转录因子POU3F3在高糖处理雪旺细胞中的作用及其对DLL1表达的调控

发布时间：2018-03-20 22:07

  本文选题：雪旺细胞高葡萄糖　切入点：S-100细胞培养　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：第一部分雪旺细胞培养方法建立及高糖的作用 目的：建立原代雪旺细胞培养模型,并观察高糖状态下雪旺细胞的生长规律,测量雪旺细胞面积,突起长度和细胞增殖活力。为高糖环境对雪旺细胞影响分子机制研究提供可用的模型。 方法：取新生1-3天的SPF级SD大鼠10只,断头处死后无菌切取双侧坐骨神经,剥离神经外膜,取神经剪碎,用低浓度双酶消化法消化神经制成细胞悬液,接种于6孔培养板。2小时差速贴壁后取上清转入另一培养板,加阿糖胞苷抑制成纤维细胞生长,传代培养,第三代后将细胞分为两组,一组应用5.5mmol/L的葡萄糖,另一组应用25mmol/L的葡萄糖分别进行培养。观察两组雪旺细胞的细胞突起,细胞面积及增殖能力。用雪旺细胞标记物S-100抗体鉴定细胞。 结果：1、应用差速贴壁法、酶消化法和化学法可以得到高纯度的雪旺细胞。建立了雪旺细胞原代培养模型。 2、分析不同糖浓度对雪旺细胞胞生长的影响,结果显示,与低糖培养组相比,在高糖培养条件下原代的雪旺细胞胞体面积减小,突起长度变长,增殖活性变大。 结论：1、应用双酶消化,差速贴壁及阿糖胞苷法可以得到纯度比较高的雪旺细胞。 2、雪旺细胞在高糖培养环境下,细胞突起变长,细胞面积减小,增殖活性变大。说明高糖明显影响体外雪旺细胞生长。本细胞模型可用于高糖环境对雪旺细胞影响的分子机制研究。 第二部分高糖对培养雪旺细胞POU3F3基因的影响及其对DLL1分子的调控 目的：分析高糖对培养雪旺细胞POU3F3基因的影响,观察其在高糖作用的SC中的表达变化规律,探讨POU3F3对DLL1的调控及其对细胞的影响。 方法：高糖条件培养SC48小时,获取高糖组(25mmol/L葡萄糖)和对照组(5.5mmol/L葡萄糖)细胞,利用RT-PCR技术扩增各组BDNF, CNTF, EGF, GMF, IGF-1, EGR-2, MMP9, NF-KB,NGF,NRG-1, POU3F3, SLC2al,SOX-10,TrkA,TrkB基因。挑出其中有统计学差异的基因进行功能研究。本实验确定POU3F3为目的基因,化学合成该基因siRNA干扰片段,用脂质体转染法将POU3F3siRNA干扰片段导入雪旺细胞株RSC96,观察干扰前、后RSC96细胞株的细胞突起,大小。 结果： 1、RT-PCR结果显示在被检测的15个基因中,与对照组(5.5mmol/L葡萄糖)相比,仅POU3F3基因在高糖(25mmol/L葡萄糖)环境下培养48h时,其mRNA表达明显升高,P0.05,差异有统计学意义,其余因子均未见明显变化,差异无统计学意义,P0.05；所有因子高糖(25mmol/L葡萄糖)培养96小时后mRNA表达与对照组(5.5mmol/L葡萄糖)相比P0.05,差异无统计学意义。 2、利用生物信息学查询发现POU3F3与DLL1密切相关,查询资料发现POU3F3可能激活DLL1转录。 3、利用RT-PCR扩增DLL1基因,观察其mRNA的表达,结果显示SC在高糖条件下培养48小时,与对照组(5.5mmol/L葡萄糖)相比,DLL1mRNA表达增加3.4倍,P0.05,差异有统计学意义；培养96小时表达增加1.5倍,P0.05,差异无统计学意义。 4、化学合成POU3F3siRNA干扰片段,利用脂质体转染的方式将片段导入雪旺细胞株RSC96,结果显示,与阴性对照组相比,将POU3F3基因干扰24小时对细胞突起长度,细胞面积均无影响,P0.05；干扰48小时后,细胞突起长度无统计学差异,P0.05,而干扰组细胞面积明显减小,P0.05。阴性对照组及只加了转染试剂组细胞面积比正常组面积减小,P0.05。 5、POU3F3与DLL1表达正相关,相关系数r=0.999,P0.05。 结论： 1、高糖培养SC,检测了15个因子的mRNA,结果显示仅POU3F3mRNA升高。提示该基因在高糖状态下的雪旺细胞中起着十分重要的作用。 2、化学合成POU3F3的siRNA干扰片段,用脂质体转染的方式将片段导入雪旺细胞株RSC96后发现细胞突起长度无变化,而细胞面积减小。表明POU3F3可能因影响雪旺细胞的体积而影响雪旺细胞形成髓鞘。 3、POU3F3可以调控DLL1的表达。在高糖环境下,POU3F3的升高导致DLL1伴随升高。
[Abstract]:The first part of the culture of Schwann cells and the effect of high sugar
Objective: to establish a primary Schwann cell culture model, observe the growth rule of Schwann cells in high glucose state, measure the area of Schwann cells, the length of neurite and cell proliferation activity, and provide an available model for studying the molecular mechanism of Schwann cell influence in high glucose environment.
