【作者】 周怡昆；

【导师】 薛耀明；

【作者基本信息】 南方医科大学 ， 内分泌与代谢病学（专业学位）， 2014， 博士

【摘要】 背景糖尿病周围神经病变是糖尿病最常见,发病机制最复杂的慢性并发症之一,在我国2型糖尿病发病率高达9.7%,而其中60-70%的患者并发周围神经病变。由于疼痛,麻木,及其继发的糖尿病足感染,截肢,导致患者生活质量明显下降和医疗费用明显增加。1,25(OH)2D3是维生素D3的体内活性代谢物,近期流行病学及临床研究显示,维生素D3不足与糖尿病周围神经病变密切相关,维生素D3的不足是糖尿病周围神经病变独立的危险因素。美国健康与营养调查研究(NHANES)的最新统计数据表明,81%的成人糖尿病患者存在维生素D3不足,且与糖尿病神经病变密切相关,即使避开人口统计因素,肥胖,并发症,神经病变药物治疗和糖尿病病程这些可能的混杂因素,这一关系仍然存在。在一项对210名糖尿病患者的临床研究中,神经病变组25-羟维生素D3(25(OH) D3:维生素D3体内活性物质的检测指标)水平比无神经病变组低,神经病变组和无神经病变组患者25(OH)D3缺乏分别占81.5%和60.4%。一项对于糖尿病痛性神经病变和维生素D3关系的前瞻性研究显示：51名痛性神经病变的糖尿病患者,均存在维生素D3不足,平均血浆25(OH)D3浓度为18ng/mL.疼痛评分与25(OH)D3浓度呈负相关。维生素D3充足后,疼痛评分明显下降。但是1,25(OH)2D3与糖尿病神经病变之间的关系尚不清楚。随着近年来对1,25(OH)2D3研究的进一步深入,其对中枢神经胶质细胞-星状细胞的保护作用已得到证实：1,25(OH)2D3抑制诱导型一氧化氮合成酶表达,减少一氧化氮的合成,减少细胞炎性反应；能增加星状细胞神经生长因子mRNA及蛋白表达,介导神经营养保护作用；增加星状细胞细胞外谷胱甘肽池,并明显减少脂多糖介导的亚硝酸盐产生,清除活性氧物质。但目前关于1,25(OH)2D3对周围神经系统影响,尤其是与雪旺细胞之间关系的研究较少。雪旺细胞(Schwann cell SC)在周围神经系统具有重要作用,例如：髓磷脂的形成,支持,营养,促进轴突和神经元的恢复,分泌多种神经营养因子(例如：神经生长因子(nerve growth factor NGF))。神经生长因子在周围神经病变神经再生中具有重要地位,降低的神经生长因子水平一定程度上导致了轴突再生失败及糖尿病周围神经病变的发病。高糖条件下,下降的神经生长因子水平降低了背根神经节神经元的出芽。许多研究显示1,25(OH)2D3介导多种细胞神经生长因子的表达,虽然仅有一篇文章报道其上调雪旺细胞神经生长因子基因表达,1,25(OH)2D。与糖尿病神经病变的关系是否也与1,25(OH)2D3介导雪旺细胞神经生长因子的分泌有关,虽然目前尚没有研究直接提及其对雪旺细胞神经生长因子分泌水平的影响。然而已经有动物研究在一定程度上支持了我们的设想,糖尿病大鼠坐骨神经神经生长因子减少,维生素D3的衍生物能剂量依赖性地增加其神经生长因子产生,防止神经生长因子的耗竭。然而,为什么高糖状态会引起神经纤维产生的神经生长因子减少,这与1,25(OH)2D3减少之间是否存在关系?1,25(OH)2D3通过维生素D受体,结合维甲酸x受体或经过ERK1等信号分子途径,激活CYP24A1基因表达,25(OH)D3-24羟化酶(CYP24A1)能将1,25(OH)2D3或25(OH)D3代谢成为没有活性的1,24(OH)2D3或24(OH)。已有研究显示CYP24A1在糖尿病动物模型的肾脏中表达增加。在多种肿瘤细胞中,发现增高的CYP24A1表达,明显地减少了1,25(OH)2D3对肿瘤细胞的抗增殖作用,而给予CYP24A1抑制剂,能增加1,25(OH)2D3的抗肿瘤增殖作用。根据以上数据,我们假设：高糖可能通过影响CYP24A1基因的表达,干扰1,25(OH)2D3正常代谢,.从而导致1,25(OH)2D3介导的雪旺细胞神经生长因子分泌功能的紊乱,影响神经纤维的正常或损伤状态下的生理病理功能。我们首先明确高糖及或1,25(OH)2D3是否影响雪旺细胞神经生长因子的分泌,雪旺细胞上是否存在代谢酶CYP24A1或其他合成酶如：CYP27A1及CYP27B1的表达,在高糖作用下,这些1,25(OH)2D3的代谢基因会有怎么的变化,是否参与到高糖与1,25(OH)2D3的关系中,是否影响雪旺细胞神经生长因子分泌。我们将以大鼠RSC96雪旺细胞作为研究模型,探讨高糖与1,25(OH)2D3对雪旺细胞分泌神经生长因子的影响,以及二者之间的相互作用关系。第一部分高糖及1,25(OH)2D3对雪旺细胞神经生长因子分泌水平的影响目的：研究高糖及1,25(OH)2D3对RSC96雪旺细胞神经生长因子分泌水平的影响。方法：1)高糖对雪旺细胞神经生长因子分泌的影响：将雪旺细胞分别培养在含5.6mmol/L葡萄糖(5.6G),17mmol/L葡萄糖(17G)和17mmol/L甘露醇(17M)DMEM培养基中培养96小时,实验分为三组：①5.6G组②17G组③17M组。应用酶联免疫吸附法(elisa)检测细胞上清液中神经生长因子分泌水平,应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。2)1,25(OH)2D3对雪旺细胞神经生长因子分泌的影响：1,25(OH)2D3(D310-7mol/1)及其相应的乙醇溶质分别作用于含5.6mmol/L葡萄糖DMEM培养液中培养的雪旺细胞48小时,实验分成三组：①5.6G组②D3+5.6G组③乙醇+5.6G组。应用酶联免疫吸附法(elisa)检测细胞上清液中神经生长因子分泌水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。3)高糖对1,25(OH)2D3作用的雪旺细胞神经生长因子分泌的影响：雪旺细胞分别在含5,6mmol/L葡萄糖(5.6G),17mmol/L葡萄糖(17G)和17mmol/L甘露醇(17M)DMEM培养基中培养48小时后,加入1,25(OH)2D3(D310-7mol/1)作用48小时,实验分成三组：①5.6G+D3组②17G+D3组③17M+D3组。应用酶联免疫吸附法(elisa)检测细胞上清液中神经生长因子分泌水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。结果：1)给予5.6mmol/L葡萄糖,17mmol/L葡萄糖和17mmol/L甘露醇DMEM培养基培养96小时,雪旺细胞分泌神经生长因子水平没有显著差异(P>0.05)(单因素方差分析F=0.447P=0.648S-N-K两两比较M±SEM5.6G组：177.33±52.40pg/ml17G组：134.56±9.65pg/ml17M组：158.30±15.52pg/ml N=6)。2)1,25(OH)2D3及其相应的乙醇溶质分别作用于含5.6mmol/L葡萄糖DMEM培养液中培养的雪旺细胞48小时后,1,25(OH)2D3组神经生长因子分泌水平明显增加(P<0.05)(单因素方差分析F=7.317P=0.006S-N-K两两比较分析M±SEM5.6G组：177.33±52.40pg/ml D3+5.6G组：340.29±30.91pg/ml乙醇+5.6G:161.28±17.90pg/ml N=6).3)雪旺细胞分别在含5.6mmol/L葡萄糖,17mmol/L葡萄糖和17mmol/L甘露醇DMEM培养基中培养48小时后,加入1,25(OH)2D3(D310-7mol/1)作用48小时,高糖组(D3+17G)神经生长因子的水平较其他两组明显降低。(P<0.05)(单因素方差分析F=15.979P=0.000S-N-K两两比较分析M±SEM:D3+5.6G组340.29±30.91pg/ml D3+17G组：151.09±10.43pg/ml D3+17M组：319.05±30.87pg/ml N=6).结论：1)高糖不直接影响雪旺细胞神经生长因子的分泌。2)1,25(OH)2D3增加雪旺细胞神经生长因子的分泌。3)首次发现高糖削弱1,25(OH)2D3刺激雪旺细胞神经生长因子的分泌的能力。第二部分1,25(OH)2D3合成代谢酶基因在雪旺细胞上的表达及高糖对其的影响目的：研究雪旺细胞上是否存在1,25(OH)：D。合成代谢酶基因表达,及高糖是否影响这些酶类的表达,寻找高糖削弱1,25(OH)2D3刺激雪旺细胞神经生长因子的分泌能力的分子机制。方法：1)1,25(OH)2D。合成代谢酶基因引物设计：包括：合成酶25(OH)D3-25羟化酶(CYP27A1),25(OH) D3-1a-羟化酶(CYP27B1)基因,代谢酶：25(OH)D3-24羟化酶(CYP24A1)基因。CYP27A1上游引物5’-AGCCCTCTACACAGATGCCTTAAC-3’,下游引物5’-CCCAGTTATTCATGTATCGCTTCC-3’.