miR-143在氧糖剥夺/再灌注引起的星形胶质细胞活化中的作用机制研究

目的 探讨miR-143/HIPK2轴在氧糖剥夺/再灌注（oxygen-glucose deprivation/reperfusion,OGD/R）引起的星形胶质细胞活化中的作用。方法 星形胶质细胞OGD/R处理0,3,6,12 h,应用Western blotting检测星形胶质细胞的活化标志物GFAP和蛋白激酶HIPK2表达,实时荧光定量PCR检测miR-143水平变化,免疫荧光染色检测OGD/R处理6 h后GFAP表达。对照组、OGD/R模型组给予Anti-Con和Anti-miR-143干预,应用Western blotting检测GFAP表达。采用荧光素酶报告基因技术验证HIPK2是否为miR-143的靶标分子。miR-143和miR-Con以及Anti-miR-143和Anti-miR-Con分别转染星形胶质细胞,应用Western blotting检测HIPK2表达。离体培养星形胶质细胞分为5个处理组,分别为Anti-Con干预的对照组、Anti-Con干预的OGD/R模型组、Anti-miR-143干预的OGD/R模型组、Anti-miR-143和siRNA Con共同干预的OGD/R模型组以及Anti-miR-143和HIPK2 siRNA共同干预的OGD/R模型组,应用Western blotting检测GFAP表达。结果 与对照组相比,OGD/R 6 h处理的星形胶质细胞GFAP表达增高至峰值（P<0.01）,该结果进一步被免疫荧光染色验证,HIPK2表达下调至最低值（P<0.001）,miR-143表达增加（P<0.01）。与Anti-Con干预的对照组相比,Anti-Con干预的OGD/R模型组星形胶质细胞GFAP表达上调（P<0.01）;与Anti-Con干预的OGD/R模型组比,Anti-miR-143干预的OGD/R模型组星形胶质细胞GFAP表达下调（P<0.05）。miR-143能明显抑制HIPK2基因活性,但对其结合位点突变体没有显著抑制作用。与Anti-miR-143和siRNA Con共同干预的OGD/R模型组比,Anti-miR-143和HIPK2 siRNA共同干预的OGD/R模型组星形胶质细胞GFAP表达水平上调（P<0.05）。结论OGD/R通过miR-143/HIPK2轴引起星形胶质细胞活化。     

丹参和三七有效组分对氧糖剥夺损伤的大鼠星形胶质细胞谷氨酸摄取的影响

目的探讨丹参、三七有效组分单独及联合使用对氧糖剥夺/复氧(OGD/R)诱导的星形胶质细胞谷氨酸摄取能力的影响。方法体外培养原代Wistar大鼠大脑皮层星形胶质细胞,分为8个组别:正常组,模型组,低、高剂量丹参多酚酸组,低、高剂量血栓通组和低、高剂量联合组。正常组和模型组均不给药;低、高剂量丹参多酚酸组分别给予12.5和25.0μg·mL^-1丹参多酚酸;低、高剂量血栓通组分别给予3.125和6.25μg·mL^-1血栓通;低、高剂量联合组分别给予12.5μg·mL^-1丹参多酚酸联合3.125μg·mL^-1血栓通,25.0μg·mL^-1丹参多酚酸联合6.25μg·mL^-1血栓通。正常组给予常规培养;模型组进行OGD 3 h/R 3 h的处理;给药组于给药2 d后进行OGD 3 h/R 3 h的处理。用酶标仪检测细胞活力和谷氨酸含量。结果正常组,模型组,低、高剂量丹参多酚酸组,低、高剂量血栓通组和低、高剂量联合组的细胞活力值分别为1.00±0.14,0.35±0.66,0.41±0.10,0.68±0.09,0.41±0.13,0.60±0.12,0.44±0.14和0.71±0.11,摄取谷氨酸量分别为(65.29±0.89),(34.96±13.27),(57.33±18.56),(52.34±13.07),(55.50±4.54),(52.94±12.30),(48.73±11.27)和(52.15±7.52)nmol·L^-1。与模型组相比,正常组和3个高剂量组的细胞活力均显著升高,差异均有统计学意义(均P<0.001);与低剂量联合组相比,3个高剂量组的细胞活力均显著升高,正常组、低剂量丹参多酚酸组和低剂量血栓通组的摄取谷氨酸量均明显升高,差异均有统计学意义(均P<0.05);与高剂量联合组相比,3个低剂量组的细胞活力均显著降低,差异均有统计学意义(均P<0.05)。结论低剂量丹参多酚酸组和低剂量血栓通组可以提高受损星形胶质细胞谷氨酸摄取能力,高剂量丹参多酚酸组、高剂量血栓通组和低、高剂量联合组对受损星形胶质细胞谷氨酸摄取能力无影响。     

