不同葡萄糖培养条件对L6骨骼肌细胞中活性氧的生成作用*☆

背景：活性氧在不同葡萄糖培养条件下的生成状况是研究2型糖尿病发病机制的基础。 目的：观察不同浓度葡萄糖培养条件下,细胞内活性氧的生成情况。 方法：取体外培养的L6骨骼肌细胞和诱导分化的肌管细胞,分别用含有3,5.5,25 mmol/L葡萄糖的DMEM培养液培养,检测细胞内活性氧的生成情况。 结果与结论：随着高糖培养时间的延长,细胞内活性氧生成有增加的趋势,其中25 mmol/L葡萄糖培养细胞4 d的活性氧生成明显高于5.5 mmol/L葡萄糖培养细胞的水平;3,5.5 mmol/L葡萄糖培养对细胞内活性氧生成无显著影响。诱导肌管细胞较成肌细胞在高糖培养条件下更易产生活性氧。说明体外培养细胞葡萄糖浓度、培养时间和培养细胞分化程度是影响细胞内活性氧生成的因素,可用于模拟体内高糖损伤的积累效应。     

人外周血内皮祖细胞增殖、凋亡及丙二醛、抗氧化因子合成与波动性高糖的影响**☆

背景：研究表明波动性高血糖较持续性高血糖对血管内皮功能的损害可能更严重。 目的：观察波动性高糖对人外周血内皮祖细胞增殖、凋亡及丙二醛、抗氧化因子合成的影响。 方法：密度梯度离心法获取人外周血单个核细胞。取经培养鉴定后的内皮祖细胞，细胞同化后分别给予5.5 mmol/L，20 mmol/L，5.5/20 mmol/L葡萄糖(5.5，20 mmol/L的葡萄糖培养液每8 h更换1次)及20 mmol/L甘露醇。干预72 h后，MTT法检测内皮祖细胞的增殖情况；流式细胞仪检测细胞凋亡率；比色法测定培养液中丙二醛水平及超氧化物歧化酶活力。 结果与结论：20 mmol/L和5.5/20 mmol/L葡萄糖作用72 h，内皮祖细胞增殖减少、凋亡率增高(P < 0.01），培养液中丙二醛水平增高、超氧化物歧化酶活力降低(P < 0.01)，均以5.5/20 mmol/L葡萄糖作用最明显(P < 0.01)。说明波动性高糖较恒定性高糖更易抑制内皮祖细胞增殖并促进其凋亡，其机制可能与波动性高糖环境下氧化应激水平增高有关。     

低糖对星形胶质细胞水通道蛋白4表达及分布的影响

目的探讨低糖体外培养对大鼠原代星形胶质细胞水通道蛋白4(aquaporin 4,AQP4)表达量及分布的影响。方法对体外培养的原代大鼠星形胶质细胞进行0mmol/L(无糖)和1mmol/L葡萄糖低糖培养,以5.5mmol/L葡萄糖作为正常糖浓度对照进行如下实验:(1)采用活细胞成像技术检测无糖培养后的星形胶质细胞的体积变化;(2)以Western blot检测无糖培养0h、3h、6h、12h和24h,以及0mmol/L、1mmol/L和5.5mmol/L葡萄糖培养24h后星形胶质细胞的AQP4表达量;(3)以免疫荧光染色检测0mmol/L、1mmol/L和5.5mmol/L葡萄糖培养6h后AQP4在星形胶质细胞上的分布。结果 (1)与0min时比较,无糖培养15min(1.14±0.03)和30min时(1.15±0.02)星形胶质细胞相对体积均明显增大(P0.01)。(2)无糖0mmol/L(含血清)培养实验中,与0h(AQP4相对表达量为1)比较,12h(1.55±0.21)、24h时(1.67±0.16)AQP4相对表达量均明显增加(P0.01);0mmol/L、1mmol/L和5.5mmol/L葡萄糖(含血清)培养24h后,与5.5mmol/L组(AQP4相对表达量为1)比较,0mmol/L组AQP4相对表达量(3.23±0.23)明显增加(P0.01)。(3)与0h时(AQP4相对表达量为1)比较,无糖(无血清)培养6h后AQP4蛋白相对表达量(1.32±0.09)明显上调(P0.05),24h时相对表达量(0.80±0.05)下降(P0.05);0mmol/L、1mmol/L和5.5mmol/L葡萄糖(无血清)培养24h后,与5.5mmol/L(AQP4相对表达量为1)组比较,0mmol/L组AQP4相对表达量(0.75±0.07)明显降低(P0.05)。无论含或不含血清,与5.5mmol/L葡萄糖组比较,0mmol/L和1mmol/L组AQP4在星形胶质细胞向膜上集中特异性分布更加明显。结论低糖处理可引起星形胶质细胞水肿,AQP4蛋白表达量发生变化和向细胞膜上集中分布。AQP4在低血糖性脑水肿中可能发挥重要作用。     