退火温度57.5℃,PCR产物长度314bp; CYP27B1上游引物5’-TACGCTCTCCTGGGCACTCTATGA-3’下游引物5’-AAGCGTCTCCCTATGCAACTTCGTT-3’,退火温度57.5℃, PCR产物长度408bp。 CYP24A1上游引物5’-GCAGTGGACGACCGCGAACA-3’,下游引物5’-CGATGCACATTCTCTTCCCGATGCC-3’,退火温度60℃,PCR产物长度412bp; β-actin上游引物5’-GAACCCTAAGGCCAACCGTGAA-3’,下游引物5’-CCGCTATTGCCGATAGTGATG-3’,退火温度55℃, PCR产物长度433bp。采用半定量PCR法检测雪旺细胞中是否有以上酶类表达。Fermatas公司RNA试剂盒提取细胞总RNA, Thermo公司试剂盒反转录成CDNA及PCR扩增。PCR反应条件：95℃3min,95℃30s、55℃30s、72℃30s共30个循环,72℃10min。跑胶,成像分析。2)1,25(OH)2D3对其代谢合成酶基因表达的影响：不同浓度1,25(OH)2D3培养雪旺细胞20h,分别检测前述已知基因的表达,每种基因分为三组：①A组：对照组②B组：1,25(0H)2D3(lO-8mol/L)③C组：无水乙醇5μL④D组：1,25(OH)2D3(10-7mol/L)⑤E组：无水乙醇50μL。行半定量RT-PCR检测。实验进行3次。3)高糖对1,25(OH)2D3对其代谢合成酶基因及蛋白表达的影响：17mmol/L葡萄糖分别作用于雪旺细胞20小时及96小时：A：高糖对CYP27A1，CYP27B1及CYP24A1基因的影响：分别检测已知基因的表达量,每种基因都分为六组：①5.6G1组②1761组③17M1组④5.6G4组⑤17G4组⑥17M4组。(①②③代表作用20小时,④⑤⑥代表作用96小时)半定量PCR检测,实验进行3次。B：高糖对CYP24A1蛋白表达的影响：对用半定量PCR法初步筛选表达有变化的基因,用western blotting方法进一步验证。实验进行3次。4)过氧化氢对CYP24A1基因及蛋白的影响：0.8mmol/L的过氧化氢作用于雪旺细胞20小时,A：过氧化氢对CYP24A1基因的影响：分为两组：①5.6G组②5.6G+H2O2组。行半定量RT-PCR检测,实验进行3次。B.过氧化氢对CYP24A1蛋白的影响：用western blotting方法进一步验证,实验进行3次。结果：1)雪旺细胞存在CYP27A1, CYP27B1, CYP24A1基因表达2)1,25(OH)2D。增强CYP24A1基因的表达：PCR电泳图进行研究基因/β-actin灰度值分析,行单因素方差分析,1,25(OH)2D。作用20小时后,呈剂量依赖性增强CYP24A1基因表达。F值：118.237P=0.000,B组(M±SEM0.99±0.07N=3),D组(M±SEM1.87±0.07N=3)与其他三组(M±SEM A组：0.52±0.05;C组：0.47±0.04;E组：0.46±0.05N=3)间比较有显著差异(S-N-K)。3)高糖上调CYP24A1基因及蛋白表达：A.17mmol/L高糖增加CYP24A1基因表达,对CYP27A1及CYP27B1基因表达没影响：①PCR电泳图进行研究基因/β-actin灰度值分析,行单因素方差分析,高糖作用20小时及96小时后,CYP27A1及CYP27B1的F值分别为0.66及0.369,两者均P>0.05,因此,17mmol/L高糖不影响雪旺细胞CYP27A1及CYP27B基因的表达。②PCR电泳图进行研究基因/β-actin灰度值分析,行单因素方差分析,高糖作用20小时及96小时后,CYP24A1基因表达增强。F值：15.261P=0.000(M±SEM5.6G1组0.07±0.01：17G1组0.15±0.01；17M1组0.06±0.02；5.6G4组0.06±0.01；17G4组0.14±0.01；17M4组0.06±0.02N=3)。B：高糖上调CYP24A1蛋白的表达：17G组CYP24A1蛋白表达明显高于其他两组(P<0.05)单因素方差分析F=6.706P=0.030(S-N-K两两比较M±SEM5.6G组：0.40±0.0517G组：0.82±0.1117M组：0.44±0.09N=3)。4)过氧化氢对雪旺细胞CYP24A1基因及蛋白表达的影响：A.过氧化氢降低雪旺细胞CYP24A1基因表达：0.8mmol/L的过氧化氢作用雪旺细胞20小时后,检测雪旺细胞CYP24A1基因表达。PCR电泳图用CYP24A1/β-actin灰度值分析。结果显示：过氧化氢降低雪旺细胞CYP24A1的基因表达。两独立样本T-检验(t=7.881, P=0.001)(M±SEM5.6G组：0.29±0.01;5.6G+H202组：0.14±0.02N=3)。B.过氧化氢不影响雪旺细胞CYP24A1蛋白表达：0.8mmol/L的过氧化氢作用雪旺细胞20小时后,检测雪旺细胞CYP24A1蛋白表达。凝胶电泳图用CYP24A1/β-actin灰度值分析。结果显示：过氧化氢不影响雪旺细胞CYP24A1的蛋白表达。两独立样本T-检验(t=0.445, P=0.679)(M±SEM5.6G组：0.40±0.05;5.6G+H202组：0.36±0.07N=3)。结论：1)首次发现雪旺细胞表达CYP27A1, CYP27B1, CYP24A1基因,表明雪旺细胞是1,25(OH)2D3的靶细胞。自身可能具有合成活性维生素D3代谢产物的能力,可能通过自分泌及旁分泌的方式增加局部1,25(OH)2D3浓度,进而影响自身及周围细胞组织。2)首次发现高糖上调CYP24A1基因及蛋白表达,这种CYP24A1基因及蛋白的上调可能干扰1,25(OH)2D3在雪旺细胞的正常分解代谢功能,从而导致对1,25(OH)2D。正常生理功能的影响。3)高糖引起CYP24A1基因及蛋白表达增加,可能与活性氧物质无关。第三部分CYP24A1抑制剂对高糖培养下1,25(OH)2D3介导的雪旺胞神经生长因子分泌能力的影响目的：研究CYP24A1抑制剂是否影响高糖培养下1,25(OH)2D3介导的雪旺细胞神经生长因子分泌的能力方法：CYP24A1抑制剂genistein对高糖培养下的雪旺细胞CYP24A1蛋白的抑制情况：雪旺细胞培养在含17mmol/L葡萄糖的培养液中48小时后,给予genistein (Gen50μmol/1)及其溶质二甲基亚砜(DMSO)作用48小时,实验分2组：①17G+Gen组②17G+DMSO组。应用western blotting检测CYP24A1蛋白表达。实验进行3次。2) Genistein对高糖作用下1,25(OH)2D3介导的雪旺细胞神经生长因子分泌的影响：雪旺细胞培养在含17mmol/L葡萄糖的培养液中48小时后,给予1,25(OH)2D3和genistein或DMSO作用48小时,实验分为2组：①D3+17G+Gen组②D3+17G+DMSO组。用ELISA检测神经生长因子蛋白的表达水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。结果：1) Genistein降低雪旺细胞CYP24A1蛋白表达：雪旺细胞培养在含17mmol/L葡萄糖的培养液中48小时后,给予genistein或DMSO作用48小时,凝胶电泳图用CYP24Al/β-actin灰度值分析,两独立样本T检验t=-3.030, P=0.039,17G+Gen组CYP24A1的表达明显低于17G+DMKO组。(Mean±SEM17G+Gen组：0.55±0.0417G+DMSO组：0.83±0.09N=6)。2) Genistein增加高糖培养下1,25(OH)2D3介导的雪旺细胞神经生长因子的分泌水平：雪旺细胞培养在含17mmol/L葡萄糖的培养液中48小时后,给予1,25(OH)2D3和genistein或DMSO作用48小时,两独立样本T检验t=2.599,P=0.027,D3+17G+Gen组神经生长因子的分泌明显高于对照组(Mean±SEM D3+17G+Gen组;446.53±68.61Pg/ml D3+17G+DMSO组：242.38±38.25Pg/mlN=6)。结论：1)首次发现Genistein抑制高糖上调的雪旺细胞CYP24A1蛋白的表达。2)首次发现Genistein能够恢复高糖作用下,1,25(OH)2D3介导雪旺细胞分泌神经生长因子水平的能力。第四部分慢病毒介导shRNA沉默CYP24A1基因雪旺细胞模型的构建目的：建立慢病毒介导shRNA沉默CYP24A1基因雪旺细胞模型。方法：应用慢病毒包装shRNA,沉默雪旺细胞的CYP24A1基因：根据公司合成的4个shRNA载体(CYP24A1-1,CYP24A1-2,CYP24A1-3, CYP24A1-4)及阴性(空载体),空白对照(没有处理的雪旺细胞),分为6组：①CYP24A1-1②CYP24A1-2③CYP24A1-3④CYP24A1-4⑤阴性对照(NC组)⑥空白对照(BC组)。