非创伤性股骨头坏死患者股骨头骨组织的差异蛋白组学研究
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摘要
研究背景股骨头坏死(Osteonecrosis of the femoral head, ONFH)是由不同原因导致的股骨头骨髓造血细胞、骨髓脂肪细胞及骨细胞变性坏死的一种疾病,为骨科临床常见病之一。主要分为创伤性(traumatic osteonecrosis of femoral haead TONFH)和非创伤性股骨头坏死(non-traumatic osteonecrosis of femoral head, NONFH)两大类。有资料表明股骨头缺血性坏死已取代了原髋关节结核的位置,居髋关节疾病的首位。股骨头坏死病因复杂,病程缓慢,治疗困难,致残率高,是长期以来困扰国内外学者的医学难题,有“不死的癌症“之称。而目前临床上由于缺乏有效的早期筛查和检测手段,很少能在股骨头坏死早期发现从而进行药物或手术干预,因此大多数患者直到出现疼痛,活动受限才来就诊,此时多是处于股骨头塌陷变形、骨性关节炎等病变晚期,只能施行人工髋关节置换手术,最终导致患者生活质量下降以及国家家庭个人的经济负担增加。因此,明确股骨头坏死的原因,弄清其发病机制,寻找有价值的生物学标志物(biomarker),以期早期筛查发现股骨头坏死并进行早期治疗,是临床和实验室专家一直努力研究的方向。
创伤性股骨头坏死病因比较明确,但非创伤性股骨头坏死确切病因和发病机制仍存在众多争论。目前激素性和酒精性股骨头缺血性坏死仍被普遍认为是非创伤性股骨头缺血性坏死最常见的原因。研究者们相继提出了10多种学说,有脂肪代谢紊乱、血管内凝血、微血管损伤等。10余种学说所涉及多种酶类、细胞因子等,它们都是蛋白质。众所周知,蛋白质是正常生理活动和疾病发生发展过程的执行者和参与者。以往对蛋白质的研究只是针对生命活动中某一或某几种蛋白质,难以从整体、系统上阐明生命活动的基本机制。因此,大规模、全方位的蛋白质研究势在必行。
蛋白质组学(Proteomics)是从整体水平上研究蛋白质表达、组成及活动规律的学科。其研究内容主要包括三个部分：表达蛋白质组学(Expression Proteomics)、结构蛋白质组学(Structural Proteomics)和功能蛋白质组学(Functional Proteomics)。表达蛋白质组学是指对一个指定的细胞、组织或生物所产生全部蛋白质的鉴定。结构蛋白质组学是指对上述全部蛋白质一级和三维结构的测定。功能蛋白质组学则是阐明蛋白质的生理功能以及机体在不同时期中蛋白质表达水平的动态变化。蛋白组学的研究可以实现与基因组的对接与确认,直接揭示人类重大疾病发生与发展的病理机制。
蛋白质组学在大多数骨科疾病的研究方面还处于初步阶段。近年来关于股骨头坏死的蛋白质组学研究多集中在患者的血清学和实验动物骨组织方面的研究。少数几项血清蛋白质组学的研究也已发现多种蛋白的高表达与股骨头缺血性坏死密切相关。但存在的问题是：血清蛋白质成分不稳定,极易受机体内外环境的背景干扰,实验数据重复性较差；动物实验的数据和人体存在一定差异。这些蛋白质组学研究大多基于常规双向凝胶电泳(2-DE)技术,存在一定的局限性：①上样量的限制使低丰度蛋白质(<1 ng)难以检测；②电泳结果需染色处理,而不同的蛋白质与染料结合的差异较大；③目前尚不能全自动处理,耗时长,不能进行高通量分析。
荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为目前最受欢迎的定量蛋白质组学研究手段之一。
骨组织蛋白的提取主要有化学法和物理法两大类。几项人体和动物实验的骨组织蛋白质组学的研究采取的是化学方法,虽然该法能够从多种骨组织中分离出蛋白质并将低丰度蛋白有效地鉴别出来,但耗时较长,约需一周时间,容易导致蛋白的分解变性,不适合做后续的Western blot研究。2009年王簕等应用锤击研磨法提取骨组织蛋白,简便易行,不破坏蛋白质的构成和理化性质,值得借鉴。
目的探讨非创伤性股骨头坏死的发病机制,为筛选可能生物学标志物和分子靶向治疗提供理论依据。本研究收集非创伤性股骨头坏死患者的坏死股骨头骨组织,以正常股骨头骨组织对照,应用液氮研磨的物理方法提取骨组织总蛋白,通过荧光差异凝胶电泳(DIGE)寻找差异蛋白,利用基质辅助激光解析离子化-飞行时间质谱技术(MALDI-TOF/TOF)进行鉴定,进行生物信息学分析,探讨其发病机制。
方法本研究共分四个部分。第一部分,骨组织样本的收集。临床上经影像学和病理确诊的非创伤性股骨头坏死实行髋关节置换术的患者,取坏死股骨头骨组织,修剪成骨粒状,置于-80℃冰箱备用,以新鲜股骨颈骨折患者的股骨头骨组织为对照。第二部分,建立骨组织的荧光差异双向电泳图谱。使用液氮研磨法提取骨组织总蛋白,利用荧光差异凝胶电泳(DIGE)技术分离蛋白质,扫描分析建立图谱,寻找差异蛋白。第三部分,明显差异蛋白质的鉴定和生物信息学分析。挖取差异表达2倍以上的蛋白质,行基质辅助激光解析离子化-飞行时间／飞行时间质谱仪(MALDI-TOF/TOF)鉴定,数据库检索,获得这些蛋白质的序列、结构和功能信息,结合文献,对感兴趣蛋白行生物信息学分析。第四部分,差异蛋白的Western blot验证。选取载脂蛋白A1和ATP合成酶(ATP5β),运用蛋白免疫印迹(Western blot)技术,再次行蛋白质定量分析,验证DIGE结果的可靠性。