N-乙酰半胱氨酸对糖氧剥夺诱导星形胶质细胞损伤的抑制作用及机制研究

目的 探讨N-乙酰半胱氨酸（N-acetyl-L-cysteine,NAC）对糖氧剥夺（oxygen and glucose deprivation,OGD）诱导的星形胶质细胞损伤过程中AKT信号通路的影响及其意义。方法 以原代培养的星形胶质细胞建立OGD模型,并分为4组：对照组,OGD组,NAC组和NAC+AKT特异性抑制剂MK-2206组（NAC+MK-2206组）;细胞培养24 h后,采用噻唑蓝（MTT）法检测细胞存活率、流式细胞仪技术分析细胞凋亡比例,试剂盒检测细胞内SOD活性和丙二醛（MDA）含量,蛋白免疫印迹法检测细胞内AKT、磷酸化AKT（p-AKT）、mTORC1、磷酸化mTORC1（p-mTORC1）、胞浆型磷脂酶A2（cLPA2）、caspase3及Bcl-2的表达水平。结果 与OGD组相比,NAC组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性增加,细胞凋亡比例,cPLA2、caspase3表达,MDA含量降低。与NAC组相比,NAC+MK-2206组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性降低,细胞凋亡比例、cPLA2、MDA含量、caspase3表达增加。结论 NAC缓解OGD诱导AKT信号通路抑制作用,降低cPLA2诱导细胞凋亡作用。     

小续命汤含药血清对氧糖剥夺模型大鼠星形胶质细胞的保护作用研究

目的:探讨小续命汤含药血清对氧糖剥夺(OGD)模型大鼠星形胶质细胞的保护作用及其机制。方法:将体外培养的大鼠星形胶质细胞随机分为对照组、模型组和小续命汤含药血清低、中、高浓度组,其中对照组细胞不作任何处理,模型组和小续命汤含药血清低、中、高浓度组细胞经OGD 2.5 h后分别在0(即模型组不加药)、2.5%、5%、10%的小续命汤含药血清中复氧0、3、6、12 h,采用比色法检测细胞中乳酸脱氢酶(LDH)含量。取OGD后复氧12 h时的对照组、模型组和小续命汤含药血清高浓度组大鼠细胞,采用荧光探针法检测其活性氧(ROS)水平,采用免疫荧光双染法检测其锰超氧化物岐化酶(MnSOD)水平。结果:对照组细胞中LDH含量始终保持在较低水平;模型组细胞分别在OGD后复氧0~12 h时,其LDH含量从(110.99±17.06)U/L逐渐上升至(436.64±55.29)U/L,均显著高于对照组(P<0.05);与OGD后复氧相同时间点的模型组比较,小续命汤含药血清低、中、高浓度组细胞中LDH含量均不同程度地降低,并表现出时间和剂量依赖趋势,其中小续命汤含药血清各浓度组细胞在OGD后复氧6、12 h时其LDH含量均较模型组显著降低(P<0.05)。OGD后复氧12 h时,模型组细胞中ROS水平显著高于对照组,MnSOD水平显著低于对照组(P<0.05);小续命汤含药血清高浓度组细胞中ROS水平显著低于模型组,MnSOD水平显著高于模型组(P<0.05)。结论:小续命汤含药血清可能通过上调MnSOD的水平,清除过量的ROS,从而减轻OGD导致的大鼠星形胶质细胞损伤,发挥其保护作用。     

