不同糖浓度培养雪旺细胞NGF和CGT表达变化及Egb761干预后的变化
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摘要
背景与目的
雪旺细胞(Schwann Cell, SC)是周围神经系统的胶质细胞,除了与周围神经的发生、发育、形态、功能等方面密切相关外,在周围神经损伤后的再生与修复过程中也起着非常重要的作用,SC还能分泌多种活性物质诱导、刺激和调控轴突的再生和髓鞘的形成。
神经生长因子(nerve growth factor, NGF)是最早发现的细胞生长调节因子,也是迄今为止研究得最清楚的一个神经营养因子。NGF在神经元的发育、轴突的生长、递质的合成及细胞的凋亡等阶段均起着重要的作用。当外周神经受损伤时,SC成为其主要来源。
半乳糖神经酰胺转移酶(Galactosylceramide transferase, CGT)是合成神经髓鞘主要成份半乳糖神经酰胺的限速酶,在髓鞘以及体外培养的SC中表达。CGT是微粒体酶,主要在神经、脑、肾脏中表达,它催化半乳糖转移到神经酰胺而合成半乳糖神经酰胺(Galactosylceramide, GalC)。GalC是髓鞘中最丰富的脂质之一,在中枢或周围神经系统中,占髓鞘干重的20%,对髓鞘的结构和功能起重要作用,但其过度的表达可诱导SC的凋亡。
银杏叶提取物具有抗脂质过氧化,对缺血-缺氧性损伤、机械损伤的神经元具有保护作用,并且具有抑制细胞凋亡的作用。
目前,国内对于髓鞘形成细胞SC的研究大多集中在组织工程方面的研究,而对于其本身分泌活性物质对于周围神经的作用研究并不多见。不同浓度葡萄糖水平对SC所分泌的NGF及CGT影响的研究颇少见及关于银杏叶提取物在糖尿病周围神经保护方面的作用研究报道较少,特别是低糖对SC分泌的活性物质的影响尚未见报道。本实验的目的就是为了观察不同的糖浓度对SC NGF mRNA.CGT mRNA的影响及银杏叶提取物干预所设定的低糖及高糖组SC NGF mRNA.CGT mRNA表达的变化。
材料与方法
1实验方法
本实验首先将所培养的雪旺细胞分别于10mmol/L、25mmol/L、50mmol/L糖浓度下培养,采用RT-PCR技术,分别于培养后72h对各组的NGF mRNA,CGT mRNA表达进行测定。之后,用银杏叶提取物Egb761分别干预10mmol/L、50mmol/L糖浓度培养的SC,分别与未用Egb761干预的10mmol/L,50mmol/L糖浓度组进行比较,各组均培养72小时,采用RT-PCR技术,对各组的NGF及CGT基因表达进行测定。
2统计学方法
采用SPSS10.0统计软件进行统计分析,数据以均数±标准差(x)表示。数据经检验符合正态分布、方差齐,两组之间的比较用t检验,组间比较采用单因素方差分析(ANOVA),三组间的两两比较用LSD检验。以α=0.05为检验水准。
结果
1 SC细胞于不同的糖浓度培养过程中的形态学观察
倒置显微镜下观察25mM糖浓度下细胞形态呈长梭形或多个细胞呈多角形连接在一起,类似成纤维细胞,细胞核位于细胞中间,呈圆形,1OmM及50mM糖浓度下细胞成类圆形,细胞核不明显。
2不同糖浓度培养SC NGFmRNA、CGTmRNA表达的变化
经过扫描及统计分析发现,高糖及低糖时雪旺细胞CGT mRNA表达明显增高,而NGF mRNA表达降低,组间进行比较得出：高糖组、低糖组与正常糖浓度组比较差异均有统计学意义,P<0.05；低糖与高糖组比较差异无统计学意义,P>0.05。
3 Egb761干预后NGF mRNA、CGT mRNA表达的变化
经过扫描及统计分析发现Egb761干预的高糖组与低糖组分别与空白对照组进行比较,NGF mRNA的表达差异均有统计学意义(P<0.05),CGTmRNA表达无统计学意义P>0.05。
结论
1、本实验于低糖及高糖浓度培养SC,与正常糖浓度相比NGF mRNA的表达减少,CGT mRNA的表达增加,且差异均具有统计学意义,提示高糖及低糖均可能导致周围神经的损伤
2、银杏叶提取物Egb761分别干预10mmol/L、50mmol/L糖浓度培养的SC72h,测定NGF mRNA的表达分别与未加Egb761干预组比较均增加,差异均具有统计学意义,提示银杏叶提取物对可能对周围神经损伤的修复具有一定促进作用。
Background and Objectives
Schwann cell (SC), which is the glial cells of peripheral nervous system, plays the most important role in the peripheral nerve regeneration. SC can also secrete a variety of active factors to induce, stimulate and regulate axon regeneration and myelin formation.
