荧光差异凝胶电泳筛选胆管癌胆汁差异表达蛋白的研究

目的 探讨双向荧光差异凝胶电泳筛选胆管癌胆汁差异表达蛋白的可行性.方法 实验组与对照组胆汁各6例,分别取自胆管癌及胆总管结石病例.实验与对照组每例各取100μg蛋白质,分别标记400 pmol的Cy3与Cy5,另每例各取50 μg混合后制备成内标样品并标记2.4 nmol的Cy2.标记后的实验组、对照组及内参组样品各取100 μg等量混合后在同一凝胶内进行双向荧光差异凝胶电泳,通过质谱分析,筛选并鉴定有统计学意义的差异表达蛋白,探讨其生物学意义.结果 实验组与对照组共筛选出55个差异表达蛋白,其表达量两组间相差1.5倍以上,t检验有统计学意义(P＜0.05).对其中15个差异表达蛋白进行质谱鉴定,共鉴定出8种差异表达蛋白.结论 双向荧光差异凝胶电泳胆汁中筛选胆管癌胆汁差异表达蛋白是可行的,可用于胆管癌早期诊断标志物的筛选.

Abstract：

Objective To determine the probability of identification of differential expression of biliary proteins induced by cholangiocarcinoma using 2D-DIGE. Methods Bile was obtained from 12patients with obstructive jaundice (including 6 cases of cholangiocarcinoma and 6 of cholelithiasis).Each sample was labeled with three different CyDyes (y3,Cy5,Cy2) including one internal standard,pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. MALDI-TOF-MS and bioinformatics were adopted to identify and elucidate the significance of differentially expressed proteins in bile induced by cholangiocarcinoma. Results 55 matched protein spots differences in abundance were detected with statistical variance of two groups(Average Volum Ratio ≥1.5, t-test, P＜0. 05). Among these proteins, 13 PMF were obtained by MALDI-TOF-MS analysis. Eight proteins were identified by searching a protein database. Conclusion The differentially displayed proteomes between the pathological bile obtained from benign and malignant obstructive jaundice indicates the potential application of 2D-DIGE to identify the biomarker of cholangiocarcinoma.     

瘢痕疙瘩与正常皮肤的蛋白质组学研究

目的 通过比较瘢痕疙瘩与正常皮肤组织的蛋白质组表达差异,筛选出与瘢痕疙瘩产生相关的蛋白质.方法 2010年1月至6月运用蛋白质组学技术,对8例瘢痕疙瘩组织和3例正常皮肤组织进行差异双向凝胶电泳(2D-DIGE),选择差异蛋白质斑点,进行基质辅助激光解离飞行时间(MALDI-TOF/TOF)质谱分析和生物学信息分析.结果 成功建立瘢痕疙瘩和正常皮肤组织的双向凝胶电泳图谱,瘢痕疙瘩和正常皮肤组织凝胶电泳图谱中平均蛋白质斑点数分别为2978和3053,其中表达差异超过4倍的斑点共有40个,质谱分析和数据库检索共鉴定出32种蛋白质,包括上调蛋白有20种,下调蛋白有12种.从功能上可分为载体蛋白(3种)、信号转导蛋白(4种)、增殖凋亡相关蛋白(2种)、细胞骨架蛋白(6种)、细胞外基质蛋白(8种)、免疫因子(3种)、肿瘤相关蛋白(2种)和未知功能蛋白(4种).结论 蛋白质组学能较好地显示瘢痕疙瘩与正常皮肤组织间的蛋白质表达差异.对这些差异蛋白质进一步深入研究,将有助于揭示瘢痕疙瘩的发病机制,也为发现新的治疗靶点提供线索和依据.

Abstract：

Objective To investigate and search correlative proteins of keloid by comparing the results of differential proteomic analysis between keloid and normal skin. Methods From January 2010 to June 2010 two-dimensional gel electrophoresis was used to define patterns of protein expression in keloid skin from 8 patients and matched normal skin from 3 patients. Differential expression protein spots were showed and analyzed by matrix-assisted laser desorption ionization-time of flying/time of flying(MALDI-TOF/TOF) mass spectrometry. Results This study succeeded to provide a two-dimensional protein profiling comparison between normal skin and keloid. Gel-analysis software identified an average of 2978 spots in keloid while 30S3 spots in normal skin and statistical filtering yielded 40 spots of a 4-fold change, 32 of which were identified by using mass spectrometry, 20 were up-regulated and 12 were down-regulated. Functional analysis revealed that these proteins could be fractionated to carrier proteins (3 proteins), signal transduction proteins (4 proteins) , proliferation and apoptosis related proteins (2 proteins) , cytoskeleton proteins(6 proteins) , extracellular matrix proteins(8 proteins) , immunity related proteins (3 proteins) , tumor related proteins (2 proteins) , and function unknown protein (4 proteins). Conclusions Proteomic analysis can identify the proteins with variance of keloid versus normal skin. The further research to these differential proteins may help reveal the pathogenesis of keloid and provide new treatments for keloid.     