实验分为以下几个步骤：1)构建CYP24shRNA载体2)质粒扩增与抽提3)质粒转染,利用western blot,PCR技术筛选有效载体4)慢病毒包装5)细胞模型制作6)利用western blot,PCR技术进行干扰效果检测。结果：1)筛选CYP24A1-4慢病毒载体作为转染的有效载体。2)293Ta细胞成功包装慢病毒。3)慢病毒成功感染雪旺细胞,并且沉默了CYP24A1基因和蛋白。结论：慢病毒介导shRNA沉默CYP24A1基因雪旺细胞模型建立。第五部分慢病毒介导RNA干扰沉默CYP24A1基因对高糖培养下1,25(OH)2D3介导雪旺细胞神经生长因子分泌的影响目的：通过沉默CYP24A1基因的雪旺细胞证明高糖对1,25(OH)2D。介导雪旺细胞神经生长因子分泌的影响与CYP24A1上调有关。方法：1) CYP24A1基因沉默对雪旺细胞神经生长因子分泌水平的影响：普通RSC96雪旺细胞(5.6G组)与CYP24A1基因沉默雪旺细胞(si5.6G组)均培养在含5.6mmol/L葡萄糖DMEM培养基中96小时。分为两组：①5.6G组②si5.6G组(siRNA干扰组)。用ELISA检测神经生长因子蛋白的表达水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。2) CYP24A1基因沉默对1,25(OH)2D3介导雪旺细胞神经生长因子分泌水平的影响：1,25(0H)2D3(D310-7mol/1)及其相应的乙醇溶质分别作用于含5.6mmol/L葡萄糖DMEM培养液中培养的雪旺细胞和CYP24A1基因沉默的雪旺细胞48小时。分为四组：①5.6G+D3组②5.6G+ET组③si5.6G+D3组④si5.6G+ET组。用ELISA检测神经生长因子蛋白的表达水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。3) CYP24A1基因沉默对高糖作用下1,25(OH)2D3介导的雪旺细胞神经生长因子分泌水平的影响：普通RSC96雪旺细胞(5.6G组)与CYP24A1基因沉默雪旺细胞(si5.6G组)均分别培养在含17mmol/L葡萄糖(17G)或17mmol/L甘露醇(17M)DMEM培养基48小时,再加入1,25(OH)2D3(D310-7mol/1)作用48小时,分别将实验分为四组：①17G+D3组②17M+D3组③si17G+D3组④si17M+D3组。用ELISA检测神经生长因子蛋白的表达水平。应用酶标仪检测吸光度,得出OD值,每次实验设置3个复孔,实验进行2次。结果：1)雪旺细胞CYP24A1基因沉默不影响雪旺细胞正常神经生长因子分泌：两独立样本T检验分析,两组间没有显著差异,T=0.931, P=0.374(Mean+SEM,5.6G组：162.08±4.13；si5.6G组：156.76±3.95N=6)。2)CYP24A1基因沉默增加1,25(OH)2D3介导雪旺细胞神经生长因子分泌水平：经单因素方差检验分析,F=22.971,P=0.000,si5.6G+D3组较其他三组增加。(Mean±SEM,5.6G+D3组：238.17±7.26；5.6G+ET组：201.16±5.28；si5.6G+D3组：278.83±9.5:si5.6G+ET组：204.56±7.55,N=6)。3)CYP24A1基因沉默减少了高糖对1,25(OH)。D3介导雪旺细胞神经生长因子分泌的影响：17G作用下基因沉默组比普通细胞组神经生长因子分泌明显增加,两独立样本T-检验,t=-8.807,P=O.000(Mean±SEM17G组：145.49±6.75；si117G组：238.28±7.57;N=6)；将17G作用下及其对照17M作用下,神经生长因子的分泌水平的差异做比较,CYP24A1基因沉默雪旺细胞比普通雪旺细胞差异显著减少。提示CYP24A1基因沉默后,高糖对1,25(OH)2D3介导的雪旺细胞神经生长因子分泌的影响明显降低了。两独立样本T检验分析,t=4.371p=O.001(Mean±SEM17M-17G组：92.79±5.49:si17M-17G组：33.50±12.4;N=6)。结论：1)CYP24A1基因沉默不影响雪旺细胞神经生长因子分泌。2)CYP24A1基因沉默增加1,25(OH)2D3介导雪旺细胞神经生长因子的分泌水平。3)CYP24A1基因沉默减少了高糖对1,25(OH)2D3介导雪旺细胞神经生长因子分泌的影响。证明高糖对雪旺细胞CYP24A1基因及蛋白的上调,在高糖对1,25(OH)2D3功能的影响中,确实起到了重要作用。更多还原

【Abstract】 BackgroudDiabetic Peripheral neuropathy is the most common and the most complicated one of chronic complications of diabetes.In our country, the incidence of type2diabetes were up to9.7%, and the patients with peripheral neuropathy were60-70%. The pain, numbness, and secondary to diabetic foot infection,amputation, resulted in the quality of life of the patients to decrease significantly and medical costs to increase significantly.1,25(OH)2D3is the active metabolite of Vitamin D3in vivo.Recent epidemiological and clinical studies have showed that Vitamin D3deficiency and diabetic peripheral neuropathy are closely related and inadequate Vitamin D3is independent risk factor of diabetic peripheral neuropathy. The latest USA Health and Nutrition Examination Survey (NHANES) statistics have showed that, insufficient Vitamin D3in81%adult patients with diabetes relates with diabetic neuropathy closely,even avoiding these potentially confounding factors,such as, the demographic factors,obesity, complications, drug treatment for neuropathy and diabetes duration. In a clinical study of210patients with diabetes, neuropathy group of25-hydroxyvitamin D3(25(OH) D3:in vivo detection index for vitamin D3active substances) levels were lower than those without neuropathy group,and25(OH) D3deficiency levels in the group with and without neuropathy were accounted for81.5%and60.4%, respectively. A prospective studies of painful diabetic neuropathy and vitamin D3had shown the relationship:vitamin D3deficiencies were present in51patients with painful diabetic neuropathy, the mean plasma25(OH) D3concentration was18ng/mL.Pain scores and25(OH) D3concentrations were negatively correlated. After supplement with adequate vitamin D3,the pain