Nerve growth factor(NGF), the first discovered neurotrophic factor which act as a key regulator in the neuron development, axonal growth, neurotransmitter synthesis and cell apoptosis.
Galactosylceramide transferase(CGT) is the necessary components of :synthesizing myelin galactosylceramide rate-limiting enzyme. It is reported that CGT is secreted by the myelin sheath and SC in vitro. CGT, a microsomal enzyme, mainly is expressed in the nerves, brain and kidney. It can catalyze the transfer of galactose to the ceramide to syntheticing galactosylceramide. GalC is one of the most abundant lipids in myelin ceramide, accounting for 20% of the dry weight of myelin in the central or peripheral nervous system. It play an important role in the myelin sheath structure and function.
Ginkgo Biloba Extract play a protective role in anti-lipid peroxidation, anti-ischemic-hypoxic injury,anti-mechanical damage neurons and inhibiting cell apoptosis.
At present, the study of myelin sheath-forming cells-SC is mainly in tissue engineering research area, however the studies of the effects and mechanism of active substances secreted by SC in peripheral nerve are rare. Although diabetic peripheral neuropathy is common but its pathogenesis is not clear. The influence of different glucose concentrations cultureing SC to NGF and CGF secretion by SC is unknown too. Especially the influence of low glucose to active substances secreted by SC has never been reported. There are few reports about protection mechanism of Ginkgo biloba extract to diabetic peripheral.The purpose of this study is to observe the influence of the NGF and CGF secretion by SC intervention with different glucose concentration, and the potential mechanism of Ginkgo biloba extract on nerve protection.
Materials and Methods
1. Experimental Methods:In this study, the cultured Schwann cells were divided into 10mmol/L,25mmol/L and 50mmol/Lglucose concentration group.Using RT-PCR, to determine the respectively expression of the NGF and the CGT gene after 72h cultured in each group. At the same time, using Ginkgo Biloba Extract to interfere 10mmol/L,50mmol/L glucose concentration group in cultured SC and using RT-PCR technique to determine the NGF and the CGT gene expression after 72h for each group were determined.
2.Statistic analysis:The experimental data were expressed by mean±standard deviation (χ±s) and the data were handled with SPSS10.0 statistic software. Significance level is judged by a=0.05.
Results
1 Observating the morphological of SC under different glucose concentrations
SC morphological were accumulated together like spindle cell morphology or more cells were polygonal to connect together, similar to fibroblasts under 25mM glucose observed by inverted microscope.the nuclei in the middle, rounded. SC was round underlOmM and 50mM glucose concentration. Cells nucleus is not obvious.
2 The changes of NGF mRNA, CGT mRNA of SC under different glucose
concentration
SC CGT mRNA expression increased significantly, while the expression of NGF mRNA decreased compared with the normal groups under high glucose and low-glucose group. Statistical differences were significance, P<0.05; low glucose compared with high glucose group showed no significant difference, P> 0.05.
3 The changing of SC NGF mRNA CGT mRNA under different glucose concentration intervented by Egb761.
SC NGF mRNA expression increased significantly intervented by Egb761,P <0.05.
Conclusions
1. Both high- glucose and low glucose out of normal range may lead to peripheral nerve injury.
2.Ginkgo biloba extract Egb761 intervent cultured SC in 10mmol/L and 50mmol/L glucose concentration, NGF mRNA expression were increased, indicated that Ginkgo biloba extract were benefite at the repairment of peripheral nerve injury.