高糖状态下小鼠肾小球足细胞凋亡和组成型光形态建成1蛋白的表达

背景：组成型光形态建成1蛋白与细胞凋亡有关。 目的：观察高糖对体外培养的小鼠肾小球足细胞凋亡和组成型光形态建成1蛋白表达的影响。 方法：将不同浓度葡萄糖溶液分别加入体外培养的条件永生性小鼠肾小球足细胞株培养液中，培养不同时间后检测肾小球足细胞凋亡指数和死亡指数，以筛选最佳剂量效应葡萄糖浓度。用30 mmol/L葡萄糖溶液干预小鼠肾小球足细胞(高糖组)，并设立对照组和采用30 mmol/L甘露醇干预的甘露醇组。 结果与结论：在一定浓度范围内，葡萄糖呈时间和剂量依赖性诱导肾小球足细胞凋亡和死亡(P < 0.05)，随着葡萄糖浓度的进一步加大，肾小球足细胞死亡指数明显升高，而凋亡指数变化不大。与对照组比较，高糖组凋亡指数显著增加(P < 0.05)，其COP1 mRNA及蛋白表达水平均明显下降(P < 0.05)。 结果证实，高糖可诱导肾小球足细胞的凋亡，其机制可能与其下调COP1的表达有关。     

光损伤诱导视网膜上皮细胞半胱天冬酶－3表达与促红细胞生成素的干预效应*☆

背景：有实验表明，促红细胞生成素对视网膜的光化学损伤有保护作用，该作用与光化学损伤视网膜色素上皮细胞中半胱天冬酶-3表达的变化有关？ 目的：采用光刺激诱导人视网膜色素上皮细胞凋亡，观察不同剂量促红细胞生成素对细胞半胱天冬酶-3表达的影响。 设计：观察对比实验。 单位：青岛大学医学院。 材料：成人视网膜色素上皮-19细胞株购自美国细胞培养收集公司。DMEM/F12混合培养基、新生牛血清、胰蛋白酶购自GIBCO生物技术公司。重组人促红细胞生成素购自Sigma生物技术公司。人半胱天冬酶-3定量EIA试剂盒购自上海西唐生物科技有限公司。半胱天冬酶-3单克隆抗体购自美国Santa Cruz 公司；PV6001免疫组织化学试剂盒和DAB显色试剂盒购自北京中山生物技术公司。 方法：实验于2006-05/2007-01在青岛大学医学院病理生理教研室完成。取成人视网膜色素上皮-19细胞株传代培养的2~5代细胞建立光损伤模型，传代细胞随机分为7组，每组4孔，①正常对照组：不加光照，不加促红细胞生成素药物干预。②光损伤模型组：光照12 h，不加促红细胞生成素药物干预。③光损伤＋10 000U/L 促红细胞生成素组、光损伤＋20 000 U/L 促红细胞生成素组及光损伤＋40000 U/L 促红细胞生成素组：光照12 h，分别加10 000、20 000及40 000 U/L促红细胞生成素。④光损伤＋40 000 U/L促红细胞生成素组＋AG490组：光照12 h，加促红细胞生成素 40 000 U/L及Jak2激酶抑制剂 50 000 U/L。⑤光损伤＋40 000 U/L促红细胞生成素组＋蛋白激酶B特异性抑制剂组：光照12 h，加促红细胞生成素 40 000 U/L及蛋白激酶B特异性抑制剂100 μmol/L。 主要观察指标：采用酶联免疫吸附实验和免疫组织化学法定量检测不同含量促红细胞生成素干预治疗前后视网膜色素上皮细胞半胱天冬酶-3表达的变化。 结果：正常对照组半胱天冬酶-3无明显表达；光损伤模型组半胱天冬酶-3表达于视网膜色素上皮细胞核上，呈大量特异性黄色着色；光损伤不同剂量促红细胞生成素组半胱天冬酶-3表达的特异性黄色着色随促红细胞生成素含量增加而减弱，以光损伤＋40 000 U/L 促红细胞生成素组最弱光损伤＋40 000 U/L促红细胞生成素组＋AG490组半胱天冬酶-3呈强阳性表达，光损伤＋40 000 U/L促红细胞生成素组＋蛋白激酶B特异性抑制剂组半胱天冬酶-3表达仍呈阳性，但较光损伤模型组稍弱。 结论：重组人促红细胞生成素可减少视网膜色素上皮细胞的光损伤后半胱天冬酶-3的表达。重组人促红细胞生成素抗光损伤诱导的视网膜色素上皮细胞凋亡作用机制之一可能是抑制半胱天冬酶-3的表达。     