score decreased significantly. However, the mechanism of which remains unclear.Schwann cells play an important role in the peripheral nervous system, such as: the formation of myelin, supportion and nutrition, promoting the recovery of the axons and neurons and secreting neuro trophic factors (e.g.:Nerve Growth Factor) Nerve growth factor plays an important role in nerve regeneration in peripheral neuropathy.Nerve growth factor levels decrease to some extent resulting the onset of axonal regeneration failure and diabetic peripheral neuropathy. Under high glucose conditions, declining levels of nerve growth factor reduces the dorsal root ganglion neurons sprouting.As further studies in recent years on1,25(OH)2D3, the glial cells of the central nervous system-stellate cells’protective effect has been confirmed:1,25(OH)2D3inhibits inducible nitric oxide synthase enzyme expression and reduces the synthesis of nitric oxide and cell inflammatory response; Increases expression of nerve growth factor mRNA and protein in stellate cells, inducing neurotrophin protective effect; increases glutathione pool outside stellate cells, and significantly reduces LPS-mediated of nitrite, scavenging reactive oxygen species.But the studies on the fluence of1,25(OH)2D3on the peripheral nervous system, especially the relationship between1,25(OH)2D3and Schwann cells is very fewer.Although many studies have shown that1,25(OH)2D3mediated expression of nerve growth factor in various cell, but only one article had shown Schwann cells up-regulated gene expression of nerve growth factor, but did not mention their secretion levels of nerve growth factor.Although studies have shown that a derivative of vitamin D3dose-dependent increases sciatic nerve growth factor in diabetic rat and prevents nerve growth factor depletion. But almost no research concerned the relationship between high glucose and1,25(OH)2D3affect on Schwann cells.1,25(OH)2D3through the vitamin D receptor,binding retinoic acid x receptor or through signaling molecules, such as the ERK1pathway activates CYP24A1gene expression.25(OH) D3-24-hydroxylase (CYP24A1) can metabolize1,25(OH)2D3or25(OH) D3into inactive the1,24(OH)2D3or24(OH).Studies have shown CYP24A1increased expression in the kidney of diabetic animal model.Whether CYP24A1expression in Schwann cells exists, how the metabolic genes of1,25(OH)2D3will change in high glucose and whether that involves in the relationship between high glucose and1,25(OH)2D3and affects on NGF secretion by Schwann cells, will be the focus of our study.To investigate relationship between diabetic neuropathy and1,25(OH)2D3and whether high glucose affects on1,25(OH)2D3metabolism, we treat RSC96rat schwann cells as a model to explore influence of the high glucose and1,25(OH)2D3on nerve growth factor secreting by Schwann cells, and the interaction relationship between the two.Chapter1Effects of high glucose and1,25(OH)2D3on secretion levels of nerve growth factor by Schwann cellsObjective:To study the effect by high glucose and1,25(OH)2D3on secretion levels of nerve growth factor by RSC96Schwann cells.Methods:1)Effect of high glucose on secretion of NGF by Schwann cells:The Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for96hours.Experiments were divided into three groups:①5.6G group②17G group③17M group.Enzyme-linked immunosorbent assay (elisa) detected supernatant for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment set three wells.The experiment was performed twice.2)Effect of1,25(OH)2D3on secretion of NGF by Schwann cells:1,25(OH)2D3(D310-7mol/1) and solute ethanol treated Schwann cells respectively in5.6mmol/L glucose DMEM medium for48hours.Experiments were divided into three experimental groups:①5.6G group②D3+5.6G group③ethano1+5.6G group.Enzyme-linked