结果通过DIGE成功分离1600±50个蛋白质点,明显差异蛋白点274个,利用EttanTM Spot picker自动工作站挖取表达差异达2倍以上的明显差异蛋白点50个,利用MALDI-TOF/TOF质谱仪对这些差异蛋白点进行蛋白质鉴定,共鉴定出9种蛋白质,分别是载脂蛋白A1 (APOA1)、热休克蛋白β-1(HSPp-1)、ATP合成酶β亚基(ATP5β)、纤维蛋白原γ链(FGG)、纤维蛋白原p链(FGB)、血清白蛋白(ALB)、硫氧蛋白过氧化物还原酶(PRDX2)、转铁蛋白(TF)肌动蛋白(ACTG1),除纤维蛋白原表达有下调外,其余蛋白均表达上调。通过多种数据库查询,这些蛋白分别涉及参与蛋白质和脂质代谢、能量代谢、细胞骨架、信号转导、氧化还原等方面的功能,并进行亚细胞定位、相互作用等生物学信息学分析。选取载脂蛋白Al (APOA1)和ATP合成酶(ATP5β)做Western blot,结果与DIGE结果相符。
结论：
1.使用液氮研磨提取骨组织总蛋白的物理方法,简便有效,可以满足后续的蛋白质组学研究,对蛋白质图谱影响小
2.运用荧光差异双向凝胶电泳(DIGE)可以高效分离骨组织蛋白质组,获得股骨头坏死的差异蛋白质表达谱。
3.通过对差异表达的蛋白质进行MALDI-TOF/TOF质谱分析鉴定,获得了9种蛋白质,通过生物学信息学分析,这些蛋白质可能与股骨头坏死的发生、发展有关,为筛选用于诊断的生物学标志物、为该疾病的分子靶向治疗提供理论依据。
4.通过Western blot对载脂蛋白A1和ATP合成酶表达量进行验证,结果和DIGE结果一致,证明了以上研究的准确性和可靠性。
创新点：经国内外文献检索,使用DIGE和MALDI-TOF/TOF质谱技术方法对非创伤性股骨头坏死患者股骨头骨组织进行蛋白组学分析的研究,未见报道。本研究直接采集股骨头坏死患者的坏死股骨头骨组织样本用于研究,数据更加客观真实；采用液氮研磨物理法提取蛋白,减少蛋白降解的风险：利用DIGE技术分离寻找差异蛋白,重复性和敏感性更好。
Background Osteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of myeloid element, adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head (TONFH) and non-traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time. it is also called "the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip'pain and limitation of hip'motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts'task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.
The pathologic process of traumatic osteonecrosis of femoral head is already clear, but the etiology and pathogenesis of non-traumatic osteonecrosis of femoral head are still unclear. Currently, hormone and alcohol are thought to be the most causes of non-traumatic osteonecrosis. Researchers have put out more than 10 theories up to now, they all involve in many kinds of enzymes, cytokines, gene, protein and so on, which are all closely related with proteins. Proteins are the performer and participant in course of disease occurrence and development, and also the signal of disease process. Previous researchs about protein only aimed at one or a few proteins in life activity, therefore, large-scale, omni-directional protein study is necessary.
