丁苯酞对K141N突变型HSPB8蛋白致线粒体异常的保护作用

发布时间：2018-04-25 07:38

  本文选题：丁苯酞 + 热休克蛋白B8　； 参考：《浙江大学》2014年硕士论文

【摘要】：研究背景： 腓骨肌萎缩症(Charcot-Marie-Tooth Disease,CMT)又称遗传性运动感觉性神经病(hereditary motor and sensory neuropathy,HMSN)是一类最常见的遗传性周围神经病。临床上依据其病理和电生理特点可分为三型：脱髓鞘型(CMT1型)、轴突型(CMT2型)和中间型(Intermediate CMT,ICMT)。我们通过定位候选基因克隆策略在致病基因定位区间发现了小分子热休克蛋白B8(HSPB8)基因的点突变(423G→T)导致氨基酸K141N替换,并最终通过共分离分析等研究表明其为CMT2L致病突变。在前期的研究中,我们发现K141N突变型HSPB8蛋白在人神经母细胞瘤细胞(SH-SY5Y)中引起线粒体的聚集,同时也有研究发现病人皮肤成纤维细胞中存在线粒体膜电位的异常。丁苯酞(dl-3-n-butylphthalide,NBP)与芹菜籽中提取的左旋芹菜甲素的结构相同,为其人工合成的消旋体。已有研究证实NBP可以改善脑缺血区微循环、保护线粒体功能、增加神经细胞线粒体ATP酶活性以及减轻神经功能损伤程度。所以我们推测丁苯酞可能会对K141N突变型HSPB8蛋白造成的线粒体异常有一定的保护作用。 研究目的： 进一步研究K141N突变型HSPB8对线粒体的影响,同时使用NBP进行干预,探讨NBP对K141N突变型HSPB8致线粒体异常的保护作用。 研究方法： (1)采用透射电镜观察携带K141N突变型HSPB8基因的CMT2L病人腓肠神经内线粒体分布情况。 (2)应用CCK-8检测不同浓度NBP对K141N突变型HSPB8蛋白诱导的SH-SY5Y细胞活力降低的保护作用。 (3)通过免疫荧光技术分别观察给予10μmol/L NBP处理前后,感染了野生型和K141N突变型HSPB8蛋白的脊髓运动神经元突起数目。 (4)采用透射电镜分别观察给予10μmol/L NBP处理前后,高表达野生型和K141N突变型HSPB8蛋白的SH-SY5Y细胞内线粒体分布情况。 (5)采用流式细胞仪分别测量给予10μmol/L NBP处理前后,高表达野生型和K141N突变型HSPB8蛋白的SH-SY5Y细胞,在受到H2O2损伤性刺激后线粒体活性氧自由基(ROS)产生的情况。 结果： (1)携带K141N突变型HSPB8基因的CMT2L病人腓肠神经内存在线粒体异常聚集。 (2) HSPB8K141N突变后导致细胞活力降低,给予不同浓度(1、10、100μmol/L)NBP处理后细胞活力增高,当NBP浓度为10μmol/L时细胞活力明显增高,给药前后存在显著差异(P0.001)。 (3)高表达K141N突变型HSPB8蛋白会减少脊髓运动神经元突起数目,给予10μmol/LNBP预处理,高表达K141N突变型HSPB8蛋白组神经元突起数目增多,给药前后存在显著性差异(P0.01)。 (4)高表达K141N突变型HSPB8蛋白的SH-SY5Y细胞存在线粒体异常聚集,给予10μmol/LNBP预处理线粒体异常聚集情况有所改善。 (5)高表达K141N突变型HSPB8蛋白导致线粒体抗氧化损伤的能力降低,ROS生成量高于高表达野生型HSPB8蛋白的细胞,两者存在显著性差异(P0.05)；给予10μmol/LNBP预处理,高表达K141N突变型HSPB8蛋白组细胞ROS生成量降低,给药前后存在显著性差异(P0.05)。 结论： NBP对K141N突变型HSPB8蛋白诱导的细胞活力降低有明显的保护作用,同时可以促进脊髓运动神经元的发育。从线粒体角度这种保护作用可能是通过抑制线粒体聚集、提高线粒体抗氧化应激的能力拮抗K141N突变型HSPB8致线粒体异常。
[Abstract]:Research background:
Charcot-Marie-Tooth Disease (CMT) (CMT), also known as hereditary motor sensory neuropathy (hereditary motor and sensory neuropathy, HMSN), is the most common type of hereditary peripheral neuropathy. It can be divided into three types according to its pathological and electrophysiological characteristics: demyelination (CMT1), axon type (CMT2 type) and intermediate type (Inte). Rmediate CMT, ICMT). We found that the point mutation of the small molecular heat shock protein B8 (HSPB8) gene (423G to T) resulted in the K141N substitution of amino acids by the location of the candidate gene cloning strategy in the location interval of the pathogenic gene, and finally through the co separation analysis, we found that it was a CMT2L pathogenic mutation. In the previous study, we found the K141N mutation. Type HSPB8 protein causes mitochondrial aggregation in human neuroblastoma cells (SH-SY5Y), and there is also a study of the abnormal mitochondrial membrane potential in the skin fibroblasts of the patient. Dl-3-n-butylphthalide (NBP) is the same as a synthetic raceme, which is the same as the structure of celery seed extracted from celery seed. Some studies have confirmed that NBP can improve the microcirculation of cerebral ischemia area, protect mitochondrial function, increase the activity of mitochondrial ATP enzyme in neural cells and reduce the degree of nerve function damage. Therefore, we speculate that phthalide may have some protective effect on the mitochondrial abnormalities caused by the K141N mutant HSPB8 protein.
The purpose of the study is:
We further studied the effect of K141N mutant HSPB8 on mitochondria, and NBP intervention to explore the protective effect of NBP on mitochondrial abnormalities induced by K141N mutant HSPB8.
Research methods:
(1) transmission electron microscopy was used to observe the distribution of mitochondria in the sural nerve of CMT2L patients carrying K141N mutant HSPB8 gene.
(2) CCK-8 was used to detect the protective effect of different concentrations of NBP on the activity of SH-SY5Y cells induced by K141N mutant HSPB8 protein.
(3) the number of protuberances of the spinal motor neurons infected with the wild type and the K141N mutant HSPB8 protein was observed by immunofluorescence technique before and after the treatment of 10 mol/L NBP.
(4) the distribution of mitochondria in SH-SY5Y cells with high expression of wild type and K141N mutant HSPB8 protein was observed by transmission electron microscopy before and after 10 mol/L NBP treatment.
(5) the flow cytometry was used to measure the SH-SY5Y cells with high expression of wild type and K141N mutant HSPB8 protein before and after 10 mol/L NBP treatment, and the mitochondrial reactive oxygen free radical (ROS) was produced after H2O2 damage stimulation.
Result锛，

本文编号：1800427