【作者】 李曼；

【导师】 高海青；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 研究背景胆固醇作为体内最丰富的固醇类化合物广泛存在于哺乳动物体内的细胞膜和髓鞘,同时也是几种重要的类固醇激素（肾上腺皮质激素、性腺激素等）、胆酸、神经甾体的前体,因此保证胆固醇的供给,维持其代谢平衡是十分重要的。胆固醇广泛存在于全身各组织中,其中约1/4分布在脑及神经组织中,占脑组织总重量的2%左右,肝、肾及肠等内脏以及皮肤、脂肪组织亦含较多的胆固醇。体内胆固醇的来源主要有两个:一是外源性的食物供给约占30%;另一部分是内源性自身合成,约占总胆固醇的70%。几乎全身各器官均可合成胆固醇,肝脏是主要合成器官,体内合成的胆固醇通过载脂蛋白运输到全身各器官。由于胆固醇不能通过血脑屏障,因此血脑屏障形成后中枢神经系统胆固醇主要来源于原位合成。众所周知,高胆固醇血症是动脉粥样硬化等心脑血管疾病主要危险因子,但几种遗传性胆固醇合成缺陷性疾病的发现表明低胆固醇同样危害人类健康及胚胎发育等。目前为止共发现9种遗传性胆固醇合成缺陷性疾病,其中2种与胆固醇合成途径鲨烯形成前的酶缺陷有关:classical mevalonic aciduria,hyper-IgD and periodic fever syndrome;7种与鲨烯形成后的酶有关,它们分别是:史-伦-奥三氏综合征（Smith-Lemli-Opitz syndrome,SLOS）,desmosterolosis,HEM发育异常（Hydrops-ectopic calcification-moth-eatenskeletal dysplasia）,Lathosterolosis,Antley-Bixler综合征,chondrodysplasia punctata 2（CDPX2）,伴有鱼鳞样痣和肢体缺陷的先天性半身发育异常（congenital hemidysplasia ichthyosiform nevous and limbdefects,CHILD）综合征。遗传性胆固醇合成缺陷性疾病通常表现为出生时多发畸形及内脏器官、骨骼和/或皮肤发育异常。其中史-伦-奥三氏综合征（SLOS）是目前最常见的遗传性胆固醇合成缺陷性疾病。史-伦-奥三氏综合征（SLOS）是一种常染色体隐性遗传性疾病,主要是胆固醇生物合成通路终反应的酶—3β-脱氢胆固醇-△7还原酶（3β-hydroxysterol-△7 reductase,DHCR7）基因突变所导致的酶缺陷,导致胆固醇水平明显降低而前体物质7-脱氢胆固醇（7-dehydrocholesterol,7DHC）大量积聚。SLOS的新生儿临床发病率约为1/10000-1/60000,人群筛查显示白色人种中大约有3%-4%的DHCR7突变基因携带者。因此,根据突变基因携带者频率推测SLOS临床发病率应约为1/2500-1/4500,两者差异提示有许多SLOS病例未获临床确诊。可能许多严重罹患胎儿在胚胎期自然流产或新生儿死亡病例未进行确诊检验。另外,无明显临床表现或仅有轻度智力障碍的轻型病例亦难以获得临床确诊。SLOS临床表现通常有不同程度的发育畸形如小头畸形、腭裂、四肢发育异常,智力发育迟缓,张力减退,喂养困难等,根据症状的轻重分为Ⅰ型（轻型）和Ⅱ型（重型）,Ⅰ型病人可无明显特异性临床表现,Ⅱ型患者常有多种畸形及器官功能衰竭,多于新生儿期因心肺功能障碍死亡。SLOS的诊断主要根据临床症状、血浆7DHC增加和胆固醇降低以及突变基因遗传分析。目前尚无有效的治疗方法,多为对症治疗如先心病、腭裂、内脏和四肢缺陷的外科修复。迄今为止共发现121个DHCR7基因突变,几乎遍布整个基因编码区,包括105个错义突变,5个无意突变,3个剪切位点突变和8个核苷酸的插入或缺失,50%的错义突变位于9个跨膜单位。基于对DHCR7基因结构和功能的分析,为进一步探究SLOS的病理生理和神经生理机制并为干预治疗实验提供模型,从基因水平上建立了两种Dhcr7基因敲除(Dhcr7^-/-)的小鼠模型。两种模型中Dhcr7^-/-小鼠都伴有明显的固醇代谢紊乱和无吸吮、呼吸困难,肺脏发育不全、轻微的中枢神经系统结构异常等,通常于出生后24h内死亡,同Ⅱ型SLOS患者类似。其中肺发育不全及神经功能障碍可能是Dhcr7^-/-小鼠死亡的主要病因。目前对遗传性胆固醇合成缺陷性疾病中胆固醇不足对胚胎发育的病理生理机制了解甚少,多数研究者认为与形态发生素Sonic Hedgehog（shh）有关。Shh是胚胎发育过程中调节细胞间相互作用的重要信号分子,其自身激活需要共价结合胆固醇。然而7DHC在正常情况下可以代替胆固醇在shh自身激活中的作用并能维持其正常功能,提示Dhcr7^-/-小鼠体内聚集的7DHC可替代胆固醇参与shh活化及信号途径。目前虽不能完全排除shh信号途径异常与SLOS发病有关,但其至少可能并非导致SLOS多发畸形及各种功能障碍的主要病理生理机制。转基因技术可以恢复组织DHCR7的表达及胆固醇合成,从而有希望成为今后治疗SLOS及其它遗传性胆固醇合成缺陷性疾病的重要方法。转基因选择性重塑Dhcr7^-/-小鼠不同组织DHCR7的功能,观察转基因前后的病理生理变化,可进一步了解胆固醇的生理功能并为器官特异性转基因治疗提供重要依据。本实验室已成功利用转基因技术部分重塑胆固醇含量最多的中枢神经系统的DHCR7功能,尽管脑组织固醇代谢改善甚小,却有12%转基因Dhcr7^-/-小鼠存活,因此中枢神经系统的胆固醇合成缺陷对Dhcr7^-/-小鼠早期死亡起了重要作用。通过对存活转基因Dhcr7^-/-小鼠的研究,可见明显的神经发育缺陷及髓鞘形成延迟。第一部分转基因选择性重塑Dhcr7^-/-小鼠肝脏胆固醇合成对其病理生理改变的影响目的利用转基因技术部分重塑中枢神经系统DHCR7表达可获得12%转基因Dhcr7^-/-小鼠存活。为进一步了解胆固醇缺乏对外周组织的影响,本研究利用转基因技术选择性重塑Dhcr7^-/-小鼠肝脏的DHCR7表达,从而了解重塑胆固醇合成后是否改善Dhcr7^-/-小鼠的早期病死率。方法首先构建肝脏特异性启动子载脂蛋白E（apolipoprotein E,ApoE）和人DHCR7基因的重组载体用于显微注射。通过PCR和DNA印迹（Southern blot）确认转基因阳性的小鼠,通过RT-PCR、蛋白印迹（western blot）和免疫组化检测转基因后DHCR7的表达,通过GC-MS分析体内各组织的固醇组成验证转基因后对胆固醇合成的影响,通过组织病理切片和免疫组化观察转基因重塑肝脏胆固醇合成对肺脏发育的影响,通过经典的1%Triton X-100（TX）制备肺脏的脂筏观察重塑胆固醇合成后对细胞膜的功能结构域的影响。