青白散对慢性软组织损伤模型大鼠骨骼肌一氧化氮合酶系统和一氧化氮的影响*☆

背景：对慢性软组织损伤后一氧化氮合酶系统和一氧化氮的研究目前较少。 目的：观察青白散对大鼠慢性软组织损伤模型骨骼肌中一氧化氮合酶系统和一氧化氮的影响。 方法：雄性 SD 大鼠随机分为对照组、模型组、氨基胍组、青白散组。后3组采用机械损伤法制备慢性骨骼肌损伤动物模型,分别予以生理盐水 10 mL/kg,0.10 g/kg氨基胍,0.54 g/kg青白散,1次/d,连续 14 d。于给药后1,2,3周,分别检测大鼠肌组织一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性。 结果与结论：骨骼肌损伤修复过程中,模型组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性较对照组显著增高;而青白散组和氨基胍组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性均较模型组显著降低。说明青白散可能通过阻抑诱导型一氧化氮合酶诱导过量一氧化氮的产生,为慢性软组织损伤的修复创造了有利条件。     

重组人促红细胞生成素后处理缺血/再灌注前后骨骼肌诱导型一氧化氮合酶及一氧化氮的变化

背景：促红细胞生成素对包括心脑在内的多种组织器官的缺血/再灌注损伤有保护作用。 目的：观察重组人促红细胞生成素后处理对家兔后肢腓肠肌缺血/再灌注前后诱导型一氧化氮合酶、一氧化氮和超微结构的影响。 方法：将家兔随机分为空白对照组、模型组和重组人促红细胞生成素组,后2组建立兔左后肢腓肠肌缺血/再灌注实验模型,其中重组人促红细胞生成素组在兔左后肢缺血2 h后静脉注射重组人促红细胞生成素。 结果与结论：再灌注后4,12 h,重组人促红细胞生成素组诱导型一氧化氮合酶表达水平及一氧化氮浓度增高幅度较模型组低(P < 0.05)。电镜下腓肠肌细胞超微结构显示,模型组内皮细胞膜溶解,肿胀明显,肌纤维内线粒体水肿。重组人促红细胞生成素组肌纤维损伤较轻,肌节内Z线及肌节内各带结构基本正常,大部分线粒体结构正常,显示重组人促红细胞生成素组超微结构损伤程度明显轻于模型组。结果证实,重组人促红细胞生成素后处理可通过抑制诱导型一氧化氮合酶蛋白的表达,减少再灌注后过量一氧化氮生成,改善骨骼肌缺血/再灌注损伤。     

