目的 观察羟基红花黄色素A(hydroxysafflor yellow A,HSYA)对脑缺血再灌注损伤小鼠皮层COX-2/PGD2/DPs途径的影响.方法 C57BL/6雄性小鼠随机分为假手术组、模型组、HSYA组,线栓法建立小鼠MCAO/R模型,术前连续5 d尾静脉注射HSYA 20 mg·kg-1,假手术组、模型组给予等量生理盐水;术后24 h对小鼠神经功能评分、脑梗死体积测定,HE染色法观察小鼠皮层组织病理学,Western blot和qRT-PCR法分别检测COX-2、DP1、DP2 mRNA和蛋白表达,ELISA检测TNF-α、IL-1β、PGD2含量.结果 与假手术组相比,模型组神经功能评分和脑梗死体积明显增加,皮层细胞损伤明显,皮层COX-2、DP1、DP2 mRNA和蛋白表达明显增加,TNF-α、IL-1β、PGD2含量明显增加;与模型组相比,HSYA组神经功能评分明显降低,脑梗死体积减少,缺血区皮层细胞损伤明显改善,COX-2、DP1、DP2 mRNA和蛋白表达明显下调,TNF-α、IL-1β、PGD2水平明显降低.结论 HSYA抑制COX-2/PGD2/DPs途径的激活,可能参与对脑缺血再灌注脑损伤保护作用.

Objective·To explore the effects of adenovirus vector-mediated small interfering RNA (siRNA) targeting phosphatase nuclear targeting subunit (PNUTS) on proliferation,invasion,migration and epithelial-mesenchymal transition (EMT) of laryngeal squamous cell carcinomas Hep-2 cells and its mechanism.Methods· Recombinant adenovirus vector expressing PNUTS siRNA was infected into laryngeal squamous cell carcinomas Hep-2 cells and the experiment was designed into PBS group,Ad-GFP group and Ad-siPNUTS group.Levels ofPNUTS mRNA and protein were detected by real-time PCR and Western blotting respectively.MTT assay was used to detect proliferation abilities of Hep-2 cells.Transwell assays were used to detect invasion and migration abilities of Hep-2 cells.The expression levels of total Rb,phosphorylated Rb (p-Rb),PI3K,phosphorylated AKT (p-AKT),E2FI,E-cadherin,N-cadherin and ZEB1 protein were detected by Western blotting.Results· Compared with Ad-GFP group,in Ad-siPNUTS group,the PNUTS mRNA and protein (both P=0.000) levels were dramatically decreased.The proliferation of Ad-siPNUTS infected Hep-2 cells were inhibited on the second day (P=0.004),the third day (P=0.001) and the fourth day (P=0.000).Meanwhile,the invasion and migration abilities of Ad-siPNUTS infected Hep-2 cells were decreased (both P=0.000).The expression levels of total Rb (P=0.000),p-Rb (P=0.000),PI3K (P=0.023),p-AKT (P=0.000),E2F 1 (P=0.000),N-cadherin (P=0.005) and ZEB 1 (P=0.000) were decreased while the E-cadherin (P=0.003) was increased.Conclusion· Ad-siPNUTS could inhibit the proliferation,invasion and migration abilities of Hep-2 cells and reverse the development of EMT,which may be related to PI3K/AKT signaling pathway and Rb signaling pathway.

目的:探讨X线电离辐射诱导鼻咽癌CNE-2细胞发生上皮-间充质转化(epithelial-mesenchymal transition,EMT)及其可能的信号通路.方法:分别采用0 Gy、2 Gy、4 Gy和8 Gy的X线照射鼻咽癌CNE-2细胞,通过倒置显微镜观察照射24 h后细胞形态的改变;应用细胞划痕实验和Transwell实验观察细胞迁移和侵袭能力的变化;分别采用real-time PCR和Western blot法检测EMT相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cad-herin)和波形蛋白(vimentin)mRNA及蛋白水平的变化,并采用Western blot法检测Akt和p-Akt蛋白水平的变化.结果:经X线照射后鼻咽癌CNE-2细胞呈典型的"鹅卵石"样或纺锤形,且伸出伪足,细胞间隙增大,细胞的侵袭和迁移能力增强(P<0.01);real-time PCR和Western blot法检测结果显示,与未照射组相比,各照射组细胞中E-cad-herin mRNA和蛋白的表达水平均显著下调(P<0.01),而N-cadherin和vimentin mRNA和蛋白的表达水平明显上调(P<0.05);PI3K/Akt信号通路中p-Akt蛋白的水平显著升高(P<0.01),而Akt的表达无明显变化.结论:X线电离辐射能够诱导鼻咽癌CNE-2细胞发生EMT,其机制可能与PI3K/Akt信号通路的激活有关.