雪旺细胞(Schwann Cell, SC)是周围神经系统的胶质细胞,除了与周围神经的发生、发育、形态、功能等方面密切相关外,在周围神经损伤后的再生与修复过程中也起着非常重要的作用,SC还能分泌多种活性物质诱导、刺激和调控轴突的再生和髓鞘的形成。
神经生长因子(nerve growth factor, NGF)是最早发现的细胞生长调节因子,也是迄今为止研究得最清楚的一个神经营养因子。NGF在神经元的发育、轴突的生长、递质的合成及细胞的凋亡等阶段均起着重要的作用。当外周神经受损伤时,SC成为其主要来源。
半乳糖神经酰胺转移酶(Galactosylceramide transferase, CGT)是合成神经髓鞘主要成份半乳糖神经酰胺的限速酶,在髓鞘以及体外培养的SC中表达。CGT是微粒体酶,主要在神经、脑、肾脏中表达,它催化半乳糖转移到神经酰胺而合成半乳糖神经酰胺(Galactosylceramide, GalC)。GalC是髓鞘中最丰富的脂质之一,在中枢或周围神经系统中,占髓鞘干重的20%,对髓鞘的结构和功能起重要作用,但其过度的表达可诱导SC的凋亡。
银杏叶提取物具有抗脂质过氧化,对缺血-缺氧性损伤、机械损伤的神经元具有保护作用,并且具有抑制细胞凋亡的作用。
目前,国内对于髓鞘形成细胞SC的研究大多集中在组织工程方面的研究,而对于其本身分泌活性物质对于周围神经的作用研究并不多见。不同浓度葡萄糖水平对SC所分泌的NGF及CGT影响的研究颇少见及关于银杏叶提取物在糖尿病周围神经保护方面的作用研究报道较少,特别是低糖对SC分泌的活性物质的影响尚未见报道。本实验的目的就是为了观察不同的糖浓度对SC NGF mRNA.CGT mRNA的影响及银杏叶提取物干预所设定的低糖及高糖组SC NGF mRNA.CGT mRNA表达的变化。
材料与方法
1实验方法
本实验首先将所培养的雪旺细胞分别于10mmol/L、25mmol/L、50mmol/L糖浓度下培养,采用RT-PCR技术,分别于培养后72h对各组的NGF mRNA,CGT mRNA表达进行测定。之后,用银杏叶提取物Egb761分别干预10mmol/L、50mmol/L糖浓度培养的SC,分别与未用Egb761干预的10mmol/L,50mmol/L糖浓度组进行比较,各组均培养72小时,采用RT-PCR技术,对各组的NGF及CGT基因表达进行测定。
2统计学方法
采用SPSS10.0统计软件进行统计分析,数据以均数±标准差(x)表示。数据经检验符合正态分布、方差齐,两组之间的比较用t检验,组间比较采用单因素方差分析(ANOVA),三组间的两两比较用LSD检验。以α=0.05为检验水准。
结果
1 SC细胞于不同的糖浓度培养过程中的形态学观察
倒置显微镜下观察25mM糖浓度下细胞形态呈长梭形或多个细胞呈多角形连接在一起,类似成纤维细胞,细胞核位于细胞中间,呈圆形,1OmM及50mM糖浓度下细胞成类圆形,细胞核不明显。
2不同糖浓度培养SC NGFmRNA、CGTmRNA表达的变化
经过扫描及统计分析发现,高糖及低糖时雪旺细胞CGT mRNA表达明显增高,而NGF mRNA表达降低,组间进行比较得出：高糖组、低糖组与正常糖浓度组比较差异均有统计学意义,P<0.05；低糖与高糖组比较差异无统计学意义,P>0.05。
3 Egb761干预后NGF mRNA、CGT mRNA表达的变化
经过扫描及统计分析发现Egb761干预的高糖组与低糖组分别与空白对照组进行比较,NGF mRNA的表达差异均有统计学意义(P<0.05),CGTmRNA表达无统计学意义P>0.05。
结论
1、本实验于低糖及高糖浓度培养SC,与正常糖浓度相比NGF mRNA的表达减少,CGT mRNA的表达增加,且差异均具有统计学意义,提示高糖及低糖均可能导致周围神经的损伤
2、银杏叶提取物Egb761分别干预10mmol/L、50mmol/L糖浓度培养的SC72h,测定NGF mRNA的表达分别与未加Egb761干预组比较均增加,差异均具有统计学意义,提示银杏叶提取物对可能对周围神经损伤的修复具有一定促进作用。
Background and Objectives
Schwann cell (SC), which is the glial cells of peripheral nervous system, plays the most important role in the peripheral nerve regeneration. SC can also secrete a variety of active factors to induce, stimulate and regulate axon regeneration and myelin formation.