吡拉格雷钠对氧糖剥夺/复氧后星形胶质细胞水肿及AQP4表达的影响

目的探讨氧糖剥夺并复氧培养后星形胶质细胞水肿及其水通道蛋白4(AQP4)表达的变化以及吡拉格雷(TSF)对其表达变化的影响。方法体外培养原代星形胶质细胞,建立氧糖剥夺/复氧细胞水肿模型,并随机分为正常组、氧糖剥夺/复氧损伤(OGD/Reox)组、奥扎格雷钠(Ozagrel)阳性对照组和TSF治疗组,在氧糖剥夺/复氧损伤后6、12、24和48 h 4个时间点测定细胞体积变化,乳酸脱氢酶(LDH)漏出率、MTT法检测细胞损伤及存活情况,Western blot法检测各组细胞膜AQP4蛋白的表达水平,RT-PCR法检测各组细胞AQP4 m RNA的转录水平。结果星形胶质细胞经缺氧再复氧处理,随着复氧时间的延长细胞损害加重(P<0.05,P<0.01);TSF治疗组的细胞体积明显低于OGD/Reox损伤组(P<0.05);给予TSF治疗后细胞在氧糖剥夺/复氧后的LDH漏出率较OGD/Reox损伤组明显降低(P<0.05);TSF治疗组与单纯氧糖剥夺/复氧相比较MTT检测细胞活力明显升高(P<0.05);给予TSF治疗组,AQP4表达明显降低(P<0.05);与Ozagrel相比较差异无显著性。Western blot及RT-PCR检测蛋白及m RNA的表达水平,给予TSF治疗组的AQP4蛋白及m RNA表达均明显降低(P<0.05)。结论TSF治疗能明显减轻体外氧糖剥夺/复氧损伤引起的星形胶质细胞水肿,可能是通过降低氧糖剥夺后AQP4的表达水平而实现。     

吡拉格雷钠对氧糖剥夺/复氧后星形胶质细胞水肿及AQP4表达的影响北大核心CSCD

目的探讨氧糖剥夺并复氧培养后星形胶质细胞水肿及其水通道蛋白4(AQP4)表达的变化以及吡拉格雷(TSF)对其表达变化的影响。方法体外培养原代星形胶质细胞,建立氧糖剥夺/复氧细胞水肿模型,并随机分为正常组、氧糖剥夺/复氧损伤(OGD/Reox)组、奥扎格雷钠(Ozagrel)阳性对照组和TSF治疗组,在氧糖剥夺/复氧损伤后6、12、24和48 h 4个时间点测定细胞体积变化,乳酸脱氢酶(LDH)漏出率、MTT法检测细胞损伤及存活情况,Western blot法检测各组细胞膜AQP4蛋白的表达水平,RT-PCR法检测各组细胞AQP4 m RNA的转录水平。结果星形胶质细胞经缺氧再复氧处理,随着复氧时间的延长细胞损害加重(P<0.05,P<0.01);TSF治疗组的细胞体积明显低于OGD/Reox损伤组(P<0.05);给予TSF治疗后细胞在氧糖剥夺/复氧后的LDH漏出率较OGD/Reox损伤组明显降低(P<0.05);TSF治疗组与单纯氧糖剥夺/复氧相比较MTT检测细胞活力明显升高(P<0.05);给予TSF治疗组,AQP4表达明显降低(P<0.05);与Ozagrel相比较差异无显著性。Western blot及RT-PCR检测蛋白及m RNA的表达水平,给予TSF治疗组的AQP4蛋白及m RNA表达均明显降低(P<0.05)。结论TSF治疗能明显减轻体外氧糖剥夺/复氧损伤引起的星形胶质细胞水肿,可能是通过降低氧糖剥夺后AQP4的表达水平而实现。     