结果1.hDHCR7特异性表达于肝脏的转基因小鼠通过显微注射法将重组载体pLiv-11-3HA-DHCR7-in5转基因片段注射到受精卵核内,通过PCR和Southern blot检测到3只转基因小鼠TgDHCR7-1,TgDHCR7-2,TgDHCR7-3,同Dhcr7^+/-小鼠配对杂交可获得转基因阳性的Dhcr7^-/-(Dhcr7^-/-Tg+)子代小鼠。2.hDHCR7特异性表达于肝脏取出生10天的转基因阳性小鼠的脑、肝脏、肺脏、心脏、胸腺、脾脏、肾脏、肠、睾丸和肌肉组织以及胚胎第11.5（E11.5）、13.5和16.5天的小鼠肝脏等组织,通过RT-PCR、Western blot和免疫组化显示hDHCR7自胚胎早期开始特异性表达于肝脏。3.转基因后的病理生理改变（1）固醇分析:GC-MS定性定量分析血浆、肝脏、肺和脑组织的固醇组成,结果显示Dhcr7^-/-Tg+小鼠肝脏和肺脏的胆固醇统计学上已恢复至正常水平,血浆胆固醇中等水平升高,而中枢神经系统的胆固醇无显著改善。因此特异性重塑肝脏的DHCR7表达能明显改善Dhcr7^-/-小鼠外周组织的胆固醇代谢,但对中枢神经系统没有影响。（2）肺脏的胚胎晚期发育:转基因Dhcr7^-/-小鼠肺脏在胚胎19.5天已经形成小的肺泡腔及肺泡前的小间隔,较Dhcr7^-/-小鼠明显改善。T1-α,PECAM-1和caveolin-1分布也明显改善。因此转基因特异性重塑肝脏胆固醇合成明显促进了胚胎晚期肺脏发育。（3）转基因后Dhcr7^-/-小鼠早期存活率:收集TgDHCR7-2,TgDHCR7-3组196只后代小鼠,部分（20只）Dhcr7^-/-Tg+小鼠存活时间超过24h,但均于48h内死亡。结果显示重塑肝脏胆固醇合成改善周围组织胆固醇代谢对Dhcr7^-/-小鼠早期死亡无明显影响,进一步证实中枢神经系统胆固醇合成障碍是Dhcr7^-/-小鼠致死性病理生理异常的主要原因。（4）肺脏脂筏的固醇分布:正常小鼠脂筏中只检测到胆固醇,而Dhcr7^-/-Tg-和Dhcr7^-/-Tg+小鼠中均检测到胆固醇和7DHC,7DHC参与脂筏结构。结论选择性重塑肝脏的DHCR7表达明显增加外周组织的胆固醇含量,改善了Dhcr7^-/-Tg+小鼠胚胎晚期的肺脏发育,但没有改善Dhcr7^-/-小鼠早期死亡率,这些现象进一步证明Dhcr7^-/-小鼠中枢神经系统缺陷是导致其早期死亡的主要病理原因。转基因后Dhcr7^-/-小鼠肺脏胆固醇含量虽然明显恢复,但是7DHC仍然存在并参与脂筏,从而可能影响脂筏的结构以及细胞及组织的功能。第二部分Dhcr7^-/-小鼠脑组织脂筏的蛋白质组学研究目的Dhcr7^-/-小鼠中枢神经系统胆固醇合成缺陷造成的功能异常可能是其早期死亡的主要原因,但其病理生理机制尚不清楚。胆固醇参与中枢神经系统发育中的多种重要生理过程如信号传导、神经细胞的生长分化、神经递质转运、髓鞘形成、突触的生长发育等。近年来研究发现,上述中枢神经系统的结构及功能的形成均与细胞膜尤其是富含胆固醇及鞘磷脂的特殊膜结构—脂筏有密切关系。因此,胆固醇合成缺陷可能通过改变脂筏结构及功能而影响中枢神经系统发育。目前,自新生小鼠脑组织分离生理性脂筏及其蛋白组谱的研究尚缺乏可靠性方法,因此本研究首先建立非离子型去垢剂脂筏分离并应用液相色谱串联质谱（LC-MS/MS）定性定量分析脂筏蛋白组谱的方法,并对Dhcr7^-/-小鼠胆固醇合成缺陷导致的中枢神经系统脂筏的蛋白质组谱改变进行初步观察。方法1.在无去污剂条件下两步法密度梯度离心分离脑组织的脂筏取出生1天的正常新生鼠和Dhcr7^-/-小鼠脑组织两步法制备脂筏。第一步利用5%-35%-45%不连续性OptiPrep密度梯度制备细胞膜及细胞器膜成份即类脂膜;第二步类脂膜在无去污剂的条件下通过5%-25%-30%-35%-40%蔗糖密度梯度超速离心制备脂筏。2.“试管胶”蛋白消化结合无标记的鸟枪法（shot-gun）蛋白质组学方法由于脂筏蛋白的低丰度高疏水特性,本实验采用“试管胶”蛋白消化结合无标记的鸟枪法蛋白质组学方法能获得准确而可重复的蛋白质组。脂筏蛋白直接同聚丙酰胺形成凝胶块,并在试管内胰蛋白酶消化成肽段,称为“试管胶”消化。消化后的肽段用于液相色谱质谱分析。用此方法比较正常新生鼠和Dhcr7^-/-小鼠脑组织脂筏的蛋白组学。结果1.无去污剂条件下制备脂筏的Optiprep-蔗糖不连续密度梯度离心法可以制备无去污剂脂筏。2.通过“试管胶”（tube-gel）消化蛋白结合液相色谱串联质谱（LC-MS/MS）即TubeGeLC-MS/MS方法获得无TX方法制备的脂筏蛋白质组同常规的TX方法类似,包含大量的细胞膜蛋白及胞浆蛋白和少量的线粒体蛋白、核蛋白等。3.比较正常新生鼠和Dhcr7^-/-小鼠脑组织脂筏蛋白质组,结果显示Dhcr7^-/-小鼠脑组织脂筏中膜蛋白的含量明显降低,其中很多蛋白是GPI锚定蛋白和脂筏相关蛋白;而非膜蛋白则呈现两面性,部分胞浆蛋白、线粒体相关蛋白和核蛋白含量明显降低,部分胞浆蛋白含量升高或无统计学差异。结论无去污剂条件下制备脂筏的Optiprep-蔗糖不连续密度梯度离心法可以制备无去污剂脂筏。与常用的非离子型去污剂分离方法相比,无去垢剂分离的脂筏可能更符合真实的生物学状态。通过比较正常和Dhcr7^-/-小鼠的中枢神经系统脂筏的蛋白组差异,初步研究结果显示Dhcr7^-/-小鼠脑组织的脂筏中,丰度明显降低的某些蛋白是神经发育在内的神经系统功能行使过程中的关键蛋白以及神经递质传递信号传导相关蛋白,进一步提示了胆固醇合成缺陷可能通过多途径影响中枢神经系统的功能及发育等。更多还原

【Abstract】 BackgroundCholesterol is an essential lipid in all mammalian cells,and is a major component of cell membranes.In addition to the structural role in cellular membranes,cholesterol is also the precursor molecule for sterol-based compounds including bile acids, oxysterols,neurosteroids,glucocorticoids,mineralocorticoids,and sex steroids. Moreover,the recognition that increased cholesterol level（hypercholesterolemia） is a major risk factor for the development of heart disease and atherosclerosis has