Nerve growth factor(NGF), the first discovered neurotrophic factor which act as a key regulator in the neuron development, axonal growth, neurotransmitter synthesis and cell apoptosis.
Galactosylceramide transferase(CGT) is the necessary components of :synthesizing myelin galactosylceramide rate-limiting enzyme. It is reported that CGT is secreted by the myelin sheath and SC in vitro. CGT, a microsomal enzyme, mainly is expressed in the nerves, brain and kidney. It can catalyze the transfer of galactose to the ceramide to syntheticing galactosylceramide. GalC is one of the most abundant lipids in myelin ceramide, accounting for 20% of the dry weight of myelin in the central or peripheral nervous system. It play an important role in the myelin sheath structure and function.
Ginkgo Biloba Extract play a protective role in anti-lipid peroxidation, anti-ischemic-hypoxic injury,anti-mechanical damage neurons and inhibiting cell apoptosis.
At present, the study of myelin sheath-forming cells-SC is mainly in tissue engineering research area, however the studies of the effects and mechanism of active substances secreted by SC in peripheral nerve are rare. Although diabetic peripheral neuropathy is common but its pathogenesis is not clear. The influence of different glucose concentrations cultureing SC to NGF and CGF secretion by SC is unknown too. Especially the influence of low glucose to active substances secreted by SC has never been reported. There are few reports about protection mechanism of Ginkgo biloba extract to diabetic peripheral.The purpose of this study is to observe the influence of the NGF and CGF secretion by SC intervention with different glucose concentration, and the potential mechanism of Ginkgo biloba extract on nerve protection.
Materials and Methods
1. Experimental Methods:In this study, the cultured Schwann cells were divided into 10mmol/L,25mmol/L and 50mmol/Lglucose concentration group.Using RT-PCR, to determine the respectively expression of the NGF and the CGT gene after 72h cultured in each group. At the same time, using Ginkgo Biloba Extract to interfere 10mmol/L,50mmol/L glucose concentration group in cultured SC and using RT-PCR technique to determine the NGF and the CGT gene expression after 72h for each group were determined.
2.Statistic analysis:The experimental data were expressed by mean±standard deviation (χ±s) and the data were handled with SPSS10.0 statistic software. Significance level is judged by a=0.05.
Results
1 Observating the morphological of SC under different glucose concentrations
SC morphological were accumulated together like spindle cell morphology or more cells were polygonal to connect together, similar to fibroblasts under 25mM glucose observed by inverted microscope.the nuclei in the middle, rounded. SC was round underlOmM and 50mM glucose concentration. Cells nucleus is not obvious.
2 The changes of NGF mRNA, CGT mRNA of SC under different glucose
concentration
SC CGT mRNA expression increased significantly, while the expression of NGF mRNA decreased compared with the normal groups under high glucose and low-glucose group. Statistical differences were significance, P<0.05; low glucose compared with high glucose group showed no significant difference, P> 0.05.
3 The changing of SC NGF mRNA CGT mRNA under different glucose concentration intervented by Egb761.
SC NGF mRNA expression increased significantly intervented by Egb761,P <0.05.
Conclusions
1. Both high- glucose and low glucose out of normal range may lead to peripheral nerve injury.
2.Ginkgo biloba extract Egb761 intervent cultured SC in 10mmol/L and 50mmol/L glucose concentration, NGF mRNA expression were increased, indicated that Ginkgo biloba extract were benefite at the repairment of peripheral nerve injury.
