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3,5.5,25mmol/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25mmol/L葡萄糖培养细胞4d的活性氧生成明显高于5.5mmol/L葡萄糖培养细胞的水平;3,5.5mmol/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。     

骨骼肌收缩介导葡萄糖转运中活性氧的作用及其机制

活性氧在骨骼肌收缩介导的葡萄糖转运中扮演着十分重要的角色.但相关机制还不明确.文章综述了活性氧在骨骼肌葡萄糖转运中的作用,包括活性氧参与骨骼肌收缩介导的葡萄糖转运的适宜浓度,其机制可能涉及多种信号转导途径,如5'-一磷酸腺苷活化蛋白激酶途径、丝裂原活化蛋白激酶途径、磷脂酰肌醇3激酶/蛋白激酶B途径和Ca2+途径等.这将有助于进一步揭示运动防治胰岛素抵抗和2型糖尿病的分子机制.     

L6细胞胰岛素抵抗的骨骼肌细胞模型

背景:在胰岛素抵抗的细胞模型中,以游离脂肪酸诱导3T3-L1脂肪细胞和高胰岛素诱导的HepG2肝癌细胞最为常见,对分布最为广泛靶组织骨骼肌的胰岛素抵抗模型报道较少.目的:拟使用L6细胞株建立胰岛素抵抗的骨骼肌细胞模型.设计、时间及地点:细胞学体外观察,于2007-06/2008-05在辽宁医学院生物化学与分子生物学教研室完成.材料:L6细胞株由中国协和医科大学细胞中心提供.α-MEM培养基为美国Gibco公司产品.方法:复苏后的L6细胞置于α-MEM培养基中诱导培养14 d,以考马斯亮蓝染色观察肌管出现来确定L6成肌细胞株分化完成.将分化后的细胞建立胰岛素抵抗模型,按1 × 108/孔接种于24孔板内,用含体积分数为0.02胎牛血清的α-MEM培养,以L6细胞贴满24孔板的板底为宜.换液后,向各孔内分别加入含体积分数为0.02牛血清白蛋白的α-MEM无血清培养基饥饿培养5 h.设立2组,实验组的板孔内加入含1×107mol/L胰岛素的HEPES缓冲液(136mmol/LNaCl,4.7mmol/LKCl,1.25 mmol/L MgSO4,1.2 mmol/L CaCl2,0.2%牛血清白蛋白,20 mmol/L HEPES,pH 7.4)孵育1 h;对照组的板孔内加入不含胰岛素的HEPES缓冲液孵育1 h.弃上清后用HBS缓冲液冲洗,每孔内分别加入含0.1 mol/L葡萄糖的HBS缓冲液100 μL,孵育10 min后取出置于冰板上快速回收上清.主要观察指标:考马斯亮蓝染色观察L6细胞出现肌管结构情况,葡萄糖氧化酶法检测上清中葡萄糖浓度.结果:诱导前L6细胞未见肌性结构出现,诱导分化后L6细胞内出现肌管结构.使用α-MEM无血清培养基饥饿培养1,3 h,两组上清中葡萄糖浓度基本相似(P>0.05);使用α-MEM无血清培养基饥饿培养5 h后,实验组上清中葡萄糖浓度明显高于对照组(P<0.05).结论:诱导分化成肌后的L6骨骼肌细胞经α-MEM无血清培养基饥饿培养5 h,在胰岛素浓度为1×10-7mol/L的刺激下可形成胰岛素抵抗的骨骼肌细胞模型.     

碱性成纤维细胞生长因子对培养的骨骼肌细胞生长作用的研究

目的：观察碱性成纤维细胞因子（bFGF)对体外培养的新生Wistar大鼠骨骼肌细胞存活、分化及促增殖分裂影响。方法：应用无血清培养技术，采用四唑盐（MTT）微量比色法，乳酸脱氢酶（LDH）活性、肌酸磷酸激酶（CK）活性测定及蛋白定量检测。结果：bFGF干预组MTT微量比色的ΔOD值、LDH活性、CK活性及蛋白质合成均高于对照组。结论：外源性bFGF明显促进细胞的存活、生长及增殖分化。     