神经胶质成熟因子-β诱导糖尿病大鼠视网膜Müller细胞炎症反应的机制研究

目的 研究神经胶质成熟因子-β(GMFB)对糖尿病大鼠视网膜Müller细胞活化的作用及相关机制。方法SPF级雄性SD大鼠60只，采用腹腔注射链脲佐菌素（STZ,55 mg/kg)制备糖尿病模型后分为STZ组、STZ+AAVGMFB组和STZ+AAV-GMFB+K252a组，每组15只。另取15只正常大鼠作为CON组。STZ+AAV-GMFB组和STZ+AAV-GMFB+K252a组大鼠于成模8周后玻璃体腔单次注射AAV-GMFB腺病毒载体5μL。STZ+AAV-GMFB+K252a组在注射腺病毒基础上给予腹腔注射25μg/（kg·d）的K252a。12周后，免疫荧光检测GMFB在Müller细胞中的表达，免疫组织化学染色检测视网膜胶质纤维酸性蛋白（GFAP）的表达，酶联免疫吸附试验检测视网膜炎性因子肿瘤坏死因子-α(TNF-α)、白细胞介素（IL）-1β、IL-6的表达，Western blot检测GMFB、脑源性神经营养因子（BDNF）及磷酸化酪氨酸激酶受体B(p-TrkB)蛋白相对表达水平。HE染色检测视网膜病理改变。结果 GMFB在Müller细胞中大量表达。与CON组比较，S...     

8-O-乙酰山栀子苷甲酸对慢性炎性痛模型大鼠脊髓背角内HDAC1～5表达的影响及与JAK_2-STAT_3信号通路的关系研究

目的:研究8-O-乙酰山栀子苷甲酸(8-OaS)对慢性炎性痛模型大鼠脊髓背角内组蛋白去乙酰化酶1～5(HDAC1～5)表达的影响及与Janus激酶2-信号转导与转录活化因子3(JAK_2-STAT3)信号通路的关系。方法:取SD大鼠随机分为正常对照组、假手术组(生理盐水)、完全弗氏佐剂(CFA)组(生理盐水)和8-OaS低、中、高剂量组(2、20、200μg/kg),每组6只,除正常对照组和假手术组外,其余各组大鼠左侧后足趾侧皮下注射CFA复制慢性炎性痛模型,建模后腹腔注射相应药物,每日1次,连续7 d。采用热辐射法检测首次给药第1、2、3、4、5、6、7、8、11、15天大鼠的缩足潜伏期。另取大鼠按上述后5组方法进行分组给药,采用Western blot法检测大鼠末次给药后腰膨大节段脊髓背角中HDAC1～5、磷酸化JAK_2(pJAK_2)、磷酸化STAT3(pSTAT3)蛋白表达情况。再另取大鼠随机分为假手术组(生理盐水)、CFA组(生理盐水)、8-OaS组(20μg/kg)和JAK_2-STAT_3的拮抗药α-氰基-(3,4-羟基)-N-苄苯乙烯胺(AG490)组(8 mg/kg),每组6只,同上法复制IP模型后腹腔注射相应药物,每日1次,连续7 d,采用免疫荧光组织化学染色观察各组大鼠HDAC5和胶质纤维酸性蛋白(GFAP)在脊髓背角的表达情况。结果:与正常对照组和假手术组比较,其余各组大鼠的缩足潜伏期均明显缩短(P<0.05);与CFA组比较,8-OaS低、中、高剂量组大鼠的缩足潜伏期均明显延长(P<0.05),且呈剂量依赖性。与假手术组比较,CFA组大鼠脊髓背角中HDAC1～5、pJAK_2、pSTAT3蛋白表达均明显增强(P<0.05);与CFA组比较,8-OaS低、中、高剂量组大鼠脊髓背角中HDAC5、pJAK_2、pSTAT3蛋白表达均明显降低(P<0.05),但HDAC1～4蛋白表达差异均无统计学意义(P>0.05)。HDAC5大量表达于星形胶质细胞上,与假手术组比较,CFA组大鼠脊髓背角中GFAP和HDAC5表达均明显增强(P<0.05);与CFA组比较,8-OaS组和AG490组大鼠脊髓背角中GFAP和HDAC5表达均明显降低(P<0.05)。结论:8-OaS可有效缓解由CFA诱导的慢性炎性痛,其机制可能与下调脊髓背角中HDAC5表达和JAK_2、STAT3的磷酸化水平有关。     

癫痫伴抑郁症患者生活质量的研究

目的:评价各种危险因素对癫痫伴发抑郁患者生活质量的影响及心理干预的治疗效果.方法:采用Beck抑郁问卷(BDl)及癫痫患者生活质量量表-3I(QO-LIE-31中文版),并自制一般情况调查表对患者进行测查,对癫痫伴抑郁症患者进行心理干预治疗.结果:120例成年癫痫患者中47.5%伴发抑郁.负性影响癫痫患者伴发抑郁的因素为发作频率,而正性影响因素根据影响程度的大小依次为职业和对癫痫相关知识的掌握情况.心理干预治疗对提高癫痫伴抑郁患者生活质量是积极、有效的.结论:癫痫患者抑郁发生率高且受多种因素的影响.加强包括药物治疗、心理治疗在内的综合治疗,真正提高癫痫患者的生活质量.     