引文
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[31]Brownlee M. The pathobiology of diaberic complications:a unifying mechanism [J]. Diabetes,2005,54(6):1615-1625.
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[30]Ziegler D. Oxidative Stress and Antioxidant Defense in Relation to the Severity of Diabetic Polyneuropathy and Cardiovascular Autonomic Neuropathy[J]. Diabetes Care,2004,27:2178-2183.
[31]Brownlee M. The pathobiology of diaberic complications:a unifying mechanism [J]. Diabetes,2005,54(6):1615-1625.
[32]Vincent AM,McLean LL,Backus C. Short term hyperglycemia produces oxidative damage and apoptosis in neurons[J]. FASEB J,1996;44(7):2503-2513.
[33]Biwenga GP,Haenen GRMM,Bast A. The pharmacology of the antioxidantlipoic acid[J]. Gen Pharco1,1997,29:315-33.
[34]Tricia SF. Efficacy and Safety of a-Lipoic Acid Supplementation in the Treatment of Symptomatic Diabetic Neuropathy[M]. The Diabetes Educator,2007,33:111-117
[35]Nakamura,Kato K,Hamada X,et al. A protein kinase betaselective inhibitor ameliorates neural dysfunction in streptozotocin—induced diabetic rats[J]. Diabetes,1999,48: 2090-2010.
[36]蒋雯巍,蒋雨平.高同型半胱氨酸血.症与糖尿病神经病变[J].中华老年医学志,2005,24(9)：708-709.
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[38]Pittenger G,Vinik A. Nerve growth factor and diabetic neuropathy[J]. Exp Diabesity Res,2003,4(4):271-285.
[39]Koisbi R,Ando Y,Ono M,et al. Angptl regulates lipid metabolism in mice[J]. J Nat Genet,2002,30:151-157.
[40]Delva P,Pastori C,Montesi G. Intralymphocyte free magnesium and cal slum and insul in tolerance test in a group of essential hypertensive patients[J]. Life Science,1998,63: 1405-1049.
[41]Gianni C,Djrck PJ. Pathologic alterations in human diabetic polyneuropathy [J]. Diabetic Neuropathy,1999,5:09-15.
[42]Kennedy JM, Zochodne DW. The regenerative deficit of peripheral nerves in experimental diabetes:its extent,timing and possible mechanisms [J]. Brain 2000,23: 2118-2129.
[43]VinikAI,Leicbter SB,Pittenger,GL. Phospholipid and glutamic acid decarboxylase autoantibodies in diabetic neuropathy [J]. Diabetes Care,1995,18:1225-1229.
[44]Jannot D,Raccah D,Tour DDDL,et al. Genetic and environmental regulation of Na/K adenosine triphosphatase activity in diabetic patients[J]. Metabolis,2002,51(3): 284-291.
[45]Dobrowsky RT,Rouen S,Yu C.. Altered Neurotrophism in Diabetic Neuropathy: Spelunking the Caves of Peripheral Nerve[J]. PharmacOl. Exp. Ther,2005,313: 485-491.
[46]Nobecourt E J acquerminet S,Hansel B,et al. Defectiveantioxidative activity ofsmall dense HDL3 particles in type 2 diabetes:relationship to elevated oxidative stress and hyperglycaemia[J]. Diabetologia,2005,48:529-538.
[47]Wang Y Schmeichel AM,Lida H,et al. Ischemia-reperfusion injury causes oxidative stress and apoptosis of Schwann cell in acute and chromc experimental diabetic neuropathy[J]. Antioxid Redox Signal,2005,7:1513-1520.
[48]Wautier MP,Chappey O,Corda S,et al. Activation of NAPDH oxidase by AGE Links oxidant stress to altered gene expression via RAGE[J]. Am J Physiol,2001,280:685-694.
[49]Sekido h,Suzuki T,Jomori T,et al. Reduced cell replication and induction of apoptosis by advanced glycation end products in rat Schwarm cells[J]. Biochem Biophys Res Commun,2004,320:241-248.