immunosorbent assay (elisa) detected supernatant for levels of nerve growth factor and then detected absorbance OD values obtained from microplate reader. Each experiment set three wells.The experiment was performed twice.3)Effect of high glucose on secretion of NGF by Schwann cells treated with1,25(OH)2D3:the Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for48hours,combination with1,25(OH)2D3for additional48huours.Experiments were divided into three groups:①5.6G group②17G group③17M group.Results:1)To give5.6mmol/L glucose,17mmol/L glucose and17mmol/L mannitol DMEM medium for96hours, there was no significant difference in secretion levels of nerve growth factor by Schwann cells (P>0.05)(ANOVA F=0.447P=0.648.S-N-K pairwise comparison analysis M±SEM5.6G group:177.33±52.40pg/ml17G group:134.56±9.65pg/ml17M group:158.30±15.53pg/ml N=6).2)1,25(OH)2D3(D310-7mol/l)and solute ethanol treated Schwann cells respectively in5.6mmol/L glucose DMEM medium for48hours.1,25(OH)2D3group of nerve growth factor secretion significantly increased.(P<0.05)(ANOVA F=7.317P=0.006S-N-K pairwise comparison analysis M±SEM5.6G group:177.3±52.40pg/ml D3+5.6G group:340.29±30.91pg/ml ethanol+5.6G:161.28±17.90pg/mlN=6).3) The Schwann cells were cultured in5.6mmol/L glucose (5.6G),17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM culture medium for48hours,combination with1,25(OH)2D3for additional48huours.Levels of nerve growth factor in high glucose group (17G) was significantly lower than the other two groups.(P<0.05)(ANOVA F=15.979P=0.000SNK pairwise comparison analysis of M±SEM:D3+5.6G group340.29±30.91pg/ml D3+17G group:151.09±10.43pg/ml D3+17M group:319.05±30.87pg/ml N=6).Conclusions: 1) High glucose does not directly affect secretion of nerve growth factor by Schwann cells.2)1,25(OH)2D3increased secretion of nerve growth factor by Schwann cells.3) This is the first time study found that high glucose weakened ability of1,25(OH)2D3to stimulate Schwann cells secreting nerve growth factor. Capter2Expression of synthesis and metabolic enzyme of1,25(OH)2D3in schwann cells and effect of high glucose on these eznymeObjective: To study whether1,25(OH)2D3anabolic genes express in schwann cells, and whether high glucose affect the expression of these enzymes to look for molecular mechanisms that high glucose weakened ability of1,25(OH)2D3to stimulate nerve growth factor secretion by Schwann cells.Methods:1) Design1,25(OH)2D3anabolic gene primers comprising:synthase25(OH) D3-24-hydroxylase (CYP27Al)gene,25(OH) D3-la-hydroxylase (CYP27B1)gene; metabolic enzymes:25(OH) D3-24-hydroxylase (CYP24A1) genes.Total RNA was extracted using RNA kit Fermatas company and then reversed transcribed and PCR amplified CDNA using the company Thermo kit. CYP27A1upstream primer5’-AGCCCTCTACACAGATGCCTTAAC-3’,downstream primer5’-CCCAGTT-ATTCATGTATCGCTTCC-3’, Annealing temperature57.5℃, PCR product length314bp;CYP27B1upstream primer5’-TACGCTCTCCTGGGCACTCTATGA-3’ downstream primer5’-AAGCGTCTCCCTATGCAACTTCGTT-3’, annealing temperature57.5℃, PCR product length of408bp;CYP24A1upstream primer5’-GCAGTGGACGACCGCGAACA-3’, downstream primer’-CGATGCACATTCTC-TTCCCGATGCC-3’,annealing temperature of60℃, PCR product length of412bp; β-actin primer5’-GAACCCTAAGGCCAACCGTGAA-3’, reverse primer5’-CCGCTATTGCCGATAGTGATG-3’,annealing temperature of55℃,PCR product length of433bp. Semi-quantitative PCR method was detected whether Schwann cells expressed these enzymes. PCR conditions:95℃3minutes,95℃30s,55℃30s,72℃30s for30cycles,72℃10minutes. Running glue and analysing image.2) Different concentrations of1,25(OH)2D3cultured Schwann cells for20hours, and the aforementioned known gene expression were detected.Each gene is divided into three groups:①A groups:control group②Group B:1,25(OH)2D3(10"8mol/L)③C group:ethanol5μL④D group:1,25(OH)2D3(10-7mol/L)⑤E group: ethanol50μL. Semi-quantitative RT-PCR analysis was used. Experiments were performed three times.3)17mmol/L glucose affect Schwann cells at20hours and96hours respectively. A. The effects of high glucose on the CYP24A1gene:the expression of known genes were detected, and each gene was divided into six groups:①5.6G1group②17G1group③17M1group④5.6G4group⑤17G4group⑥17M4group.