Objective To investigate the effects of chronic intermittent hypoxia and on GLUT-2, IRS-2 expression in the rat kidney .Methods Rats were randomly divided into control group ( control) , chronic intermittent hypoxia group (CIH), chronic intermittent hypoxia reoxygenation group (RH).The chronic intermittent hypoxia animal models were developed , arterial blood gas analysis was immediately carried out .Serum glucose was measured by peroxidase and serum insulin was detected by radioimmunoassay .After Removing the kidney tissue of rats ,protein expression of GLUT-2, IRS-2 were detected with Western blot and immunohistochemical , mRNA expression of GLUT-2,IRS-2 were observed by qPCR .Results Oximetry in the control group was ≥95%, oxygen saturation in the CIH group was ≤85%, oxygen saturation in the RH group was ≥86%; fasting blood glucose , serum insulin and insulin resistance index in the CIH group were significantly higher those of control group and RH group ( P<0.05);RH group was higher than control group (P<0.05).Protein and mRNA expression of GLUT-2, IRS-2 in&nbsp;the CIH group were higher than the control group and RH group ( P<0.05 ); RH group was higher than control group (P<0.05).Conclusions Chronic intermittent hypoxia can increase blood glucose ,upregulate the expres-sion of GLUT-2 , IRS2 in the rat kidney and enhance insulin resistance and decrease insulin sensitivity .

Objective To explore the clinical features and NEMO gene mutation in a boy with ectodermal dysplasia and immunodeficiency (EDA-ID).Methods According to the patient's clinical presentation and family history,the integrity of the IL-12/IFN-γ axis was detected by Q-RT-PCR after ruling out the common primary immunodeficiency disease (PID),and then NEMO gene was sequenced.Results The patient showed recurrent fever,pale skin,sparse hair,mild frontal bossing,and fungus and BCG infections.The X-ray of the head revealed no obvious tooth germ.The expression of IL-12B mRNA in the whole blood cells was reduced more after stimulation with BCG plus IFN-γ than BCG alone (P ＜ 0.05).NEMO gene sequencing analysis found 1241T ＞ A substitution mutation in exon 10,predicting valine-to-aspartic acid missense mutation at the 414th amino acid site (p.V414D).The mother and one of her sisters had heterozygous mutations at the same site.Cornclusion Male children with BCG infections and recurrent fever should be considered to have mutation of NEMO gene,especially when there are some ectodermal dysplasia phenotypes such as pale and dry skin without sweat,sparse hair and no teeth.Matrilineal family history of incontinentia pigmenti is helpful to diagnosis.EDA-ID can be diagnosed early by combining the integrity test of IL-12/IFN-γ axis and genetic analysis.

Objective To investigate the effects of ciliary neurotrophic factor ( CNTF) on the expression of choline acetyltransferase ( ChAT) in hippocampus and cognitive function of diabetic mellitus ( DM) , and explore its possible mechanism .Methods 50 SD male rats were meanly divided into normal , DM, DM+DMSO, DM+CNTF, DM+CNTF+AG490 groups randomly .Streptozotocin-diabetic rats model were established , diabetic rats with depression were screened by open-field test.Escape latency and frequency of entrance into the target zone were measured by Morris water maze .The mRNA and protein levels of ChAT were examined with RT-PCR,Western blot and immuno-histochemistry.Results Compared to that of normal group ,escape latency was increased and frequency of entrance into the target zone was decreased significantly in DM,DM+DMSO and DM+CNTF+AG490 groups(P<0.05),the mRNA and protein levels of ChAT in hippocampal were decreased significantly (P<0.05) .The scores of escape latency and frequency of entrance into the target zone were highly improved in DM+CNTF group,the mRNA and protein expressions of ChAT in hippocampal were increased markedly than those of DM group (P<0.05) . Conclusions CNTF could improve cognitive ability of DM rats by increasing the mRNA and protein expressions of ChAT through the JAK 2/STAT3 pathway .

The rate of complete surgical resection is still low in hilar cholangiocarcinoma,which greatly affects the curative effect.Radiotherapy,one kind of treatment for tumors,has not been widely adopted in the past years.In recent years,with the development of radiotherapy technology,research of treating hilar cholangiocarcinoma through radiotherapy has become a spot.Many studies have shown that radiotherapy,as an adjuvant therapy for surgery and non-surgical treatment,can be the major means for treatment,and it could bring benefit for patients' survival extension and improvement of life quality.

