Objective To investigate the clinical manifestation of convulsive status epilepticus (CSE) associated with the outcome of children contracted this illness in order to make an effort to prevent and treat those children. Method The patients with CSE admitted from October 1998 to October 2008 were prospectively and consecutively enrolled. All the patients met the diagnostic criteria set by International Anti-epilepsy Union (ILAE) in 1993. The prognosis predictors of outcome were analyzed with the Rank sum test, Chi-square analysis and Logistic regression analysis. Results A total of 169 patients with CSE were enrolled. Of them, 92 patients were from rural areas (54.4% ). The primary cause was central nervous system infection, and the minor ones were a reduction of dose or withdrawal of the antiepileptics, and cerebral hemorrhage. The duration of CSE before treatment was longer in rural patients than in urban residents ( P < 0.01) . The duration of CSE before treatment and the total persistence time of CSE was significantly associated with the mortality 14.2% . The source of patients, the duration before treatment, and respiratory depression were independent prognostic predictor variables of CSE (P < 0.05) .Conclusions The rural patients account for the major ratio of convulsive status epilepticus. Central nervous system infection is the primary cause. The duration before treatment and total persistence time of CSE are highly related to the mortality and morbidity.

Objective:To establish Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines using CRISPR/Cas9 technology and 3D retinal organoid culture.Methods:The target site sequence of H9 cell line was verified by polymerase chain reaction (PCR). SgRNAs were designed by CRISPR/Cas9 technique and their activity was detected.The most optimal sgRNA was selected according to the factors such as activity and specificity.After identification of the target vectors by restriction enzyme and sequencing, the target vectors were transferred to the H9 cell line by electroporation.P2A-tdTomato-P2A-iCreERT2 was inserted between Exon4 and 3’-untranslated region of hES-ZLM-001 gene.Knockin positive clones were obtained after drug treatment, enrichment of positive clones.Primers were designed to perform PCR on the target region, and homozygous de-resistant knockin positive cell clones were selected according to the sequencing results and peaks.The 1-A07 cell line was cultured, and then flow cytometry for the proportion of OCT4 positive cells, immunofluorescence for three stem cell molecular markers including SOX2, NANOG, SSEA4, karyotype analysis were carried out to confirm whether the 1-A07 cell line could be used for further experiments.Retinal organoids were obtained by three-dimensional (3D) culture technology and the expression of molecular markers was detected by immunofluorescence at different developmental stages of retinal organoids. Results:The target site sequence of H9 cell line was consistent with that given by Genebank and Ensembl.Sixteen sgRNAs were designed according to the target site sequence of H9 cell line, and finally sgRNA8 and sgRNA12 were selected.The sgRNAs and recombinant plasmids were transfected into the H9 cell line by electroporation, and four homozygous de-resistant knockin positive cell clones were obtained by PCR.Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were successfully obtained.In 1-A07 cell line, the proportion of OCT4 positive cells was about 98.7% by flow cytometry, and the expression of three stem cell markers was positive by immunofluorescence, and the karyotype was normal 46, XX.The results showed that the 1-A07 cell line could be used for further experiments.The Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were differentiated into tdTomato positive retinal organoids by 3D culture technology.BRN3A positive ganglion cells, CALBINDIN positive horizontal cells and CHAT positive amacrine cells appeared on day 30 of differentiation.RECOVERIN positive photoreceptors arose on day 45 of differentiation.PKCα positive bipolar cells presented on day 90 of differentiation.Ganglion cells were shown in the deep layer of retinal organoids, and horizontal cells, amacrine cells and bipolar cells in the middle layer, and photoreceptors in the top layer.Conclusions:Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines are successfully established and can be differentiated into retinal organoids that express tdTomato red fluorescence through 3D culture technology.Those retinal organoids contain the same types of neurons as normal human retinas, and follow a certain temporal and spatial developmental sequence similar to the developmental rules of normal human retinas.Crx-iCreERT2 fluorescent reporter human embryonic stem cell line is a powerful tool for researching retinal development and diseases and can be applied in treatments for blindness.