(①②③represented at20hours,④⑤⑥represented at96hours) Semi-quantitative PCR experiments carried out three times. B. Effects of high glucose on the expression of CYP24A1protein:after Semi-quantitative PCR initial had screened changes of gene expression,and further used western blotting method. Experiments were performed three times.4)0.8mmol/L hydrogen peroxide acts on Schwann cells for20hours. A. Effect of hydrogen peroxide on the CYP24A1gene:divided into two groups:①5.6G group②5.6G+H2O2group. Using Semi-quantitative RT-PCR analysis, the experiments were performed twice. B.Effects of Hydrogen Peroxide on CYP24A1protein:the change was validated further by western blotting method, and experiments carried out three times.Results:1)CYP27A1, CYP27B1, CYP24A1genes express in Schwann cells.2)1,25(OH)2D3enhanced expression of CYP24A1gene:using PCR electrop hero grams to study gene/(3-actin gray value, One way ANOVA analysis,enhanced CYP24A1gene expressed in schwann cells treated with1,25(OH) 2D3for20hours. F value:118.237P=0.000, there was a significant difference between group B (0.99±0.07N=3), D group (1.87±0.07N=3) and the other three groups (A group:0.52±0.05; C group:0.47±0.04; E group:0.46±0.05N=3)(S-N-K).3) High glucose increases CYP24A1gene and protein expression:A.17mmol/L high glucose increased CYP24A1gene expression. using PCR electropherograms to study gene/β-actin gray value. One way ANOVA analysis, high glucose increased CYP24A1gene expression after20hours and96hours, F value:15.261P=0.000(5.6G1group0.07±0.01;17G1group0.15±0.0117M1group0.06±0.02;5.6G4group0.06±0.01;17G4group0.14±0.01;17M4group0.06±0.02N=3).B. High glucose upregulated CYP24A1protein.17G group was significantly higher than the other two groups (P＜0.05). One way ANOVA F=6.706P=0.030(S-N-K pairwise comparison5.6G group:0.40±0.0517G group:0.82±0.1117M group:0.44±0.09N=3).4) Hydrogen peroxide affected gene and protein expression of CYP24A1in Schwann cells.A. Hydrogen peroxide reduced CYP24A1gene expression in Schwann cells:after0.8mmol/L H2O2had affected Schwann cells for20hours, the CYP24A1genee expression were detected in Schwann cells. Electropherograms of PCR was used to analyse CYP24Al/β-actin gray value. The results showed that:hydrogen peroxide reduced CYP24A1gene expression in Schwann cells.Two independent samples T-test (t=7.881, P=0.001)(5.6G group:0.29±0.01;5.6G+H2O2group:0.14±0.02N=3).B. Hydrogen peroxide does not affect CYP24A1protein expression in Schwann cells: after0.8mmol/L H2O2had affected Schwann cells for20hours, Schwann cells CYP24A1protein was detected by western blot. Gel electropherograms was used to analyse CYP24Al/β-actin gray value. The results showed that:hydrogen peroxide does not affect the expression of CYP24A1protein of Schwann cells. Two independent samples T-test (t=0.445, P=0.679)(5.6G group:0.40±0.05;5.6G+H2O2group:0.36±0.07N=3). Conclusions:1)This is first time study discovered that CYP27A1, CYP27B1, CYP24A1genes expressed in Schwann cells, indicating that Schwann cells is target cells of1,25(OH)2D3. Furthermore itself may have a synthesis capability of metabolizing active vitamin D3, possibly through autocrine and paracrine manner increasing local1,25(OH)2D3concentration,and thereby affecting themselves and the surrounding tissue.2)This is first time study found that high glucose upregulated genes and protein expression