骨骼肌再生和骨骼肌肌卫星细胞相关研究的进展

在我们的日常生活和体育锻炼中,发生肌肉损伤不可避免,肌肉发生损伤以后,需要经历修复和再生的过程.而对骨骼肌的修复和再生的发生、发展、转归的研究在骨骼肌损伤后的治疗和康复领域有很重要的临床和现实意义.但以往组织病理学的观点认为,人体成熟的骨骼肌细胞属于终末分化细胞,已经不再具有分裂和再生的能力,一旦因为某种原因遭受破坏,就会成为永久性的功能和组织的缺失,被纤维化的瘢痕组织所代替,不再具有收缩能力,使其运动能力减弱或丧失.这种观点一直影响着后来的医学和体育工作者.Carlson在1973年发表了一篇综述,其中对当时有关骨骼肌再生的文献进行了综述报道.从那以后,人们对骨骼肌再生的关注程度逐渐提高,有关的研究逐渐增多,很多文章对骨骼肌损伤后再生的病理学以及形态学的变化有所报道,但有关再生过程的机制还没有明确指出.     

骨骼肌细胞葡萄糖运载体4调控的研究进展

组织细胞外的葡萄糖跨膜转运进入细胞内 ,依赖于细胞膜上的葡萄糖运载体(ｇｌｕｃｏｓｅｔｒａｎｓｐｏｒｔｅｒ ,ＧＬＵＴ )转运 ,通过易化扩散作用 ,顺浓度差进入或输出细胞。目前为止 ,已发现 7种ＧＬＵＴ异构体 ,它们具有特定的组织分布。骨骼肌中存在 2种 ,即ＧＬＵＴ1和ＧＬＵＴ4。研究发现成年大鼠骨骼肌细胞中ＧＬＵＴ1仅占ＧＬＵＴ总量的 5 %～ 10 % ,而ＧＬＵＴ4是骨骼肌细胞中主要的葡萄糖运载体。由于人类通过骨骼肌代谢的葡萄糖在基础状态和高胰岛素条件下分别占机体葡萄糖代谢的 2 0 %和 75 %～ 95 % ,尤其在高胰岛素状态下 ,…     

地塞米松对骨骼肌细胞蛋白降解的调节及其机制研究

目的研究地塞米松对体外培养的骨骼肌肌管内长寿命蛋白降解的影响及其可能的作用机制。方法无菌分离（2日龄）Wistar大鼠双下肢肌肉．组织块法培养、增殖、传代纯化成肌细胞．融合形成肌管，使用L—C3，5-^3H]-酪氨酸标记肌管长寿命蛋白后，随机分成两组。A组：分别用不含地塞米松（对照组）或含地塞米松10nmol／L（A1组）、100nmol／L（A2组）、1000nmol／L（A3组）的培养液培养肌管，12、24、36和48h后使用液体闪烁计数仪测定培养液和细胞内L-[3，5-^3H]-酪氨酸的含量，计算肌管长寿命蛋白的降解率。B组：分别用含蛋白酶体抑制剂MG132（50μmol／L，B1组）或含MG132（50μmol／L）和地塞米松100nmol／L（B2组）的培养液培养肌管24h，相同方法测定肌管长寿命蛋白降解率。结果A1、A2和A3组长寿命蛋白降解率均较对照组显著增加，差异有显著性（P均〈0．01）。A2组和A3组长寿命蛋白降解率均较A1组显著增加，差异有显著性（P均〈0．01）。A2组和A3组比较．长寿命蛋白降解率差异无显著性（P〉0．05）。B1组长寿命蛋白降解率较对照组（24h）降低明显，B2组长寿命蛋白降解率较A2组（24h）显著降低，差异均有显著性（P均〈0．01）。结论泛素-蛋白酶体途径是骨骼肌肌管长寿命蛋白的重要降解途径之一。地塞米松能通过激活肌管内泛素-蛋白酶体途径而增强长寿命蛋白的降解，此效应在一定范围内呈量效依赖关系。     

骨骼肌损伤和修复中炎症细胞因子作用的研究进展

骨骼肌损伤在高强度的运动训练,超过习惯性的活动或体力劳动中经常发生,骨骼肌损伤后的修复一直是运动医学界长期关注和研究的基础性问题,众多研究者从不同角度展开了多方面的研究,但许多机制性问题并未完全解决。最新     

