本文選題：丁苯酞 + 熱休克蛋白B8　； 參考：《浙江大學》2014年碩士論文

【摘要】：研究背景： 腓骨肌萎縮癥(Charcot-Marie-Tooth Disease,CMT)又稱遺傳性運動感覺性神經(jīng)病(hereditary motor and sensory neuropathy,HMSN)是一類最常見的遺傳性周圍神經(jīng)病。臨床上依據(jù)其病理和電生理特點可分為三型：脫髓鞘型(CMT1型)、軸突型(CMT2型)和中間型(Intermediate CMT,ICMT)。我們通過定位候選基因克隆策略在致病基因定位區(qū)間發(fā)現(xiàn)了小分子熱休克蛋白B8(HSPB8)基因的點突變(423G→T)導(dǎo)致氨基酸K141N替換,并最終通過共分離分析等研究表明其為CMT2L致病突變。在前期的研究中,我們發(fā)現(xiàn)K141N突變型HSPB8蛋白在人神經(jīng)母細胞瘤細胞(SH-SY5Y)中引起線粒體的聚集,同時也有研究發(fā)現(xiàn)病人皮膚成纖維細胞中存在線粒體膜電位的異常。丁苯酞(dl-3-n-butylphthalide,NBP)與芹菜籽中提取的左旋芹菜甲素的結(jié)構(gòu)相同,為其人工合成的消旋體。已有研究證實NBP可以改善腦缺血區(qū)微循環(huán)、保護線粒體功能、增加神經(jīng)細胞線粒體ATP酶活性以及減輕神經(jīng)功能損傷程度。所以我們推測丁苯酞可能會對K141N突變型HSPB8蛋白造成的線粒體異常有一定的保護作用。 研究目的： 進一步研究K141N突變型HSPB8對線粒體的影響,同時使用NBP進行干預(yù),探討NBP對K141N突變型HSPB8致線粒體異常的保護作用。 研究方法： (1)采用透射電鏡觀察攜帶K141N突變型HSPB8基因的CMT2L病人腓腸神經(jīng)內(nèi)線粒體分布情況。 (2)應(yīng)用CCK-8檢測不同濃度NBP對K141N突變型HSPB8蛋白誘導(dǎo)的SH-SY5Y細胞活力降低的保護作用。 (3)通過免疫熒光技術(shù)分別觀察給予10μmol/L NBP處理前后,感染了野生型和K141N突變型HSPB8蛋白的脊髓運動神經(jīng)元突起數(shù)目。 (4)采用透射電鏡分別觀察給予10μmol/L NBP處理前后,高表達野生型和K141N突變型HSPB8蛋白的SH-SY5Y細胞內(nèi)線粒體分布情況。 (5)采用流式細胞儀分別測量給予10μmol/L NBP處理前后,高表達野生型和K141N突變型HSPB8蛋白的SH-SY5Y細胞,在受到H2O2損傷性刺激后線粒體活性氧自由基(ROS)產(chǎn)生的情況。 結(jié)果： (1)攜帶K141N突變型HSPB8基因的CMT2L病人腓腸神經(jīng)內(nèi)存在線粒體異常聚集。 (2) HSPB8K141N突變后導(dǎo)致細胞活力降低,給予不同濃度(1、10、100μmol/L)NBP處理后細胞活力增高,當NBP濃度為10μmol/L時細胞活力明顯增高,給藥前后存在顯著差異(P0.001)。 (3)高表達K141N突變型HSPB8蛋白會減少脊髓運動神經(jīng)元突起數(shù)目,給予10μmol/LNBP預(yù)處理,高表達K141N突變型HSPB8蛋白組神經(jīng)元突起數(shù)目增多,給藥前后存在顯著性差異(P0.01)。 (4)高表達K141N突變型HSPB8蛋白的SH-SY5Y細胞存在線粒體異常聚集,給予10μmol/LNBP預(yù)處理線粒體異常聚集情況有所改善。 (5)高表達K141N突變型HSPB8蛋白導(dǎo)致線粒體抗氧化損傷的能力降低,ROS生成量高于高表達野生型HSPB8蛋白的細胞,兩者存在顯著性差異(P0.05)；給予10μmol/LNBP預(yù)處理,高表達K141N突變型HSPB8蛋白組細胞ROS生成量降低,給藥前后存在顯著性差異(P0.05)。 結(jié)論： NBP對K141N突變型HSPB8蛋白誘導(dǎo)的細胞活力降低有明顯的保護作用,同時可以促進脊髓運動神經(jīng)元的發(fā)育。從線粒體角度這種保護作用可能是通過抑制線粒體聚集、提高線粒體抗氧化應(yīng)激的能力拮抗K141N突變型HSPB8致線粒體異常。
[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錛，

本文編號：1800427