gained enormous attention not only in medicine,medical and pharmacological research,but also from the general public.The discovery of a crucial role of cholesterol in human embryogenesis and the recent identification of a number of inherited disorders of cholesterol biosynthesis also show that low cholesterol level（hypocholesterolemia） may have severe consequences for human health and development.To date,nine inherited disorders of cholesterol biosynthesis have been described.Two disorders have been linked to the enzyme defects in the pre-squalene segment of the pathway: classical mevalonic aciduria and hyper-IgD and periodic fever syndrome.Remaining seven are associated with the enzymatic defects of post-squalene pathway: Smith-Lemli-Opitz syndrome（SLOS）,Desmosterolosis,Hydrops-ectopic calcification-moth-eaten（HEM） skeletal dysplasia,Lathosterolosis,Antley-Bixler syndrome,Chondrodysplasia punctata 2（CDPX2）,Congenital hemidysplasia ichthyosiform nevous and limb defects（CHILD） syndrome.Patients with these disorders are characterized by multiple congenital anomalies including internal organ, skeletal and/or skin abnormalities.SLOS is the first described and by far the most common disorder of post-squalene cholesterol biosynthesis.SLOS is an autosomal recessive multiple congenital anomaly/mental retardation disorder caused by enzymatic deficiency of 3β-hydroxysterol-Δ7reductase（DHCR7）,an enzyme in the terminal enzymatic step of cholesterol biosynthesis.DHCR7 catalyzes both the reduction of 7-dehydrocholesterol（7DHC） to cholesterol and the reduction of 7-dehydrodesmosterol to desmosterol.Desmosterol can be converted to cholesterol by 3β-hydroxysterol-Δ24 reductase（DHCR24）.Thus SLOS patients usually have low plasma cholesterol levels and invariably elevated levels of cholesterol precursors such as 7-dehydrocholesterol（and its spontaneous isomer 8-dehydrocholesterol） and absent desmosterol.The incidence of SLOS is estimated to range from 1/10,000 to 1/60,000 of live birth.Population screens for mutant DHCR7 alleles suggest a 3—4%carrier frequency in Caucasian populations,giving a hypothetical birth incidence about 1/2500-1/4500,assuming no fetal loss.Patients with SLOS display a large and variable spectrum of morphogenic and congenital anomalies,including dysmorphic craniofacial features,microcephaly,multiple internal organ,limb/skeletal and urigenital malformations,（intrauterine） growth and mental retardation,and behavioral problems.SLOS is now diagnosed by increased blood levels of 7-DHC and low cholesterol levels combination with the clinical features.There is no known cure for SLOS.Most of treatments are supportive such as surgical repair of physical anomalies of congenital heart defects,cleft palate,genital anomalies,craniofacial, gastrointestinal and limb defects.To date,a total of 121 mutations in DHCR7 have been identified