西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用

目的研究西洛他唑对大鼠脑微血管内皮细胞缺血再灌注损伤的保护作用机制。方法以原代方法培养大鼠脑微血管内皮细胞并传代,将第3代传代细胞随机分为正常对照组、西洛他唑组、溶剂对照组和缺血再灌注模型组(再灌模型组)4组,对后3组大鼠建立脑微血管内皮细胞糖氧剥夺后复糖氧模型,模拟"缺血再灌注"过程,并对西洛他唑组和溶剂对照组在造模同时分别以终浓度1×10-5mmol/L西洛他唑〔二甲基亚砜(DMSO)为溶剂〕和0.05%(体积分数)DMSO进行干预。糖氧剥夺3 h复糖氧24 h后,测定各组细胞上清液中诱导型一氧化氮合酶(iNOS)和内皮型一氧化氮合酶(eNOS)水平及细胞内环磷腺苷酸(cAMP)水平,并以四唑盐(MTT)比色实验测定细胞活力。结果西洛他唑组与再灌模型组比较,eNOS水平明显提高,iNOS水平明显降低,cAMP水平及细胞存活率显著提高(均P<0.05)。结论西洛他唑对大鼠脑微血管内皮细胞的缺血再灌注损伤有保护作用,其作用机制可能与促进eNOS水平升高和iNOS水平降低有关。     

大黄酚对神经细胞缺氧损伤的保护作用

目的 观察大黄酚对缺氧PC12细胞损伤的保护作用;方法 培养PC12细胞,建立缺氧致神经细胞损伤模型;缺氧前后四甲基偶氮唑盐法（MTT）检测PC12细胞增殖活性、光镜观察PC12细胞形态、测定上清液中乳酸脱氢酶（LDH）活性、早期癌基因表达产物（c-fos）荧光免疫组化表达和逆转录酶聚合酶链反应（RT-PCR）分析神经型一氧化氮合酶（nNOS）、诱导型一氧化氮合酶（iNOS）mRNA的表达;结果 大黄酚明显改善缺氧PC12细胞的活力,对损伤细胞形态有改善作用,使损伤细胞的培养上清液中的LDH明显减少;c-fos表达减少,PCR结果分析显示nNOS mRNA在缺氧早期表达.iNOSmRNA主要在缺氧晚期表达.结论 本缺氧模型能够引起PC12细胞凋亡现象,大黄酚能减轻PC12细胞缺氧损伤,对神经元细胞有保护作用.     

西洛他唑对糖氧剥离大鼠脑微血管内皮细胞的保护作用

目的探讨磷酸二酯酶(PDE)3抑制剂西洛他唑对大鼠脑微血管内皮细胞糖氧剥离损伤的影响及作用机制。方法原代培养大鼠皮层微血管内皮细胞,建立糖氧剥离细胞模型模拟"缺血"过程,分5组:正常对照组、西洛他唑组、依达拉奉组、溶剂对照组、模型组。糖氧剥离6h后,测定细胞上清中内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)水平、细胞内环磷腺苷酸(cAMP)浓度,及采用四唑盐(MTT)比色实验测定细胞活力。结果西洛他唑及依达拉奉均可明显提高缺血细胞模型上清中eNOS水平,同时降低iNOS水平,提高细胞内cAMP水平及细胞存活率(均P0.05),且西洛他唑作用更为显著。结论西洛他唑对培养大鼠皮层微血管内皮细胞在缺血损伤中具有保护作用,其作用机制可能是通过选择性抑制PDE3从而增加cAMP水平,间接增加eNOS水平、降低iNOS水平来实现的。     

3-N-Butylphthalide mitigates high glucose-induced injury to Schwann cells:association with nitrosation and apoptosis

A high glucose state readily causes peripheral axon atrophy, demyelination, loss of nerve fiber function, and delayed regeneration. However, few studies have examined whether nitration is also critical for diabetic peripheral neuropathy. Therefore, this study investigated the effects of high glucose on proliferation, apoptosis, and 3-nitrotyrosine levels of Schwann cells treated with butylphthalide. In addition, we explored potential protective mechanisms of butylphthalide on peripheral nerves. Schwann cells were cultured in vitro with high glucose then stimulated with the peroxynitrite anion inhibitors uric acid and 3-n-butylphthalide for 48 hours. Cell Counting Kit-8 and flow cytometry were used to investigate the effects of uric acid and 3-n-butylphthalide on proliferation and apoptosis of Schwann cells exposed to a high glucose environment. Effects of uric acid and 3-n-butylphthalide on levels of 3-nitrotyrosine in Schwann cells were detected by enzyme-linked immunosorbent assay. The results indicated that Schwann cells cultured in high glucose showed decreased proliferation, but increased apoptosis and intracellular 3-nitrotyrosine levels. However, intervention with uric acid or 3-n-butylphthalide could increase proliferation of Schwann cells cultured in high glucose, and inhibited apoptosis and intracellular 3-nitrotyrosine levels. According to our data, 3-n-butylphthalide may inhibit cell nitrification and apoptosis, and promote cell proliferation, thereby reducing damage to Schwann cells caused by high glucose.     