Methods: the newborn 1-3 days in SPF SD 10 rats were decapitated after aseptic cut bilateral sciatic nerve, nerve membrane peeling, the nerve was cut, with a low concentration of double enzyme digestion of nerve cell suspension were seeded in 6 well plates.2 hours after differential adhesion to the supernatant in another culture plate, ara2c inhibited fibroblast growth, subculture, after third generations of cells were divided into two groups, one group with 5.5mmol/L glucose, another group using 25mmol/L glucose were cultured. Cell protrusions Schwann cells were observed in the two groups, the area and the cell proliferation ability. With antibody identification cells of Schwann cell marker S-100.
Results: 1, high purity Schwann cells could be obtained by differential adherence method, enzyme digestion and chemical method. The primary culture model of Schwann cells was established.
2, the effects of different sugar concentrations on the growth of Schwann cells were analyzed. The results showed that compared with the low sugar culture group, the cell area of primary Schwann cells decreased, the length of neurite grew longer and the proliferative activity increased.
Conclusion: 1, the high purity Schwann cells can be obtained by double enzyme digestion, differential adherence and cytarabine method.
2, Schwann cells in the high glucose environment, the cell protuberance increased, the cell area decreased, and the proliferative activity increased. This indicates that high glucose significantly affects the growth of Schwann cells in vitro. This cell model can be used to study the molecular mechanism of Schwann cells in high glucose environment.
The effect of high sugar on the culture of POU3F3 gene of Schwann cells in the second part and its regulation on DLL1
Objective: to analyze the effect of high glucose on the expression of POU3F3 gene in Schwann cells, observe its expression and change rule in high glucose SC, and explore the regulation of POU3F3 on DLL1 and its effect on cells.
Methods: high glucose cultured SC48 hours, to obtain the high glucose group (25mmol/L glucose) and control group (5.5mmol/L glucose) cells, BDNF was amplified by RT-PCR technology, CNTF, EGF, GMF, IGF-1, EGR-2, MMP9, NF-KB, NGF, NRG-1, POU3F3, SLC2al, SOX-10, TrkA, TrkB gene. Pick one function study on difference gene. POU3F3 gene was identified in this experiment, the chemical synthesis of the gene siRNA interference fragment by liposome transfection POU3F3siRNA interference fragment into Schwann cell line RSC96, observe the interference, cell processes, RSC96 cell line size.
Result锛，

本文编号：1640993