[50]Cellek S,Qu W,Schmidt AM,et al. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptpsis in votro:a new insight into selective nitrergic neuropathy in diabetes[J]. Diabetologia,2004,47:331-339.
[51]Song Z,Fu DT,Chan YS,et al. Transgenic mice over expressing aldose reductase in Schwann cells show more severe nerve conduction velocity deficit and oxidative stress under hyperglycemic stress[J]. Mol Cell Neurosci,2003,23:638-647.
[52]Babior BM. NADPH oxidase Curr Opin Immunol[C].2004,16:42-47.
[53]Hashi K,Maruyama W. Isobe KP eroxynitrite induces GADD34,45and 153 VLA p38 MAPK.in human neuroblastoma SH-SY5Y cells[J]. Free Radic Biol Med,2001,30: 213-221.
[54]Rosen P,Nawroth PP,King G, et al. The role of oxidative stress in the.onset and progression of diabetes and its complications:a summary of a Congress Series sponsored by UNESCO-MCBN,the American Diabetes Association and the German Diabetes Society[J]. Diabetes Memb Res Rev,2001,17:189-212.
[55]Aragno M,Mastrocola R,Bringnardello E,et al. Dehydroepiandrosterone modulates nuclear factor-Kappa B activation in hippocampus of diabetic rats[M]. Endocrinology 2002,143:3250-3258.
[56]Young IS,Woodside Ⅳ. Antioxidants in health and disease[J]. J ClinPathol,2001,54(3): 176-186.
[57]LeeS. Cardiovascular responses and endurance during isometric exercise in patients with Type 2 diabetes compared to control subjects[J]. Med Sei Monit.2005,11(10): 470-477.
[58]Bruunsgaard H. Physical activity and modulation of systemic low level inflammation[J]. J Leukoc Biol,2005,78:819-835.
[59]Masaaki Ii,Nishimura H,Kusano K F,et al. Neuronal nitric oxide synthase mediates statin-induced restoration of vasa nervorum and reversal of diabetic neuropathy[J]. Circulation,2005,112(1):93-102.
[60]Strokov I,Manukhina E,Bakhtina L,et al. The function of endogenous protecti—vesystems in patients with insulin-dependent diabetes mellitus and polyneuropathy:effect of antioxidant therapy[J]. Bull Exp Biol Med,2000,130 (10): 986-990.
[61]Skyler JS. Effect of glycemic control on diabetes complications and on the prevention of diabetes[J]. Clinical Diabetes,2004,22:162-166.
[62]Lasker RD. The diabetes control and complications trial implications for policy and practice[J]. NEngl J Med,1993,329:1035-1036.
[63]Thomas PK. Diabetic neuropathy:Mechanisms and future treatment options[J]. J Neurol Neurosurg Psychiatry,1999,67:277-279.
[64]Singleton JR,Smith AG. Increased prevalence of impaired glucose tolerance in patients with painful sensory neumpathy[J]. Diabetes Care,2001,24:1448-1453.
[65]Brussee V,Cunningham FA,Zochodne DW. Direct insulin signaling of neuronsl verses diabetic neuropathy[J]. Diabetes,2004,53:1824-1830.
[66]SchmeichelAM,Schmeher JD. Oxidativeinjuryand apoptosisofdotsal root ganglion neurons in chronic experimental diabetic neuropathy [J]. Diabetes,2003,52:165-171
[67]Sheetz MJ,King GL. Molecular understanding of hyperglycemia's adversedfectsfordiabetic ecxnplieations[J]. JAMA,2002,288:2579-2588.
[68]Vincent AM. Short term hyperglycemia produces oxidative damageand apoptosis in neurons[J]. FASEB J,2005,19:638-640.
[69]Russell JN,GolovoyD. High glucose inducedoxidative stressandmi tochondrial dysfunction in neurons[J]. FASEB J,2002,16:1738-1748.
[70]Greene DA,Stevens MJ. Glucose induced oxidative stress and programrned cell death in diabetic neuropathy[J].1999,375:217-223.