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.     

热损伤角质形成细胞外泌蛋白的差异表达

目的 初步探索热损伤表皮角质形成细胞(keratinocytes,KC)培养上清蛋白质的整体变化规律.方法 选用人源性表皮角质形成细胞建立KC热损伤模型,收集正常培养及热损伤后12 h的KC培养上清,超滤、冻干,获得蛋白样品.运用固相pH梯度双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)分离正常及热损伤后KC培养上清中的总蛋白质,凝胶银染显色后选用ImageMaster 2D图像分析软件,进行比较分析识别差异表达的蛋白质.结果 ①正常培养及热损伤后KC培养上清凝胶的平均蛋白质点数分别为1 898±113和1 877±97,经匹配每张胶上用于统计学分析的点为1118个.②正常培养及热损伤后KC的双向凝胶电泳图谱中差异表达蛋白点数为26个,其中16个点在正常KC培养上清中高表达,10个点在热损伤后KC培养上清中高表达.③经质谱分析、数据库搜索,成功鉴定了16个差异蛋白质点,包括10种蛋白.热损伤后表达下调的蛋白质点有:alpha-enolase,actin cytoplasmic2,peroxiredoxin-4,phosphoglycerate mutase 1,G proteinregulated inducer of neurite outgrowthl;表达上调的蛋白质点有:purine nucleoside phosphorylase,tumor necrosis factor ligand superfamily member10,proteasome subunit alpha type-7,UDP-glucose 6-dehydrogenase.结论 通过建立正常及热损伤KC培养上清的双向凝胶电泳图谱,发现两者间存在一些差异表达的蛋白,为进一步从中优选调控组织损伤修复的主要作用分子,明晰创面修复过程中修复细胞间的调控机制提供了一定的理论依据.

Abstract：

Objective To compare the difference of protein expression in the supernatant of heat injured keratinocytes ( KC) and normal KC. Methods A model of heat injured KC was produced in vitro. The supernatant of normal KC and heat injured KC was collected after culture for 12 hours, and was ultrafiltered and lyophilized to get the protein. The protein sample was separated by immobilized pH gradient based two dimensional gel electrophoresis (2-DE). The gel was stained and the different expression of protein was analyzed using ImageMaster 2D analysis software. Results ①Average protein spots were 1 898 ±113, 1 877 ±97 in the supernatant of normal and heat injured KC and 1118 protein spots could be used for statistical analysis. ②Statistical result showed that 26 protein spots were significantly different between the two groups. 16 protein spots were higher in the supernatant of normal KC and then 10 protein spots were lower in the normal group. @16 protein spots, which included 10 kinds of proteins, were identified successfully as different spots. Lower expression proteins were alpha-enolase, actin cytoplasmic2, peroxiredoxin-4, phosphoglycerate mutase 1, G protein-regulated inducer of neurite outgrowth 1 in the supernatant of heat injured KC. Higher expression proteins in heat KC were purine nucleoside phosphorylase, tumor necrosis factor ligand superfamily memberl0, proteasome subunit alpha type-7, UDP-glucose 6-dehydrogenase in the supernatant of heat injuryed KC. Conclusions The result indicated that there are some significant different expression proteins in the supernatant of normal KC and heat injured KC.These findings provide new data for screening major molecules of tissue repair and finding the mechanism of wound repair.     

