维甲酸相关基因HA117抗神经元分化的初步研究

发布时间：2018-06-12 02:45

本文选题：HA117 + ATRA　；参考：《重庆医科大学》2016年硕士论文

【摘要】：第一部分HA117对人脐带间充质干细胞(hUMSCS)向神经元样细胞分化过程的影响目的:HA117是ATRA小剂量、多次刺激HL-60细胞株后通过抑制消减杂交及基因芯片等技术得到的一个多药耐药基因。前期课题组通过实验研究及生物信息学分析,推测HA117可能是一个抗分化相关基因。因此,本研究目的是观察HA117对hUMSCS向神经元样细胞分化过程的影响。方法:获取3代hUMSCS,流式检测细胞表面特异性抗原,通过成骨、成脂定向分化检测hUMSCS多向分化能力。然后,转导HA117过表达慢病毒建立HA117过表达hUMSCS,流式测定转染率,神经元分化液培养诱导HA117过表达hUMSCS向神经元样细胞方向分化,并设置空白对照组、阳性对照组及空载对照组,通过观察各组分化时间,神经元样细胞形态及Western、q-PCR、免疫荧光技术检测各组定向诱导分化后神经元标志物的表达水平来评价各组神经元样细胞分化率。结果:HA117过表达慢病毒转导hUMSCS的转染率为68.9%,hUMSCS加入神经元诱导液诱导后,细胞体积变大,未发现明显树突及轴突,神经元标志物β-tubulin III、NSE和Nestin的mRNA及蛋白在空白对照组中表达较少,甚至不表达,其表达水平在HA117组明显低于阳性对照组及空载对照组,P0.05,免疫荧光观察到HA117组荧光明显弱于阳性对照组及空载对照组,强于空白对照组。结论:HA117在hUMSCS向神经元定向分化过程中起到抗分化作用。第二部分HA117及SHH通路在先天性巨结肠中的表达及意义目的:研究HA117及SHH信号通路(SHH、Ptch1、Gli1)在先天性巨结肠(Hirschsprung's disease,HSCR)中的表达情况,以探讨其与HD发生发展中的关系。方法:收集重庆医科大学附属儿童医院2014-1015年经手术确诊为HSCR患者的巨结肠组织标本30例,肠段分吻合段(正常对照组)、扩张段和痉挛段(实验组),运用实时荧光定量PCR(quantitative real—time PCR,q RT-PCR)、蛋白质印迹(Western blot)和免疫组织化学方法检测HA117、SHH信号途径在各段中的表达情况,对其进行定量和定性并比较。结果:Q-PCR结果提示:痉挛段HA117 m RNA表达水平为1.52±0.17,明显高于吻合段(0.28±0.04),差异具有统计学意义(P0.05)。痉挛段SHH、Ptch1、Gli1 m RNA表达水平分别为0.53±0.11、0.48±0.11、0.62±0.13,明显低于吻合段(1.23±0.26、1.20±0.22、1.71±0.46),差异具有统计学意义(P0.05);Western-Blot结果提示:SHH、Ptch1、Gli1蛋白相对表达水平与m RNA表达水平一致,差异均具有统计学意义(P0.05);免疫组织化学(DAB染色)结果提示:痉挛段SHH、Ptch1、Gli1染色明显弱于吻合段。结论:HA117及SHH信号通路可能与肠神经系统(ENS)发育有着密切的联系,这可能是HSCR病理发生的重要作用机制之一。
[Abstract]:Part I effects of HA117 on differentiation of human umbilical cord mesenchymal stem cells into neuron-like cells objective: HA117 is a multidrug resistant gene obtained by suppression subtractive hybridization and gene chip technique after stimulation of HL-60 cell line with low dose ATRA. Through experimental study and bioinformatics analysis, we speculated that HA117 might be an anti differentiation related gene. Therefore, the aim of this study was to observe the effect of HA117 on the differentiation of hUMSCS into neuron-like cells. Methods: three generations of hUMSCSs were obtained and the cell surface specific antigens were detected by flow cytometry. The multidirectional differentiation ability of hUMSCS was detected by osteogenesis and lipopolysaccharide differentiation. Then, HA117 overexpression lentivirus was transduced to establish HA117 overexpression hUMSCSs, transfection rate was measured, neuronal differentiation fluid culture induced HA117 overexpression hUMSCS to differentiate into neuron-like cells, and set up blank control group, positive control group and no-load control group. The differentiation rate of neuron-like cells in each group was evaluated by observing the time of differentiation, morphology of neuron-like cells and the expression level of neuronal markers after induction by immunofluorescence technique. Results the transfection rate of the lentivirus transduction hUMSCS was 68.9%. After the cells were induced with neuronal inducer fluid, the cell volume became larger and no obvious dendrites and axons were found. The mRNA and protein of 尾 -tubulin II I NSE and nestin were less expressed in the control group. The expression level in HA117 group was significantly lower than that in the positive control group and the no-load control group (P0.05). The fluorescence of HA117 group was significantly weaker than that of the positive control group and the no-load control group, and was stronger than that of the blank control group. Conclusion: Hal 117 plays an anti-differentiation role in the process of neuronal directional differentiation of hUMSCS. Part two: expression and significance of HA117 and SHH pathway in Hirschsprungs disease of Hirschsprungs disease in Hirschsprungs disease of Hirschsprungs Methods: thirty histopathological specimens of Hirschsprung's disease (HSCR) from 2014 to 1015 in Children's Hospital affiliated to Chongqing Medical University were collected. Intestinal segments were divided into anastomosed segments (normal control group, dilated segment and spastic segment (experimental group, real-time quantitative real-time PCRQ RT-PCRR, Western blot) and immunohistochemical method to detect the expression of HA117-SHH signal pathway in each segment. Quantitative and qualitative analysis and comparison were carried out. Results the expression level of HA117 m RNA in spastic segment was 1.52 卤0.17, which was significantly higher than that in anastomotic segment 0.28 卤0.04, and the difference was statistically significant (P 0.05). The expression level of Gli1 mRNA in spastic segment was 0.53 卤0.110.48 卤0.110.62 卤0.13, which was significantly lower than that in anastomosed segment (1.23 卤0.261.20 卤0.221.20 卤0.221.71 卤0.46). The difference was statistically significant. The results of Western-Blot showed that the relative expression level of Gli1 protein was the same as the level of mRNA expression. The differences were statistically significant (P 0.05) and immunohistochemical staining (DAB). The results showed that the staining of SHHtch1 Gli1 in spastic segment was significantly weaker than that in anastomotic segment. Conclusion the development of the intestinal nervous system (ENS) may be closely related to the signal pathway of 1: HA117 and SHH, which may be one of the important mechanisms in the pathogenesis of HSCR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R714.5

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