碱性成纤维细胞生长因子对培养的骨骼肌细胞生长作用的研究

目的观察碱性成纤维细胞生长因子(bFGF)对体外培养的新生Wistar大鼠骨骼肌细胞存活、分化及促增殖分裂影响。方法应用无血清培养技术,采用四唑盐(MTT)微量比色法,乳酸脱氢酶(LDH)活性、肌酸磷酸激酶(CK)活性测定及蛋白定量检测。结果bFGF干预组MTT微量比色的ΔOD值、LDH活性、CK活性及蛋白质合成均高于对照组。结论外源性bFGF明显促进细胞的存活、生长及增殖分化。     

甘丙肽对骨骼肌细胞葡萄糖转运蛋白4的影响

背景：甘丙肽能促进骨骼肌细胞葡萄糖转运蛋白4膜转位和葡萄糖清除,葡萄糖转运蛋白4是运动促进骨骼肌细胞摄取葡萄糖的主要载体,从而增加骨骼肌摄取葡萄糖而降低血糖。目的：探讨甘丙肽与胰岛素联用对糖尿病大鼠骨骼肌细胞葡萄糖转运蛋白4膜位移的作用。方法：由第一作者检索1991年1月2011年12月PubMed数据库（http：//www.ncbi.nlm.nih.gov/PubMed）及万方数据库（http：//www.wanfangdata.com.cn）。英文检索词为＂Galanin;insulin＂,中文检索词为＂甘丙肽;葡萄糖转运蛋白4＂。检索文献量总计143篇,选择甘丙肽与胰岛素在改善骨骼肌摄取葡萄糖的特点及其临床应用方面的文献,排除陈旧及重复实验文章,同一领域文献则选择近期发表或发表在权威杂志的文章,最终纳入37篇符合标准的文献。结果与结论：甘丙肽是一种广泛分布在神经系统的神经肽,具有广泛的神经生物学功能。运动能提高血浆甘丙肽浓度以及甘丙肽对2型糖尿患者的保护机制已经得到认可。甘丙肽及胰岛素都有降低血糖的功效。糖尿病是甘丙肽和胰岛素共同障碍的结果,那么甘丙肽与胰岛素联用的疗效自然成为关注的焦点,即两者联用对糖尿病大鼠骨骼肌细胞葡萄糖转运蛋白4膜位移的作用,是否比单独运用甘丙肽或胰岛素对骨骼肌细胞葡萄糖转运蛋白4膜位移的作用更强是目前研究的趋势。     

Reactive oxygen species mediated apoptosis of esophageal cancer cells induced by marine triprenyl toluquinones and toluhydroquinones

Marine invertebrates, algae, and microorganisms are prolific producers of novel secondary metabolites. Some of these secondary metabolites have the potential to be developed as chemotherapeutic agents for the treatment of a wide variety of diseases, including cancer. We describe here the mechanism leading to apoptosis of esophageal cancer cell lines in the presence of triprenylated toluquinones and toluhydroquinones originally isolated from the Arminacean nudibranch Leminda millecra. Triprenylated toluquinone-induced and toluhydroquinone-induced cell death is mediated via apoptosis after a cell cycle block. Molecular events include production of reactive oxygen species (ROS), followed by induction and activation of c-Jun (AP1) via c-Jun-NH2-kinase-mediated and extracellular signal-regulated kinase-mediated pathways. Partial resistance to these compounds could be conferred by the ROS scavengers Trolox and butylated hydroxyanisol, a c-Jun-NH2-kinase inhibitor, and inhibition of c-Jun with a dominant negative mutant (TAM67). Interestingly, the levels of ROS produced varied between compounds, but was proportional to the ability of each compound to kill cells. Because cancer cells are often more susceptible to ROS, these compounds present a plausible lead for new antiesophageal cancer treatments and show the potential of the South African marine environment to provide new chemical entities with potential clinical significance.     