Mutations are located throughout the coding region（exons 3-9）,including 105 missense,five nonsense,three splice-site and eight nucleotide insertions or deletions.Fifty percent of the missense mutations are located in one of the nine predicted transmembrane domains.Based on those,to further characterize the pathophysiology and neurophysiology underlying SLOS and to provide a model system for testing therapeutic intervention,two genetically manipulated murine models by disruption of the mouse Dhcr7 gene have been generated.Dhcr7^-/- pups mimic the biochemical and phenotypic hallmarks of the SLOS. Although the genetic defects and biochemical consequences in the genetic disorders of cholesterol biosynthesis have now been identified,the pathophysiology underlying the neurodevelopmental abnormalities is poorly understood.Currently,it is not known why defects in cholesterol synthesis cause neurodevelopmental defects but a number of hypotheses have been proposed.The most popular theory is that the key morphogen,Sonic Hedgehog（Shh and its related proteins Indian and Desert Hedgehog）,is affected since this protein needs covalently attached cholesterol to regulate developmental signaling processes.However,features that have been shown to be caused by defective Shh signaling in the mouse embryo are absent in Dhcr7^-/- mice.Furthermore,Shh post-translational autoprocessing and expression in brain and lung in Dhcr7^-/- embryos are not abnormal,validating the previous conclusions that precursor sterols participate as efficiently as does cholesterol in the Shh processing reaction and can also substitute for cholesterol for structural requirements such as incorporation into bilayer membranes.Thus,the data collected indicate that the pathogenic defects in cholesterol biosynthesis disorders can not be explained,at least solely at the level of disturbed hedgehog signaling cascades.We have generated transgenic mice expressing human DHCR7 cDNA（hDHCR7） driven by a brain-specific nestin promoter.Though small and subtle changes in brain sterol metabolism,we still got a stochastically partial rescue（12%） and found a delayed postnatal myelination and defective neurogenesis from the rescued mutant animals,indicating that normal central nervous system（CNS） cholesterol synthesis is critical for postnatal survival.PartⅠ.Pathophysiological consequences of transgenic reconstitution of liver-specific cholesterol biosynthesis in Dhcr7 null mouseObjectiveTargeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene（Dhcr7）,an animal model of Smith-Lemli-Opitz syndrome（SLOS）,leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis.To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology,we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 in the liver.MethodWe first constructed a plasmid with ApoE promoter and human DHCR7 for microinjection.Transgenic