of CYP24A1, and upregulation of CYP24A1may interfere with catabolism function of1,25(OH)2D3in the normal of Schwann cells, leading to1,25(OH)2D3normal physiological function disrupted.3) Increasing expression of CYP24A1gene and protein in high glucose condition, may be unrelated to reactive oxygen species. Chapter3CYP24A1inhibitors affect secretion of nerve growth factor by Schwann cells cultured in high glucose medium with1,25(OH)2D3suplementationObjective:To study whether CYP24A1inhibitor affect ability of1,25(OH)2D3stimulating nerve growth factor secretion in high glucose medium in Schwann cells.Methods:1) Effect of CYP24A1inhibitor genistein on CYP24A1protein in Schwann cells cultured in high glucose medium:the Schwann cells cultured in medium containing17mmol/L glucose for48hours, then schwann cells were treated with genistein (Gen50μmol/L) or solute dimethyl sulfoxide (DMSO) for an additional48hours.The experiments were divided into two groups:①17G+Gen group②17G+DMSO group. Using western blotting to detect CYP24A1protein expression. Experiments were performed three times.2) Effect of genistein on secretion of nerve growth factor by Schwann cells treated with1,25(OH)2D3in high glucose condition:Schwann cells were cultured in the medium containing17mmol/L glucose for48hours,then schwann cells were treated with1,25(OH)2D3and genistein or DMSO for an additional48hours.The experiments were divided into2groups:①D3+17G+Gen group,②D3+17G+DMSO group. The expression level of NGF protein was detected by ELISA and then to detect absorbance OD values obtained from microplate reader.Each experiment was set three wells.The experiment was performed twiceResults:1) Genistein reduced CYP24A1protein expression in schwann cells:after schwann cells were culcured in17mmol/L Glucose culture media for48hours, then schwann cells were treated with genistein (Gen50μmol/1) or solute dimethyl sulfoxide (DMSO) for anadditional48hours, gel electrophoresis analysis with CYP24Al/β-actin gray value (t=-3.030, P=0.0.039)(Independent samples T-test Mean±SEM17G+Gen group:0.55±0.0417G+DMSO group:0.83±0.09N=6).2) Genistein group (D3+17G+Gen) nerve growth factor secretion induced by1,25(OH)2D3increased significantly. Schwann cells cultured in the medium containing17mmol/L glucose for48hours,then schwann cells were treated with1,25(OH)2D3, and genistein or DMSO for an additional48hours (T=-2.99, P=0.027). Independent samples T-test(Mean±SEM D3+17G+Gen group;446.53±68.61pg/ml D3+17G+DMSO group:242.38±38.25pg/ml N=6).Conclusions:1)This is first time study found that genistein inhibited the expression of upregulated CYP24A1protein by high glucose in Schwann cells.2)For the first time,the study found that genistein could recover NGF secretion by high glucose-cultured SCs stimulated with1,25(OH)2D3. Capter4To construct lentivirus-mediated shRNA silencing CYP24A1gene Schwann cell model Objective:To construct lentivirus-mediated shRNA silencing CYP24A1gene Schwann cell modelMethods:Using lentiviral packaging shRNA,silence CYP24A1gene of Schwann cells: Synthesis of four shRNA vectors by company (CYP24A1-1,CYP24A1-2, CYP24A1-3,CYP24A1-4) and negative (empty vector),blank (no treatment Schwann cells) were divided to6groups:①CYP24A1-1②CYP24A1-2③CYP24A1-3④CYP24A1-4⑤negative control (NC group)⑥blank control (BC group). Step:1)To construct CYP24shRNA carrier2)To amplify and extract plasmid3)To transfect plasmid and screen effective carrier (western blot, PCR)4)To package Viral5)To construct cell model6)To detect interference effects (western blot, PCR).Results:1)CYP24Al-4,a transfected lentivirus vector,was screened for effective carrier.2)293Ta cells packaged with lentiviral were successful.3)Lentivirus infected Schwann cells and silenced the CYP24A1gene and protein were