人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧在高糖状态下的表达

背景:高血糖导致的自由基损伤是糖尿病视网膜病变发病机制的中心环节.目的:观察高糖对体外培养的人视网膜色素上皮细胞的氧化损伤作用以及高糖对人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧表达的影响.方法:将培养人视网膜色素上皮细胞,分为对照组、高糖组和甘露醇组,分别用含5.5 mmol/L葡萄糖,33 mmol/L葡萄糖及5.5 mmol/L葡萄糖和27.5 mmol/L甘露醇的DMEM培养液培养.采用相差倒置显微镜观察细胞生长形态,采用免疫荧光染色研究诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达的变化,用氯甲基二氯二氢荧光素二乙酯荧光染色检测视网膜色素上皮细胞中活性氧的产生量.结果与结论:与对照组相比,应用含33 mmol/L葡萄糖的DMEM培养基处理视网膜色素上皮细胞48 h可见细胞胞体变薄,形态表现多样,不规则细胞增多;高糖培养的视网膜色素上皮细胞诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达增加,活性氧产生明显增多.说明高浓度葡萄糖培养可造成人视网膜色素上皮细胞氧化损伤,使细胞形态发生变化,并导致细胞中3-硝基酪氨酸产生增多.     

人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧在高糖状态下的表达

背景：高血糖导致的自由基损伤是糖尿病视网膜病变发病机制的中心环节。目的：观察高糖对体外培养的人视网膜色素上皮细胞的氧化损伤作用以及高糖对人视网膜色素上皮细胞诱导型一氧化氮合酶和活性氧表达的影响。方法：将培养人视网膜色素上皮细胞,分为对照组、高糖组和甘露醇组,分别用含5.5mmol/L葡萄糖,33mmol/L葡萄糖及5.5mmol/L葡萄糖和27.5mmol/L甘露醇的DMEM培养液培养。采用相差倒置显微镜观察细胞生长形态,采用免疫荧光染色研究诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达的变化,用氯甲基二氯二氢荧光素二乙酯荧光染色检测视网膜色素上皮细胞中活性氧的产生量。结果与结论：与对照组相比,应用含33mmol/L葡萄糖的DMEM培养基处理视网膜色素上皮细胞48h可见细胞胞体变薄,形态表现多样,不规则细胞增多;高糖培养的视网膜色素上皮细胞诱导型一氧化氮合酶和3-硝基酪氨酸蛋白表达增加,活性氧产生明显增多。说明高浓度葡萄糖培养可造成人视网膜色素上皮细胞氧化损伤,使细胞形态发生变化,并导致细胞中3-硝基酪氨酸产生增多。     

Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification

Chondrocyte hypertrophy during endochondral ossification is a well-controlled process in which proliferating chondrocytes stop proliferating and differentiate into hypertrophic chondrocytes, which then undergo apoptosis. Chondrocyte hypertrophy induces angiogenesis and mineralization. This step is crucial for the longitudinal growth and development of long bones, but what triggers the process is unknown. Reactive oxygen species (ROS) have been implicated in cellular damage; however, the physiological role of ROS in chondrogenesis is not well characterized. We demonstrate that increasing ROS levels induce chondrocyte hypertrophy. Elevated ROS levels are detected in hypertrophic chondrocytes. In vivo and in vitro treatment with N-acetyl cysteine, which enhances endogenous antioxidant levels and protects cells from oxidative stress, inhibits chondrocyte hypertrophy. In ataxia telangiectasia mutated (Atm)-deficient (Atm(-/-)) mice, ROS levels were elevated in chondrocytes of growth plates, accompanied by a proliferation defect and stimulation of chondrocyte hypertrophy. Decreased proliferation and excessive hypertrophy in Atm(-/-) mice were also rescued by antioxidant treatment. These findings indicate that ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes.     