The proteome can entirely research the expression, composition and activity discipline of protein. It include three main parts:Expression Proteomics,Structural Proteomics and Functional Proteomics. Expression Proteomics refers to identify all the protein produced by specified cell, tissue or organ. Structural Proteomics refer to determine the first level and 3D structure of all-above metioned proteins. Functional Proteomics illuminate the physiological function of the protein and the dynamic change of the expression level in its different periods. Proteomics research can realize the docking and confirm with the genome, directly reveal the pathological mechanism of occurrence and development of the human major disease.
Proteomics research on most orthopaedic disease is still in the initial stage. In recent years, proteomics research about osteonecrosis of femoral head is mainly on serology and experimental animal tissue. Some research has found that a few high expression proteins are closely related with osteonecrosis. But problems are:serum protein component is not stable, easily influenced by other factors, repeatability of experimental data is poor; and the data from animal experiment is different.
Moreover, these proteomics research mostly based on conventional two-dimensional gel electrophoresis (2-DE) technology which has certain limitation: (1) the sample weight restrictions on that low abundance protein (<1 ng) hard to detection; (2) the results of electrophoresis-need dyeing and the results of dyeing combined with different proteins are different; (3) this technology is no-automatic processing, time-consuming, cannot undertake high-throughput analysis.
Extracting proteins from bone tissue generally have two methods:chemical method and physical method. Several experiments are taken with chemical methods, although this method can isolate a variety of bone protein and low abundance protein can be effectively identified, but it needs about a week' time, and easily led to the protein denaturation,so it is not suited for Western blot study. In 2009, WangLe used hammering grinding method to extract proteins from bone tissue, easily and do not destroy the structure and physical and chemical properties of proteins, worthy of using for reference.
At present there are many new proteomics analysis methods, among which the difference in-gel electrophoresis (DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. Because of these advantages, DIGE has become the most popular research means on proteomics.
Objective In order to find different proteins, explore its pathogenesis, provide theory'basis for screening markers and targeted therapy. Based on above background, we designed this experiment. We collected femoral head from the patients with non-traumatic osteonecrosis of the femoral head and femoral neck' fracture, used liquid nitrogen grinding to extract total proteins from bone tissue, separated and identified protein with DIGE and MALDI-TOF/TOF, then made a bioinformatic analysis,
Methods Our research is divided into four parts. The first part, collecting bone tissue sample. The second part, establishing a electrophoregram of DIGE. The third part, obvious difference protein identification and bioinformatics analysis. The fourth part, confirming expression level of some protein through Western blot test.
Results 1600±50 protein points were successfully separated, and 274 points were obviously different. We dug 50 points which expressed differently more than two times with EttanTM Spot picker automatic workstations, identified with MALDI TOF/TOF mass spectrometer. Finally,9 different proteins were identified, which are apolipoprotein Al (APOA1), heat shock proteinβ-1 (HSPβ-1), ATP synthaseβsubunit (ATP5β), fibrinogenγchain (FGG), fibrinogenβchain (FGB), serum albumin (ALB), sulfur oxygen protein peroxide oxidoreductase (PRDX2), transferrin (TF) and actinl (ACTG1) respectively. They are all up-regulation except fibrinogen. Through a variety of database, these proteins involved in lipids metabolism, energy metabolism, cytoskeleton, signal transduction, redox etc. Results of Western blot showed the expressions of APOA1 and ATP5βwere consistent with DIGE.
Conclusions:
1. Extracting total protein of bone tissue by liquid nitrogen grinding can meet the require of subsequent proteomics research and proteins have little degradation.
2. DIGE can separate total proteins of bone tissue and find different proteins efficiently.
3. By identifying different proteins with MALDI-TOF/TOF mass spectrometry, we obtained 9 proteins. By bioinformatics analysis, these proteins are all related to occurrence and development of osteonecrosis. So our results provide theoretical basis for screening diagnosis biomarkers and molecular target therapy for ONFH.
4. Through Western blot, we proved the reliability of our research.
Innovations:to our knowledge, there are no reports on proteomic analysis of bone tissue from patients of ONFH with using DIGE and MALDI-TOF/TOF. In our study, we directly use bone tissue from patients of ONFH for research, data is more objective; use physical method of liquid nitrogen grinding to extract protein to reduce protein degradation; use DIGE to find different proteins,the result is more repetitive and sensitive.