successfulConclusion:Lentivirus-mediated shRNA silencing Schwann cells CYP24A1gene model had construced successful Capter5RNA interference mediated by lentivirus silencing CYP24A1gene affected secretion levels of NGF stimulated by1,25(OH)2D3in Schwann cell Objective:To confirm that effect of high glucose on1,25(OH)2D3stimulating NGF secretion in Schwann cells is related to CYP24A1gene upregulation through CYP24A1gene silencing Schwann cells.Methods:The experiments were divided into groups:1)To research wether silencing CYP24A1gene will affect NGF secretion by Schwann cells:ordinary RSC96Schwann cells (5.6G group) and CYP24A1gene silencing Schwann cells (si5.6G group) were cultured in5.6mmol/L glucose DMEM medium for96hours, and then the supernatant was detected for NGF protein levels. The experiments were divided into two groups:①5.6G group②si5.6G group (siRNA interference group).2)To study whether silencing CYP24A1gene will affect NGF secretion by Schwann cells treated with1,25(OH)2D3:1,25(OH)2D3(D310-7mol/L) and the ethanol treated Schwann cells and CYP24A1gene silencing Schwann cells cultured in5.6mmol/L glucose DMEM medium for48hours,and then the supernatants were detected for NGF protein levels. The experiments were divided into four groups:①5.6G+D3group②5.6G+ET group③si5.6G+D3group④si5.6G+ET groups. Enzyme-linked immunosorbent assay (elisa) detected supernatants for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment seted three wells.The experiment was performed twice.3) To study whether the silencing CYP24A1gene affected NGF secretion by Schwann cells cultured in high glucose with1,25(OH)2D3suplementation:Ordinary Schwann cells (5.6G group) and silencing CYP24A1gene of Schwann cells (si5.6G group) were cultured in17mmol/L glucose (17G) and17mmol/L mannitol (17M) DMEM medium for48hours and then added1,25(OH)2D3(D310-7mol/L) and the corresponding solute ethanol (ET) for an additional48hours, respectively. Experiments were divided into four groups:①17G+D3group②17M+D3group③si17G+D3group④si17+D3group.Enzyme-linked immunosorbent assay (elisa) detected supernatants for levels of nerve growth factor and then detected absorbance OD values obtained from microplate readerfor. Each experiment seted three wells.The experiment was performed twice.Results:1) Silencing CYP24A1gene Schwann cells did not affect the normal secretion of NGF by Schwann cells:using analysis of two independent samples Ttest, there was no significant difference between the two groups.T=0.931, P=0.374(Mean±SEM, group5.6G:162.08±4.13; si5.6G group:156.76±3.95N=6).2) CYP24A1gene silencing increased secretion levels of NGF mediated by1,25(OH)2D3in Schwann cells:using One-Way ANOVA test, si5.6G+D3group increased significantly compared with the other three groups.(P=0.000)(Mean±SEM,5.6G+D3group:238.17±7.26;5.6G+ET group:201.16±5.28; si5.6G+D3group:278.82±9.5; si5.6G+ET group:204.56±7.55, N=6).3) Silencing CYP24A1gene reduces the effect of high glucose on the NGF secretion mediated by1,25(OH)2D3in Schwann cells:using analysis of Independent samples T-test,NGF level in si17G schwann cells increase significantly compared with17G schwann cells, t=-8.807P=0.000(17G group:145.49±6.75; si17G group:238.28±7.57; N=6); comparing differences between17G and17M in the secretion level of NGF, the differences had a significant reduction in silencing gene of Schwann cells compared with in ordinary Schwann cells., t=4.371p=0.001(17M-17G group:92.79±5.49; si17M-17G group:33.50±12.4; N=6).Conclusions:1) Silencing CYP24A1gene does not affect secretion of NGF by Schwann cells.2) Silencing CYP24A1gene increases secretion of NGF mediated by1,25(OH)2D3in Schwann cells.3) Silencing CYP24A1gene reduces the effect of high glucose on the NGF secretion mediated by1,25(OH)2D3in the Schwann cells.更多还原