mice identified by PCR and Southern blot were called founders.The expression of human DHCR7 was detected in different tissues by RT-PCR,western blot and immunohistology.We can get transgenic Dhcr7^+/- mice (Dhcr7^+/-tg+) by mating founder with Dhcr7^+/- mouse.Dhcr7^-/-/tg+ mice were generated by mating between Dhcr7^+/-tg+ mice.To examine the changes in the transgenic mouse we used GC-MS to measure the concentration of cholesterol and 7DHC of plasma,liver,lung and brain.Lipid rafts were extracted using the classic 1% Trion X-100 to investigate the possible mechanism of cholesterol deficiency.Results1.Generation of liver-specific hDHCR7 transgenic mouseWe generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E（ApoE） promoter and its associated liver-specific enhancer.Three mice（one female and two males）,referred as to TgDHCR7-1,TgDHCR7-2 and TgDHCR7-3,respectively,were identified as transgenic on the basis of PCR and Southern blot analyses of tail samples of genomic DNA.These mice were then crossed with Dhcr7^+/- mutants to generate Dhcr7^-/- mice bearing a human DHCR7 transgene.2.Liver-specific expression of hDHCR7 transgeneMultiple tissues were collected from N2 transgenic progeny from the founder mice crossed with C57B1/6J at postnatal day（P） 10 and analyzed for transgene expression by RT-PCR and immunoblotting.Both TgDHCR7-2 and TgDHCR7-3 transgenic lines expressed human DHCR7 mRNA robustly in the postnatal livers,but not in the other tissues and western blotting analyses showed expression of HA-tagged protein. TgDHCR7 mRNA transcript was detected in transgenic liver at E11.5（the earliest we could dissect liver tissue） by RT-PCR determination and was quantitatively～5 fold higher than endogenous murine Dhcr7 mRNA in the transgenic liver at E16.5 as judged by real-time PCR.Immunofluorescence staining with anti-HA antibody showed that TgDHCR7 was highly expressed in hepatocytes in liver sections,but such signal was not detectable in control liver.3.Pathophysiological effects of transgene expressionTo determine the physiological consequences of selective restoration of DHCR7 expression in liver,we bred the transgene onto Dhcr7 null background.Sterol metabolic profile,late gestational lung development,and postnatal survivability were evaluated and compared between transgenic and non-transgenic Dhcr7 knockout mice.（1） Sterol analysis:Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80～90%of normal levels in liver and lung.However,cholesterol deficiency in brain was not altered.These data indicated that selective reconstitution of DHCR7 expression in liver improved significantly cholesterol homeostasis in liver,lung and circulation of Dhcr7 null animals during embryogenesis,but did not affect metabolism in the brain.