创伤性股骨头坏死病因比较明确,但非创伤性股骨头坏死确切病因和发病机制仍存在众多争论。目前激素性和酒精性股骨头缺血性坏死仍被普遍认为是非创伤性股骨头缺血性坏死最常见的原因。研究者们相继提出了10多种学说,有脂肪代谢紊乱、血管内凝血、微血管损伤等。10余种学说所涉及多种酶类、细胞因子等,它们都是蛋白质。众所周知,蛋白质是正常生理活动和疾病发生发展过程的执行者和参与者。以往对蛋白质的研究只是针对生命活动中某一或某几种蛋白质,难以从整体、系统上阐明生命活动的基本机制。因此,大规模、全方位的蛋白质研究势在必行。
蛋白质组学(Proteomics)是从整体水平上研究蛋白质表达、组成及活动规律的学科。其研究内容主要包括三个部分：表达蛋白质组学(Expression Proteomics)、结构蛋白质组学(Structural Proteomics)和功能蛋白质组学(Functional Proteomics)。表达蛋白质组学是指对一个指定的细胞、组织或生物所产生全部蛋白质的鉴定。结构蛋白质组学是指对上述全部蛋白质一级和三维结构的测定。功能蛋白质组学则是阐明蛋白质的生理功能以及机体在不同时期中蛋白质表达水平的动态变化。蛋白组学的研究可以实现与基因组的对接与确认,直接揭示人类重大疾病发生与发展的病理机制。
蛋白质组学在大多数骨科疾病的研究方面还处于初步阶段。近年来关于股骨头坏死的蛋白质组学研究多集中在患者的血清学和实验动物骨组织方面的研究。少数几项血清蛋白质组学的研究也已发现多种蛋白的高表达与股骨头缺血性坏死密切相关。但存在的问题是：血清蛋白质成分不稳定,极易受机体内外环境的背景干扰,实验数据重复性较差；动物实验的数据和人体存在一定差异。这些蛋白质组学研究大多基于常规双向凝胶电泳(2-DE)技术,存在一定的局限性：①上样量的限制使低丰度蛋白质(<1 ng)难以检测；②电泳结果需染色处理,而不同的蛋白质与染料结合的差异较大；③目前尚不能全自动处理,耗时长,不能进行高通量分析。
荧光差异凝胶电泳(difference gel electrophoresis, DIGE)技术由于继承了二维凝胶电泳(two-dimensional electrophoresis,2-DE)的高分辨率,同时又具备高重现性、高灵敏度、高通量和高动态范围等优势,使得DIGE日益受到关注,成为目前最受欢迎的定量蛋白质组学研究手段之一。
骨组织蛋白的提取主要有化学法和物理法两大类。几项人体和动物实验的骨组织蛋白质组学的研究采取的是化学方法,虽然该法能够从多种骨组织中分离出蛋白质并将低丰度蛋白有效地鉴别出来,但耗时较长,约需一周时间,容易导致蛋白的分解变性,不适合做后续的Western blot研究。2009年王簕等应用锤击研磨法提取骨组织蛋白,简便易行,不破坏蛋白质的构成和理化性质,值得借鉴。
目的探讨非创伤性股骨头坏死的发病机制,为筛选可能生物学标志物和分子靶向治疗提供理论依据。本研究收集非创伤性股骨头坏死患者的坏死股骨头骨组织,以正常股骨头骨组织对照,应用液氮研磨的物理方法提取骨组织总蛋白,通过荧光差异凝胶电泳(DIGE)寻找差异蛋白,利用基质辅助激光解析离子化-飞行时间质谱技术(MALDI-TOF/TOF)进行鉴定,进行生物信息学分析,探讨其发病机制。
方法本研究共分四个部分。第一部分,骨组织样本的收集。临床上经影像学和病理确诊的非创伤性股骨头坏死实行髋关节置换术的患者,取坏死股骨头骨组织,修剪成骨粒状,置于-80℃冰箱备用,以新鲜股骨颈骨折患者的股骨头骨组织为对照。第二部分,建立骨组织的荧光差异双向电泳图谱。使用液氮研磨法提取骨组织总蛋白,利用荧光差异凝胶电泳(DIGE)技术分离蛋白质,扫描分析建立图谱,寻找差异蛋白。第三部分,明显差异蛋白质的鉴定和生物信息学分析。挖取差异表达2倍以上的蛋白质,行基质辅助激光解析离子化-飞行时间／飞行时间质谱仪(MALDI-TOF/TOF)鉴定,数据库检索,获得这些蛋白质的序列、结构和功能信息,结合文献,对感兴趣蛋白行生物信息学分析。第四部分,差异蛋白的Western blot验证。选取载脂蛋白A1和ATP合成酶(ATP5β),运用蛋白免疫印迹(Western blot)技术,再次行蛋白质定量分析,验证DIGE结果的可靠性。
结果通过DIGE成功分离1600±50个蛋白质点,明显差异蛋白点274个,利用EttanTM Spot picker自动工作站挖取表达差异达2倍以上的明显差异蛋白点50个,利用MALDI-TOF/TOF质谱仪对这些差异蛋白点进行蛋白质鉴定,共鉴定出9种蛋白质,分别是载脂蛋白A1 (APOA1)、热休克蛋白β-1(HSPp-1)、ATP合成酶β亚基(ATP5β)、纤维蛋白原γ链(FGG)、纤维蛋白原p链(FGB)、血清白蛋白(ALB)、硫氧蛋白过氧化物还原酶(PRDX2)、转铁蛋白(TF)肌动蛋白(ACTG1),除纤维蛋白原表达有下调外,其余蛋白均表达上调。通过多种数据库查询,这些蛋白分别涉及参与蛋白质和脂质代谢、能量代谢、细胞骨架、信号转导、氧化还原等方面的功能,并进行亚细胞定位、相互作用等生物学信息学分析。选取载脂蛋白Al (APOA1)和ATP合成酶(ATP5β)做Western blot,结果与DIGE结果相符。
结论：
1.使用液氮研磨提取骨组织总蛋白的物理方法,简便有效,可以满足后续的蛋白质组学研究,对蛋白质图谱影响小
2.运用荧光差异双向凝胶电泳(DIGE)可以高效分离骨组织蛋白质组,获得股骨头坏死的差异蛋白质表达谱。