Reactive oxygen species generation by leukocytes in populations at risk for atherosclerotic disease

The aim of this study was to investigate intracellular levels of reactive oxygen species (ROS) in circulating leukocytes in populations at risk for atherosclerosis compared with in healthy individuals. The study populations consisted of 27 non-diabetic men (aged 40-69 years) with untreated hypercholesterolemia (HC), 13 individuals (aged 39-56 years) with well-controlled insulin-dependent diabetes mellitus (DM), and 20 healthy individuals (aged 26-61 years) (REF). Citrated whole blood was collected in fasting condition. Using flow cytometric techniques, the resting levels and the response upon phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) stimulation of ROS were measured in circulating monocytes (MO) and granulocytes (GR). The relative mean fluorescence intensity (rMFI) in 10(4) leukocytes of fluorochromes mainly reflecting the levels of peroxynitrite (ONOO-) hydrogen peroxide (H2O2) and superoxide anion (O2-) was recorded. Significantly, higher basal levels of ONOO- in GR from the combined risk population compared with REF were found (1.4 vs. 1.5 rMFI, p<0.05). Upon PMA stimulation, significantly lower levels of O2 in GR in the risk populations compared to REF (119 vs. 90 Si, p<0.001) were observed. In conclusion, increased resting levels of ROS in circulating granulocytes, but reduced response to PMA stimulation could be demonstrated in populations at risk for atherosclerosis compared with in healthy individuals. This might indicate a higher degree of resting oxidative reactions, with partly exhausted cells and less capacity to host defence.     

Reactive oxygen species involved cancer cellular specific 5-aminolevulinic acid uptake in gastric epithelial cells

Photodynamic therapy and photodynamic diagnosis using 5-aminolevulinic acid (ALA) are clinically useful for cancer treatments. Cancer cells have been reported that 5-aminolevulinic acid is incorporated via peptide transporter 1, which is one of the membrane transport proteins, and has been reported to be significantly expressed in various gastrointestinal cancer cells such as Caco-2. However, the mechanism of this protein expression has not been elucidated. Concentration of reactive oxygen species (ROS) is higher in cancer cells in comparison with that of normal cells. We have previously reported that ROS derived from mitochondria is likely related to invasions and proliferations of cancer cells. Since 5-aminolevulinic acid is the most important precursor of heme which is necessary protein for cellular proliferations, mitochondrial ROS (mitROS) may be also related to peptide transporter 1 expressions. In this study, we used a rat gastric mucosal cell line RGM1 and its cancer-like mutated cell line RGK1, and we clarified the ALA uptake mechanism and its relations between mitROS and peptide transporter 1 expression in RGK1. We also used our self-established stable clone of cell which over-expresses manganese superoxide dismutase, a mitROS scavenger. We studied differences of the photodynamic therapy effects in these cells after ALA administrations to clear the influence of mitROS.     

Reactive oxygen species contribute to neuropathic pain by reducing spinal GABA release

Although both a loss of spinal inhibitory neurotransmission and the involvement of oxidative stress have been regarded as important mechanisms in the pathogenesis of pain, the relationship between these 2 mechanisms has not been studied. To determine whether reactive oxygen species (ROS) involvement in pain mechanisms is related to the diminished inhibitory transmission in the substantia gelatinosa (SG) of the spinal dorsal horn, behavioral studies and whole-cell recordings were performed in FVB/NJ mice. Neuropathic pain was induced by a tight ligation of the L5 spinal nerve (SNL). Pain behaviors in the affected foot were assessed by behavioral testing for mechanical hyperalgesia. Pain behaviors developed by 3 days and lasted more than 8 weeks. Both systemic and intrathecal administration of an ROS scavenger, phenyl-N-tert-butylnitrone (PBN), temporarily reversed mechanical hyperalgesia up to 2 hours, 1 week after SNL. In nonligated mice, an intrathecal injection of an ROS donor, tert-butyl hydroperoxide (t-BOOH), dose-dependently induced mechanical hyperalgesia for 1.5 hours. In whole-cell voltage clamp recordings of SG neurons, perfusion with t-BOOH significantly decreased the frequency of mIPSCs, and this effect was reversed by PBN. Furthermore, t-BOOH decreased the frequency of GABA_A receptor-mediated mIPSCs without altering their amplitudes but did not affect glycine receptor-mediated mIPSCs. In SNL mice, mIPSC frequency in SG neurons was significantly reduced as compared with that of normal mice, which was restored by PBN. The antihyperalgesic effect of PBN on mechanical hyperalgesia was attenuated by intrathecal bicuculline, a GABA_A receptor blocker. Our results indicate that the increased ROS in spinal cord may induce pain by reducing GABA inhibitory influence on SG neurons that are involved in pain transmission.