（2） Late gestational lung development:lungs from Dhcr7 null mice expressing DHCR7 in the liver showed an improved sac space formation and thinning of the prealveolar septae by histopathology,compared with non-transgenic mutant lungs. The transgene expression led to an improvement in T1-α,PECAM-1 and caveolin-1 of immunohistochemistry of lung.These all suggested that transgenic-induced improvement of cholesterol homeostasis in non-brain tissues normalized the architecture of the distal lung sacculation.（3） Postnatal survivability of Dhcr7^-/-Tg+ mice:one hundred and ninety-six pups from Dhcr7^+/-Tg+ females bred with Dhcr7^+/-Tg+ males were scored for viability.20 Dhcr7^-/-Tg+ survived the first postnatal day,but died within 48 h after birth. Lethality was minimally delayed.（4） Association of cholesterol and 7/8DHC with lipid membranes of lung tissues: cholesterol and precursor sterols were analyzed in the raft and non-raft fractions isolated by sucrose-gradient ultracentrifugation.In WT lungs,cholesterol was detected as the only major sterol,and showed a bimodal profile,with one sharp peak corresponding to the raft fractions,the second peak in high-density non-raft fractions, 7/8DHC levels were undetectable,In Dhcr7^-/-Tg- lungs,sterols composed of a mixture of 7/8DHC and cholesterol,also exhibited bimodal profiles,and 7/8DHC constituted the major sterol in both raft and non-raft fractions.In Dhcr7-/-Tg+ lungs,although cholesterol was dramatically increased in both raft and non-raft fractions, considerable amount of 7/8DHC was still detected.ConclusionThe reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues,but failed to rescue the neonatal lethality of Dhcr7 null mice.These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice,with the peripheral organs contributing the morbidity.PartⅡQuantitative proteomic analysis of brain lipid rafts in SLOS mouse modelObjectiveCentral nervous system（CNS） defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of defective cholesterol synthesis(Dhcr7^-/-) mice. Development of the brain involves many complex cholesterol-regulated events,such as membrane trafficking,signal transduction,myelin formation,and synaptogenesis. In all these developmental events,cell surface properties,determined by membrane components,are of primary importance.Deviations from normal sterol concentrations and compositions can perturb the feature of specialized lipid membrane domains （lipid rafts） and compromise cellular functions.We postulated that alterations in membrane lipid raft domains especial in cholesterol-deficient brain tissue underlay much of the pathophysiology of SLOS and determines the progression of the disease. However,the identity of possible signaling proteins had not yet been fully determined. Therefore,the objective of this study is to determine the alterations of raft protein composition in Dhcr7^-/- brains by quantitative proteomics approaches and identify the potential