3.通过对差异表达的蛋白质进行MALDI-TOF/TOF质谱分析鉴定,获得了9种蛋白质,通过生物学信息学分析,这些蛋白质可能与股骨头坏死的发生、发展有关,为筛选用于诊断的生物学标志物、为该疾病的分子靶向治疗提供理论依据。
4.通过Western blot对载脂蛋白A1和ATP合成酶表达量进行验证,结果和DIGE结果一致,证明了以上研究的准确性和可靠性。
创新点：经国内外文献检索,使用DIGE和MALDI-TOF/TOF质谱技术方法对非创伤性股骨头坏死患者股骨头骨组织进行蛋白组学分析的研究,未见报道。本研究直接采集股骨头坏死患者的坏死股骨头骨组织样本用于研究,数据更加客观真实；采用液氮研磨物理法提取蛋白,减少蛋白降解的风险：利用DIGE技术分离寻找差异蛋白,重复性和敏感性更好。
Background Osteonecrosis of the femoral head (ONFH) was a pathologic process caused by multi-pathogenic factors that damaged the blood supply of the femoral head, which led to the death of myeloid element, adipocyte and osteocytes. It is one of the common clinic diseases and mainly divided into two groups:traumatic osteonecrosis of femoral head (TONFH) and non-traumatic osteonecrosis of femoral head(NONFH). Some data indicates that ONFH has replaced the hip tuberculosis as the first place in hip diseases. Osteonecrosis of the femoral head have troubled domestic and foreign scholars for a long time. it is also called "the deadless cancer". But now, due to a lack of effective early screening and detection methods, it is rarely detected and intervented in early time. So most patients have a feel of hip'pain and limitation of hip'motion for a long time, then, think about the possibility of osteonecrosis of the femoral head. At this time, these patients had to be operated with THA because of the collapse and defomation of femoral head and osteoarthritis. It is the clinical and laboratory experts'task to make clear its pathogenesis, search valuable biomarkers for early screening and treatment.
The pathologic process of traumatic osteonecrosis of femoral head is already clear, but the etiology and pathogenesis of non-traumatic osteonecrosis of femoral head are still unclear. Currently, hormone and alcohol are thought to be the most causes of non-traumatic osteonecrosis. Researchers have put out more than 10 theories up to now, they all involve in many kinds of enzymes, cytokines, gene, protein and so on, which are all closely related with proteins. Proteins are the performer and participant in course of disease occurrence and development, and also the signal of disease process. Previous researchs about protein only aimed at one or a few proteins in life activity, therefore, large-scale, omni-directional protein study is necessary.