Biomedical Research ; Histones ; Humans ; Neoplasms

Objective To explore the feasibility of 256-slice whole-brain CT perfusion (CTP) in evaluate graft reperfusion after surgical revascularization and hemodynamic alterations before and after surgery in Moyamoya disease. Methods Twenty-five cases with Moyamoya disease were scanned on a 256-slice CT.CTP was performed pre- and post- surgical revascularization. The wolumetric CT angiographic ( CTA ) images were generated from volumetric data acquired at the arterial phase of CTP. CBF, CBV, TTP and MTT were measured in functional maps at the operated side within middle cerebral artery perfusion areas and contralateral mirroring areas. Relative CBF( rCBF), relative CBV (rCBV), relative TTP (rTTP), relative MTT (rMTT) were also obtained. Differences in perfusion CT values pre- and post operation were assessed with the paired t test or matched-pairs signed-ranks test. Data with normal distribution was present as : (x-)± s,while those with the non-normal distribution were present as M ( P25-P75 ). Results All the direct graft patencies were displayed on volumetric CTA. No significant differences were found between volumetric CTA and conventional CTA. Postoperative CBF, rCBF and rCBV values of the operated side [ 72. 86 (55.54-112. 19) ml · 100 g-1 · min-1 , 1. 31 ( 1.05-1.73), 1.45 ±0. 62] were significantly higher than those before operation [46.72(28.57-57.67) ml · 100 g-1 · min-1, 0.53(0.33-0.82), 1.01 ±0.36](Z=- 2.72, - 2. 98, t = - 2. 85, P ＜ 0. 05 ). Postoperative MTT, TTP and rTTP values of the operated side [ (3.98 ± 2. 36 ) s, ( 17.56 ± 4. 38 ) s, 1.01 ± 0. 09 ] were significantly lower than those before operation [(5.43±2.07) s,(19.40±3.87) s,1.14±0.28] (t=2.41,2.17,2.17, respectively, P＜0.05).However, no significant differences were detected for changes of CBV and rMTT after revascularization ( P ＞0. 05). Conclusion 256-slice CT has the potential value for the non-invasive assessment of both the graft patency and cerebral hemodynamics changes in moyamoya disease after surgery with administration of one contrast medium bolus in a single examination.

Objective To investigate the effect of ultrashort wave therapy on edema and inflammatory reaction after spinal cord injury (SCI) in rats. Methods 56 Sprague-Dawley rats were divided into sham-operated group (n=8), model group (n=24) and ultrashort wave group (n=24). The model was established with Allen's method. The sham-operated group was exposed endorhachis without hit. The ultra-short wave group was exposed to ultrashort wave radiation 7 minutes, once a day, 24 hours after modeling until the animals were sacrificed. Locomotors functional recovery was assessed every week post operation period by BBB score. Immunohistochemical staining was per-formed to observe the expression of the aquaporin-4(AQP-4) and ED-1. Results The BBB scores increased in the model group and the ultra-short wave group after treatment, and was higher in the ultrashort wave group than in the model group from 1 week after treatment (t>3.368, P<0.01). The expressions of AQP-4 and ED-1 were higher in the model group and ultrashort wave group than in the sham-operated group, and decreased as time went on. They were lower in the ultrashort wave group than in the model group(t>3.156, t>4.466, P<0.05). Conclu-sion Ultrashort wave therapy can alleviate the edema after SCI, reduce the activation and infiltration of inflammatory cells, and promote the recovery of neurological function.

Aim By establishing mouse acute myocardial infarction model,to observe the protection of isoflavones on ischemic myocardium and research the mechanism.Methods Mouse acute myocardial infarction model was established by ligating the left anterior descending(LAD)coronary artery.Danshen was used as the positive control.The effect of isoflavones on myocardial infarct area,serum myocardium creatase and serum levels of SOD and MDA was observed.By Real Time PCR,it was found that isoflavones could affect the expression of β-adrenergic receptor kinase(β-ARK_1).Results Isflovones could obviously reduce the myocardial infarct area and lower the levels of serum myocardial creatase and MDA.It could downregulate the expression of β-ARK_1 as the doses are increased.Conclusions Isoflavones can protect the myocardium of acute myocardial infarction mouse.The mechanism is related to the reduction of the oxidative damage,and the downregulation of the expression of β-ARK_1.

Objective To study the structural features and characteristics of a novel gene Schistosoma japonicum 79(Sj79), and observe its effect of RNA interference(RNAi),so as to provide the experimental basis for its further function study and mechanism study of anti reproductive development of schistosome. Methods The gene structure and characteristics of Sj79 were analyzed by bioinformatics methods. Then the expressions of Sj79 messenger RNA(mRNA)during the different develop?mental stages of schistosome were analyzed and the effects of RNAi silencing were observed by the soaking method. The tran?scriptional levels of Sj79 after RNAi were detected by real time PCR. Results The open reading frame of Sj79 contained 696 base pairs with an exon structure. The gene had obvious stage specificity,and its transcriptional level in mature female worms was the highest. After soaking for 3 d,the Sj79 mRNA level[(41.0 ± 12.3)%]in the siRNA?1 group with low dosage(20 nmol/L) was lower than that in the siRNA?NC group[(103.2 ± 14.4)%],the difference was statistically significant(t=3.28,P<0.05). When with high dosage(200 nmol/L ),both the Sj79 mRNA levels in the siRNA?1 group[(15.8 ± 10.9)%]and siRNA?2 group [(11.1 ± 8.8)%]were significantly lower than that in the siRNA?NC group[(100.1 ± 6.3)%](t=13.44,27.84,both P<0.01). After soaking for 7 d,only the Sj79 mRNA levels in the siRNA?1group[(43.4 ± 4.5)%]and siRNA?2 group[(62.5 ± 5.4)%]with low dosage were lower than that in the siRNA?NC group[(100.4 ± 5.2)%],and the differences had statistical sig?nificance(t=8.33,5.07,both P<0.01). Conclusion Through this study,we have improved the mRNA sequence and genom?ic information of Sj79 gene,and understood its structural features,as well as selected out two effect fragments siRNA?1and siR? NA?2 which will provide the basic evidences for the further study on egg laying interference of the female adult worm of schisto?some in vitro.