disease-associated biomarkers.To achieve the goals,we have developed a new method to isolate lipid rafts by detergent-free preparation,and established a tube-gel protein digestion combined with label-free tandem MS/MS（shotgun proteomics） to quantitatively analyze raft proteome.We have applied these methods to analyze the alterations of protein compositions in the lipid rafts isolated from the brains of SLOS mouse model.Methods1.Preparation of raft-enriched membranes from neonatal mouse brain by a newly devised detergent-free discontinuous Optiprep-sucrose gradients.Neonatal（postnatal day 1） mouse brains from wild-type（WT） and mutant （Dhcr7-/-） animals were used for preparation of raft-enriched membranes（lipid rafts）. Cellular membranes including plasma membrane and intracellular organelle membranes were initially isolated from the crude postnuclear supernatants in 5%-35%-45%discontinuous Optiprep gradients,and they were defined to lipid membranes（LM）.Subsequently,lipid rafts were purified from LM in absence of any detergent by 5%-25%-30%-35%-40%discontinuous sucrose gradients.2.Quantifying raft proteins by ’tube-gel’ protein digestion label-free shotgun proteomics.The low concentration and highly hydrophobic nature of proteins in lipid raft samples present significant challenges for the sensitive and accurate proteomic analyses of lipid raft proteins.We have devised a simple protocol using a ’tube-gel’ protein digestion that,when combined with mass spectrometry,can be used to obtain comprehensive and reproducible identification and quantitation of the lipid raft proteome prepared from neonatal mouse brain.Lipid rafts prepared from neonatal mouse brain were directly incorporated into a polyacrylamide tube-gel matrix without prior protein separation.After in-gel digestion of proteins,nanospray LC-MS/MS,was used to analyze the extracted peptides,and the resulting spectra were searched to identify the proteins present in the sample.Relative abundances of raft proteins between WT and mutant brains were compared.Results1.Establishment a new method for isolation of lipid rafts by detergent-free approach2.Development of a new approach to quantify raft proteome by a ’tube-gel’ protein digestion and label-free shotgun proteomics3.Quantitative proteomic analysis revealed multiple alterations in the lipid rafts of Dhcr7-/- brainConclusionLipid raft can be isolated efficiently from neonatal brains without TX,closer to native state of lipid rafts in cell membranes.’Tube-gel’ protein digestion and label-free shotgun proteomics can get comprehensive and reproducible identification and quantitation of the lipid raft proteome.Compared with WT,many membrane proteins decreased evidently in Dhcr7-/- mouse,some involved in the developmental process of neonatal brain,including signal transduction,neurotransmitter release,and membrane trafficking.更多还原