMA binding test was 29.97% (16.09%-47.34%) and the average intensity was (29.70±6.28) % of 258 HS patients. The decreased EMA binding fluorescence intensity correlated with MCV (r=-0.343, P<0.001) and MCHC (r=0.223, P<0.001). There was no relationship between EMA fluorescence intensity and absolute reticulocyte count (r=0.080, P=0.198) , reticulocyte percentile (r=-0.015, P=0.813) , IBIL levels (r=-0.009, P=0.902) , HGB levels (r=-0.067, P=0.280). Evaluated as a quartile variable, EMA fluorescence intensity was not correlated with anemia severity (C=0.150, P=0.746). Conclusion EMA binding test does not related to anemia levels and has no major clinical implications for disease severity in HS.

Clinical data of 19 patients with congenital pyruvate kinase deficiency were analyzed. Insufficient pyruvate kinase confirmed the diagnosis. Laboratory parameters of hemolysis were summarized. In cases of neonatal hyperbilirubinemia and unexplained hemolytic anemia, pyruvate kinase activity and next generation sequencing test may help the early diagnosis.

Objective: To evaluate the impact of the targeted next-generation sequencing (NGS) assay for difficult congenital anemias. Methods: Blood Disease Hospital Anemia Panel 2014 (BDHAP-2014) including 217 known genes of congenital anemias was developed. NGS and parental verification were performed for patients who were suspected diagnosed with congenital anaemia from August 2014 to July 2017. Results: A total of 46 patients were enrolled in this study, the clinical suspection were 11 cases Fanconi anemia (FA), 8 cases congenital dyserythropoietic anemia (CDA), 6 cases congenital sideroblast anemia (CSA), 12 cases congenital hemolytic anemia (CHA), 1 case dyskeratosis congenital (DC), 4 cases iron-refractory iron deficiency anemia and 4 cases unexplained cytopenia (Uc), respectively. 28 (60.9%) of 46 patients became confirmed cases after targeted NGS, corresponding to 44 mutations of which 33 were new. 26(56.5%) patients with results of the assay matching to clinical suspection, including FA (5/11, 45.5%), CSA (6/6, 100.0%), CDA (3/8, 37.5%) and CHA (12/12, 100.0%). 2 (4.3%) cases not matching to clinical suspection, including dyskeratosis congenital (DC) was made in 1(2.2%) patients with suspected FA and familial hemophagocytic lymphohistiocytosis (FHL) was made in 1(2.2%) patients with suspected unexplained cytopenia (Uc). In 12 CHA patients, the hemolytic type was further clarified by the NGS. The remaining 18 cases were not clearly diagnosed. Conclusion Targeted NGS assay is of major impact on congenital anemias. The assay should be used routinely in congenital anemias.

Objective: To reveal the genetic characteristics of erythrocyte membrane protein in hereditary spherocytosis (HS) in China. Methods: Next-generation sequencing technology was used to detect mutations in genes of erythrocyte membrane proteins in 51 clinically diagnosed HS patients. The relationship between gene mutations and clinical phenotypes was analyzed. Results: Mutations in erythrocyte membrane protein genes were detected in 37 patients, including 17 with ANK1 mutations (17/37, 45.9%), 14 with SPTB mutations (14/37, 37.8%), and 5 with SLC4A1 mutations (5/37, 13.5%). One patient carried both heterozygous ANK1 mutation and SPTB mutation (1/37, 2.7%). SPTA1 and EPB42 mutation was not fou nd in any patient. Nonsense mutations (36.8%) and missense mutations (31.6%) were most common. Of the 38 mutations detected, 34 were novel mutations and have not been reported elsewhere (89.5%). Sixteen HS patients underwent parental genetic validation, 6 patients (37.5%) inherited gene mutation from parents and 10 (62.5%) were de novo. The peripheral blood cell parameters of HS patients were not related to the mutant genes and gene mutation types. However, it seems that HS patients with mild clinical status are prone to carry SPTB mutations while more patients with severe clinical status have ANK1 mutations. Conclusions ANK1 and SPTB are the most common mutant genes in Chinese HS patients, mainly with missense mutations and nonsense mutations. There was no significant correlation between the mutation of HS related genes and the severity of HS.