目的 探讨1例疑似孟德尔遗传易感分枝杆菌病(Mendelian susceptibility to mycobacterial disease,MSMD)患儿的临床特征,检测IL-12/23-IFN-γ通路的完整性,并进行IFN-γ受体1(IFNGR1)基因分析.方法 根据患儿的临床表现及常规的免疫学筛查试验排除常见原发性免疫缺陷病,Q-RT-PCR在mRNA水平检测患儿及健康对照者IL-12/23-IFN-γ通路的完整性,通过PCR及RT-PCR分别对患儿及其父母的IFNGR1基因进行扩增并测序.结果 患儿有播散性卡介苗(BCG)病及全身多系统损害的表现.患儿全血经BCG和IFN-γ共同刺激48 h后IL-12B的表达水平与单独BCG刺激后比较明显降低(P＜0.05);基因测序发现患儿IFNGR1第6外显子818～ 821位杂合缺失4个碱基(c.818-821 delTTAA),使第276位脯氨酸突变为终止密码(p.Asn274fsX276),父母此位点正常.结论 MSMD患儿临床特征为BCG病及全身多系统损害.Q-RT-PCR检测IL-12/23-IFN-γ通路完整性是一种有效的筛查MSMD的手段;IFNGR1突变是引起本例患儿发生上述临床特征的根本原因.

Insulin-like growth factor-1 (IGF-1) is a kind of protein polypeptide which widely exists in the body tissue and its structure and functions are similar to insulin.IGF-1 involves in many pathological and physiological process.Through a variety of ways,IGF-1 affects the occurrence and development of heart,brain,kidney,nerves and other chronic complications of diabetes.Exploring the mechanism of IGF-1 provides new ideas for the treatment of diabetic chronic complications.

Objective:To analyze expression of glutamic acid decarboxylase-65(GADs65) in brain and therapeutic effects of GAD65-engeneered neural stem cells after the cells being transplanated into hippocampus of kainic acid-induced epilepsy model rats.Methods: Kainic acid-induced epilepsy model rats were divided into 3 groups completely randomly: GAD-NSCs transplantation group(n = 20),NSCs transplantation group(n = 20),saline control group(n = 20).The sensitivity in epilepsy models was detected by small dosed pentetrazole-induced test corresponding to 1,2 and 3 days,1,2,4 and 8 weeks after transplantation respectively.GAD65 expression in hippocampus was examined by Western Blotting at 1 week post-transplantation.Hematoxylin-eosin(HE) staining and BrdU,GAD65 immunohistochemistry were performed in rat hippocampal slices at 8 weeks post-transplantation.Results: The sensitivity of epilepsy in GAD-NSCs transplantation group was decreased at 2 days post-transplantation while that in NSCs transplantation group was decreased after 2weeks.The scores according to the scale of Racine in saline control group and NSCs transplantation group were higher than that in GAD-NSCs transplantation group.High-level GAD65 expression in hippocampus was demonstrated only in GAD-NSCs transplantation group.BrdU positive cells and GAD65 positive ceils of GAD-NSCs transplantation group were significantly higher than those in the other two groups.Conclusion: Transplantation of GAD65-engineered neural stem cells into hippocampus of epilepsy model rats,which induced High-level GAD65 expression in hippocampus,could cotinuously inhibit sensitivity of epilepsy,protect the host NSCs of post-transplanation and improve the neuropathologic changes of model animals.

氟西汀(Fluoxetine)是一种高特异性,高选择性的5-羟色胺(5-HT)再摄取抑制剂(Selective reuptake inhibitors,SSRIs),临床上主要用于治疗抑郁性精神障碍.传统抑郁症的"单胺学说"认为,抑郁的发生主要与单胺类神经递质如5-HT及去甲肾上腺素(NE)水平低下有关.近年提出的"神经营养学说"认为,抑郁的发生除与NE、5-HT等神经递质的变化有关外,可能与神经突触可塑性的损害有关.近年来国外有研究发现氟西汀能加快卒中患者的康复治疗过程.

Objective:Toexplore a safe and effective method for dissociatingneural stemcell neurospheres cultured in vitro.Methods:Neurospheres were dissociated by using of four methods,that digestion of trypsin,triturating only with pasteur pipette,grinding with stainless steel mesh and controlling volume of nerospheres with short-time digestion of trypsin.The four methods were evaluated by comparing their discrete capacity and cell survivals.Results:Trypsin digestion time prolonged led to dissociation of neurospheres,but neural stem cells(NSCs)were hardly survival.Neurospheres could not be dissociated completely by triturating only with pasteur pipette.Grinding with stainless steel mesh would damage neurospheres severely induced lowsurvival rates of NSC.In contrast,controlling volume of nerospheres with short-time digestion of trypsin could separate neurospheres better than the other methods and higher NSC survival rate(93.2%,P0.05).Conclusion:Controlling volume of nerospheres with short-time digestion of trypsin is a safe and effective method for dissociating neurospheres of NSCs cultured in vitro better than other three methods.