Early maternal deprivation affects dentate gyrus structure and emotional learning in adult female rats

Rationale  

Stress elicits functional and structural changes in the hippocampus. Early life stress is one of the major risk factors for stress-related pathologies like depression. Patients suffering from depression show a reduced hippocampal volume, and in women, this occurs more often when depression is preceded by childhood trauma. However, the underlying mechanisms that account for a reduced hippocampal volume are unknown.     

Neonatal maternal deprivation modifies feeding in response to pharmacological and behavioural factors in adult rats

Neonatal maternal deprivation permanently modifies the hypothalamo-pituitary-adrenal (HPA) axis and other neurobiological and behavioural parameters in rats. The HPA axis plays a central role in the control of feeding, and participates in the anorexigenic action of dexfenfluramine and restraint stress, and in the orexigenic action of a cafeteria diet. Therefore, we investigated whether maternal deprivation modifies feeding responses to these factors. Experimental pups were separated for 24h from the mother 5 or 14 days after birth. The anorexigenic response to both dexfenfluramine and restraint stress was increased, and body weight as well as subcutaneous adipose tissue gain induced by cafeteria diet was higher in early deprived adult rats. However, these effects were dependent on the time of maternal deprivation. According to our predictions, the feeding response of maternally deprived rats to anorexigenic and orexigenic agents was altered, which is probably partly due to an altered HPA function, but the participation of the serotonergic, the opioid and/or the dopaminergic system cannot be ruled out. Additional studies are needed to detail precisely the neurobiological substrates of modified feeding behaviour of maternally deprived animals. This early stress paradigm altering feeding behaviour could become an interesting model for research into human eating disorders.     

Early maternal deprivation alters hippocampal levels of neuropeptide Y and calcitonin-gene related peptide in adult rats

Stressful events early in life are reported to be more prevalent among patients with an adult life psychiatric disorder. Early maternal deprivation is considered an animal model of early life stress. Maternally deprived adult rats display long-term alterations in the neuroendocrine system, brain and behavior that are in many ways analogous to depressive and schizophrenic symptomatology. Neuropeptide Y (NPY) and calcitonin-gene related peptide (CGRP) have been implicated in both disorders and also been suggested to play a role in the neuroadaptational response to stress. Consequently, male Wistar rat-pups were subjected to early maternal deprivation or control handling, on postnatal day (pnd) 9. On pnd 21, pups were weaned and split into two groups that were reared either on a saw-dust floor or on a grid-floor, considered to be a mild stressor. On pnd 67, all animals were subjected to the prepulse inhibition test. One week later, the animals were sacrificed, the brains removed and dissected on ice. Levels of NPY-like immunoreactivity (LI) and CGRP-LI were quantified by radioimmunoassay in brain regional extracts. Maternal deprivation led to a significant reduction in basal startle amplitude and disruption of prepulse inhibition. These findings were paralleled by significantly reduced levels of NPY and CGRP in the hippocampus and occipital cortex. It is hypothesised that these changes may be of relevance to aspects of schizophrenic and affective symptomatology. The present study further shows that brain NPY and, in particular, CGRP are sensitive to long-term mild stress and further implicate the involvement of these peptides in the neuroendocrine stress response.     

Effects of early maternal separation on ethanol intake,GABA receptors and metabolizing enzymes in adult rats