The effects of galanin on dorsal root ganglion neurons with high glucose treatment in vitro

The exposure of neurons to high glucose concentrations is considered a determinant of diabetic neuropathy. The extracellular high concentration of glucose can cause neuronal cellular damage. Galanin (Gal) not only plays a role in processing of sensory information but also participates in energy homeostasis and glucoregulation. However, the effects of Gal on dorsal root ganglion (DRG) neurons with high glucose are not clear. Using an in vitro model of high glucose-treated DRG neurons in culture, the effects of Gal on intracellular reactive oxygen species (ROS) expression, cell viability, apoptosis, expression of Gal and its receptors (GalR1 and GalR2) of DRG neurons were investigated. Neurons were dissociated from embryonic day 15 (E15) rat DRG and cultured for 48 h and then maintained in serum-free neurobasal medium containing high glucose (45 mmol/L) or normal glucose (25 mmol/L) for 24 h. Mannitol (20 mmol/L) was also used to create a high osmotic pressure mimicking the high glucose condition. The results showed that high glucose caused a rapid increasing of intracellular ROS, decreases of cell viability, and upregulation of Gal and its mRNA. Exogenous Gal (1 μmol/L) inhibited the above effects caused by high glucose. Interestingly, high glucose caused downregulation of GalR1 and its mRNA and administration of exogenous Gal could further decrease their expression, whereas expression of GalR2 and its mRNA was not affected at different experimental conditions. The results of the present study indicate for the first time that Gal and its receptor system are involved in high glucose-induced DRG neuronal injury. The contribution of exogenous Gal on neuroprotection appears to be quite significant. These results provide rationale and experimental evidence for development and further studies of Gal on therapeutic strategy for improving diabetic neuropathy.     

High glucose changes extracellular adenosine triphosphate levels in rat retinal cultures

Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol‐treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high‐glucose‐treated cells. It was also observed that, in retinal cells cultured under high‐glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol‐treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR. © 2008 Wiley‐Liss, Inc.     

高糖及高糖条件培养液对血管内皮细胞的影响

目的探讨高糖对血管内皮细胞分泌高迁移率族蛋白B1(HMGB1)的影响,以及高糖条件培养液对血管内皮细胞氧化应激水平的影响。方法 1.采用不同糖浓度的培养基(5.6mmol/L,20mmol/L,33mmol/L)体外培养人脐静脉血管内皮细胞株(Ea.hy926),台盼兰排斥法检测48h以内细胞的存活率,使用Western blot检测培养液上清中HMGB1的表达水平。2.分别用33mmol/L高糖培养基、混合液(33mmol/L组上清液与33mmol/l高糖培养基混合),刺激血管内皮细胞,采用分光光度比色法检测血管内皮细胞内丙二醛(MDA)和超氧化物岐化酶(SOD)的水平。结果 1.与正常对照组相比,高糖组培养液上清中HMGB1表达均明显增加(P<0.05)。台盼蓝排斥法检测不同糖浓度48h内对细胞生存率影响较小。2.与单纯高糖培养基刺激组相比,混合液刺激组的血管内皮细胞内MDA、SOD水平有显著差异(P<0.05)。结论高糖可诱导体外血管内皮细胞表达分泌HMGB1。高糖环境下,机体可能通过增加分泌HMGB1,加强氧化应激反应而促进脑血管病的发生。     

Gamma-aminobutyric acid A receptor gamma 2 subunit following Mg-free-induced seizures in cultured developing neurons

BACKGROUND:Studies have demonstrated that in vitro cultured cortical neurons from embryonic rats can produce spontaneous recurrent epileptiform discharges following transient Mg2+-free extracellular solution culture. OBJECTIVE: To explore gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit expression follow-ing Mg2+-free-induced seizures in cultured developing neurons. DESIGN,TIME AND SETTING: Cellular and molecular biology. The in vitro experiment was performed at the Department of Pediatrics,Second Xia...