Objective To investigate the effects of bone marrow stromal cells(BMSCs)transplantation combined with low dose ultrashort wave (USW)radiation on functional recovery and the expression of glial fibrillary acidic protein(GFAP)and ED?1 after spinal cord injury(SCI)in rats,and further discuss its action mechanism. Methods Female Sprague?Dawley rats(n=30)were randomly divided into 5 groups:sham?oper?ated,as well as control,USW,BMSCs,and USW+BMSCs that were subjected to spinal cord injury(SCI). Basso?Beattie?Bresnahan(BBB)tests were carried out before the operation and at 1 d,1 week,2 weeks,3 weeks,4 weeks after SCI. 4 weeks later,animals were sacrificed and tissues were collected to make paraffin section. Immunohistochemical staining was performed to observe the expression of GFAP and ED?1. Results 4 weeks after SCI,BBB scores were significantly higher in the USW and USW+BMSCs groups than in the control group(both P<0.001). No signifi?cant difference was observed between the BMSCs group and control group. On the expression of GFAP ,only USW+BMSCs group showed signifi?cantly decreased compared with the control group(P<0.05). All treatment groups exhibited lower ED?1 expression than the control group(all P<0.05). Conclusion Our results indicate that USW radiation alone can obviously improve neural functional recovery after SCI. The USW radi?ation and BMSCs transplantation treatment can reduce inflammation ,and USW radiation is more effective. The combination therapy did not show a synergistic action on promoting functional recovery ,but do have an effect on reducing the inflammatory response and glial scar formation.

Objective To investigate the expression of neuron-specific enolase(NSE) and bone morphogenic protein 4(BMP4) in different hippocampal areas of pentylenetetrazol(PTZ) kindled epilepsy rats and explore their relationship with the pathogenesis of epilepsy and brain injury.Methods Fifty male SD rats were divided into experimental group(n=40) and control group(n=10).The rats in experimental group were kindled into epilepsy by chemical method,and according to the kindling process,subdivided into four groups(grade Ⅰ,Ⅲ,Ⅳ,Ⅴ).Immunohistochemistry,in situ hybridization labeled with Dig-oligonucleotide probe and the image analyzing system were used to observe the expressions of NSE and BMP4 in rat hippocampus.Results In PTZ kindled epilepsy rats,the number of cells positive for NSE and BMP4 was increased in many regions of hippocampal formation.Compared with control group,the expressions of NSE and BMP4 in CA3 and DG was elevated obviously in the grade Ⅲ group and grade Ⅳ group(P

Objective: To explore the effects of macrophages with high expression of TL1A on the activation and proliferation of HSCs in vitro. Methods: The Bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PMs) from wild type (WT) and myeloid-overexpressed TL1A transgenic mice were isolated, differentiated and activated. HSCs were harvested from activated macrophages culture supernatant (CM). HSCs were detected by immunofluorescence and real-time Q-PCR. And the proliferation was detected by CCK-8 and BrdU assay kit. The levels of IL-1β and PDGF-BB in macrophage culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Results: BMMs-derived CM-intervention HSCs were used to detect the expression of α-smooth muscle actin (α-SMA) on the 2nd, 4th and 6th day respectively by immunofluorescence method. There was no significant difference between the two groups on the 2 nd and the 6th day, P > 0.05; On day 4, the CM/Tg group was significantly higher than that of CM/WT group, P < 0.01; the results of CMs derived from PMs were consistent with the above trend. The expression of α-SMA mRNA on the 2nd, 4th and 6th day was detected by real-time Q-PCR method using BM-derived CMs. No significant difference was found between the groups on the 2nd day (P > 0.05).α-SMA mRNA increased further on the 4th and 6th day, and the level of CM/Tg in CM/Tg group was significantly higher than that in CM/WT group (P < 0.05). The detection results of CMs derived from PMs were consistent with the above trend. The results of CCK-8 assay and BrdU assay showed that the proliferation rate of HSCs in CM Tg group was significantly higher than that in CM/WT group (P < 0.01). The CMs derived from PMs were used to interfere with HSCs. And the results were consistent with the above trend. For BMMs, the levels of IL-1β and PDGF-BB in the lipopolysaccharide (LPS) + IFNγ/Tg culture supernatant were significantly higher than those in the LPS+IFNγ/WT group (P < 0.01). For the culture supernatants of PMs Liquid test results consistent with the above trend. Conclusion Macrophages with high expression of TL1A could enhance the activation and proliferation of HSCs by increasing the secretion of IL-1β and PDGF-BB.