The proteome can entirely research the expression, composition and activity discipline of protein. It include three main parts:Expression Proteomics,Structural Proteomics and Functional Proteomics. Expression Proteomics refers to identify all the protein produced by specified cell, tissue or organ. Structural Proteomics refer to determine the first level and 3D structure of all-above metioned proteins. Functional Proteomics illuminate the physiological function of the protein and the dynamic change of the expression level in its different periods. Proteomics research can realize the docking and confirm with the genome, directly reveal the pathological mechanism of occurrence and development of the human major disease.
Proteomics research on most orthopaedic disease is still in the initial stage. In recent years, proteomics research about osteonecrosis of femoral head is mainly on serology and experimental animal tissue. Some research has found that a few high expression proteins are closely related with osteonecrosis. But problems are:serum protein component is not stable, easily influenced by other factors, repeatability of experimental data is poor; and the data from animal experiment is different.
Moreover, these proteomics research mostly based on conventional two-dimensional gel electrophoresis (2-DE) technology which has certain limitation: (1) the sample weight restrictions on that low abundance protein (<1 ng) hard to detection; (2) the results of electrophoresis-need dyeing and the results of dyeing combined with different proteins are different; (3) this technology is no-automatic processing, time-consuming, cannot undertake high-throughput analysis.
Extracting proteins from bone tissue generally have two methods:chemical method and physical method. Several experiments are taken with chemical methods, although this method can isolate a variety of bone protein and low abundance protein can be effectively identified, but it needs about a week' time, and easily led to the protein denaturation,so it is not suited for Western blot study. In 2009, WangLe used hammering grinding method to extract proteins from bone tissue, easily and do not destroy the structure and physical and chemical properties of proteins, worthy of using for reference.
At present there are many new proteomics analysis methods, among which the difference in-gel electrophoresis (DIGE) technology becomes one of the most popular one, because it's not only have the high-resolution feature, inherited from two-dimensional gel electrophoresis(2-DE), but also have high reproducibility, high sensitivity, high throughput and high dynamic range. Because of these advantages, DIGE has become the most popular research means on proteomics.
Objective In order to find different proteins, explore its pathogenesis, provide theory'basis for screening markers and targeted therapy. Based on above background, we designed this experiment. We collected femoral head from the patients with non-traumatic osteonecrosis of the femoral head and femoral neck' fracture, used liquid nitrogen grinding to extract total proteins from bone tissue, separated and identified protein with DIGE and MALDI-TOF/TOF, then made a bioinformatic analysis,
Methods Our research is divided into four parts. The first part, collecting bone tissue sample. The second part, establishing a electrophoregram of DIGE. The third part, obvious difference protein identification and bioinformatics analysis. The fourth part, confirming expression level of some protein through Western blot test.
Results 1600±50 protein points were successfully separated, and 274 points were obviously different. We dug 50 points which expressed differently more than two times with EttanTM Spot picker automatic workstations, identified with MALDI TOF/TOF mass spectrometer. Finally,9 different proteins were identified, which are apolipoprotein Al (APOA1), heat shock proteinβ-1 (HSPβ-1), ATP synthaseβsubunit (ATP5β), fibrinogenγchain (FGG), fibrinogenβchain (FGB), serum albumin (ALB), sulfur oxygen protein peroxide oxidoreductase (PRDX2), transferrin (TF) and actinl (ACTG1) respectively. They are all up-regulation except fibrinogen. Through a variety of database, these proteins involved in lipids metabolism, energy metabolism, cytoskeleton, signal transduction, redox etc. Results of Western blot showed the expressions of APOA1 and ATP5βwere consistent with DIGE.
Conclusions:
1. Extracting total protein of bone tissue by liquid nitrogen grinding can meet the require of subsequent proteomics research and proteins have little degradation.
2. DIGE can separate total proteins of bone tissue and find different proteins efficiently.
3. By identifying different proteins with MALDI-TOF/TOF mass spectrometry, we obtained 9 proteins. By bioinformatics analysis, these proteins are all related to occurrence and development of osteonecrosis. So our results provide theoretical basis for screening diagnosis biomarkers and molecular target therapy for ONFH.
4. Through Western blot, we proved the reliability of our research.
Innovations:to our knowledge, there are no reports on proteomic analysis of bone tissue from patients of ONFH with using DIGE and MALDI-TOF/TOF. In our study, we directly use bone tissue from patients of ONFH for research, data is more objective; use physical method of liquid nitrogen grinding to extract protein to reduce protein degradation; use DIGE to find different proteins,the result is more repetitive and sensitive.
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