Rationale Maternal separation (MS) in neonatal rats affects ethanol self-administration (SA) in adulthood; however, the conditions and mechanisms need to be clarified. Objectives The goal of this study was to determine the effect of MS on ethanol SA in adulthood in different groups of rats, which control for time of separation, handling, and rearing conditions and, for mechanistic assessment, to examine GABA-A receptors in the central nucleus of the amygdala (CeA) and levels of liver metabolizing enzymes. Methods Newborn, male Long–Evans rats were randomly assigned to different groups and treated over postnatal days 2–14. The rats were picked up by their tails and put back down with no separation (MS0), separated from their mother for 15 min/day (MS15), separated from their mother for 180 min/day (MS180), handled once for a bedding change (NH), or were animal facility reared (AFR). In adulthood, these rats were allowed 5-day continuous access to ethanol, and GABA-A receptors and liver enzymes were measured. Results The MS15 group consumed and preferred significantly less ethanol (about one third) than the MS180 group; however, neither group was different from the MS0 or the AFR group. The NH group consumed and preferred significantly more ethanol than all other groups, at least twice that of the MS180s. GABA-A receptors were increased in the CeA in MS15s, which could help explain the effects. Alcohol dehydrogenase may have been altered in the AFRs. Conclusions Various treatments in neonates affect ethanol intake and GABA-A receptors, and possibly ethanol metabolism, in adulthood. These changes were not simply related to time of separation but were also due to the degree of handling.     

心理护理对产后抑郁的影响

目的观察心理护理对初产妇产后抑郁的影响。方法将138例初产妇随机分为观察组和对照组各69例。对照组给予常规护理。观察组在对照组护理基础上给予心理护理。采用爱丁堡产后抑郁量表(EPDS)和焦虑自评量表(SAS)对患者进行心理评估,并比较2组产后抑郁的发生情况。结果 2组护理后EPDS和SAS评分均低于护理前,且观察组低于对照组,差异有统计学意义(P<0.05)。观察组护理后28、42d抑郁症发生率低于对照组,差异有统计学意义(P<0.05)。结论心理护理可显著减少初产妇产后抑郁的发生。     

Paralytic shellfish toxins in bivalves which are not associated with dinoflagellates

Paralytic shellfish toxins (PSP toxins) were detected in the freshwater bivalve Corbicula sandai collected from Lake Biwa, Shiga Prefecture, Japan, and marine mussel Septifer virgatus from Mutsu Bay where known causative dinoflagellates and their cysts have never been observed. The toxin profile of C. sandai and S. virgatus was considerably different from suspected causative organisms Aphanizomenon flos-aquae and Protogonyaulax spp., respectively. The causative organism(s) responsible for PSP toxins in these waters is at present unknown.     

The impaired coping induced by early deprivation is reversed by chronic fluoxetine treatment in adult fischer rats

The symptoms of depression include feelings of reduced coping ability and increased helplessness. Early life adversity increases vulnerability to depression. In rats, the quantification of ability to cope with adverse challenge can be achieved using preexposure to an inescapable aversive stimulus and subsequent assessment of escape or avoidance deficits in the same environment. Here we investigated the predictive validity of a model in which, in the Fischer rat strain, postnatal isolation leads in adulthood to a state of increased sensitivity to develop an escape or avoidance deficit. On days 1-14 rat pups were isolated for 4 hours (early deprivation, ED) or for 15 minutes (early handling, EH), or were left completely undisturbed (non-handling, NH). In adulthood, subjects were placed in a shuttle box and half were exposed to brief, mild foot shocks (preexposure, PE) and the other half were non-preexposed (NPE). Half of the PE and NPE subjects were then treated for 21 days with fluoxetine and the other half with vehicle. In males, although there was no overall preexposure effect on avoidance behaviour, ED-PE and ED-NPE and EH-PE and EH-NPE demonstrated an avoidance deficit relative to NH. Fluoxetine attenuated this deficit and most notably in ED-PE. In females, vehicle ED-PE demonstrated an avoidance deficit relative to NH-PE; fluoxetine attenuated this ED effect. These findings provide supportive evidence for the predictive validity of this depression model.     

预见性护理干预对初产妇产后抑郁的影响

目的 观察预见性护理干预对初产妇产后抑郁的影响.方法 70例初产妇随机分为观察组和对照组各35例,对照组给予常规产后护理,观察组给予预见性护理干预.比较2组产后14d爱丁堡产后抑郁量表(EPDS)和焦虑自评量表(SAS)评分及临床疗效.结果 护理后对照组EPDS和SAS评分高于护理前,观察组低于护理前,且观察组低于对照组,差异均有统计学意义(P<0.05).观察组总有效率为97.1%高于对照组的57.1%,差异有统计学意义(P<0.05).结论 预见性护理干预可显著改善初产妇产后抑郁状态.