金黄色葡萄球菌侵袭烫伤家兔致淋巴细胞蛋白质组的变化

Objective To study the proteomic change in Iymphocytes of rabbits with scald injury and Staphylococcus aureus (SA) invasion. Methods Twenty-four rabbits were divided into four groups as follows: control group, scald group, scald with SA invasion 2 hs group, and scald with SA invasion 6 hs group, according to random number table, with 6 rabbits in each group. Except for rabbits in control group (sham scald at 37 ℃ ) , rabbits in the other 3 groups were subjected to 30% TBSA full-thickness scald. Rabbits in SA invasion 2 and 6 hs groups were injected with 2 mL (1.0×108 CFU/mL) SA suspension, which was in the log growth phase, via auricle vein 18 hs and 22 hs after injury. Whole blood samples were collected from carotid artery of rabbits in 4 groups 24 hs after scald. Lymphocytes were isolated and its ex-tracted proteins were analyzed by two-dimensional gel electrophoresis coupled with mass spectroscopy. Re-suits About 1030 protein spots of lymphocytes were detected in each group. Compared with that of control group, 19 protein spots were found to be differentially expressed in the other 3 groups, and 11 spots (10 pro-teins) were identified. Expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate ki-nase, glutamate dehydrogenase and annexin 1 were down-regulated, but expression level of peroxiredoxin was up-regulated obviously. Conclusions There is obvious proteornic change in lymphocytes of scalded rabbits or of scalded rabbits invaded by SA, and it may relate to immune suppression and sepsis after injury.     

金黄色葡萄球菌侵袭烫伤家兔致淋巴细胞蛋白质组的变化

Objective To study the proteomic change in Iymphocytes of rabbits with scald injury and Staphylococcus aureus (SA) invasion. Methods Twenty-four rabbits were divided into four groups as follows: control group, scald group, scald with SA invasion 2 hs group, and scald with SA invasion 6 hs group, according to random number table, with 6 rabbits in each group. Except for rabbits in control group (sham scald at 37 ℃ ) , rabbits in the other 3 groups were subjected to 30% TBSA full-thickness scald. Rabbits in SA invasion 2 and 6 hs groups were injected with 2 mL (1.0×108 CFU/mL) SA suspension, which was in the log growth phase, via auricle vein 18 hs and 22 hs after injury. Whole blood samples were collected from carotid artery of rabbits in 4 groups 24 hs after scald. Lymphocytes were isolated and its ex-tracted proteins were analyzed by two-dimensional gel electrophoresis coupled with mass spectroscopy. Re-suits About 1030 protein spots of lymphocytes were detected in each group. Compared with that of control group, 19 protein spots were found to be differentially expressed in the other 3 groups, and 11 spots (10 pro-teins) were identified. Expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate ki-nase, glutamate dehydrogenase and annexin 1 were down-regulated, but expression level of peroxiredoxin was up-regulated obviously. Conclusions There is obvious proteornic change in lymphocytes of scalded rabbits or of scalded rabbits invaded by SA, and it may relate to immune suppression and sepsis after injury.     

金黄色葡萄球菌侵袭烫伤家兔致淋巴细胞蛋白质组的变化

Objective To study the proteomic change in Iymphocytes of rabbits with scald injury and Staphylococcus aureus (SA) invasion. Methods Twenty-four rabbits were divided into four groups as follows: control group, scald group, scald with SA invasion 2 hs group, and scald with SA invasion 6 hs group, according to random number table, with 6 rabbits in each group. Except for rabbits in control group (sham scald at 37 ℃ ) , rabbits in the other 3 groups were subjected to 30% TBSA full-thickness scald. Rabbits in SA invasion 2 and 6 hs groups were injected with 2 mL (1.0×108 CFU/mL) SA suspension, which was in the log growth phase, via auricle vein 18 hs and 22 hs after injury. Whole blood samples were collected from carotid artery of rabbits in 4 groups 24 hs after scald. Lymphocytes were isolated and its ex-tracted proteins were analyzed by two-dimensional gel electrophoresis coupled with mass spectroscopy. Re-suits About 1030 protein spots of lymphocytes were detected in each group. Compared with that of control group, 19 protein spots were found to be differentially expressed in the other 3 groups, and 11 spots (10 pro-teins) were identified. Expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate ki-nase, glutamate dehydrogenase and annexin 1 were down-regulated, but expression level of peroxiredoxin was up-regulated obviously. Conclusions There is obvious proteornic change in lymphocytes of scalded rabbits or of scalded rabbits invaded by SA, and it may relate to immune suppression and sepsis after injury.     

金黄色葡萄球菌侵袭烫伤家兔致淋巴细胞蛋白质组的变化

Objective To study the proteomic change in Iymphocytes of rabbits with scald injury and Staphylococcus aureus (SA) invasion. Methods Twenty-four rabbits were divided into four groups as follows: control group, scald group, scald with SA invasion 2 hs group, and scald with SA invasion 6 hs group, according to random number table, with 6 rabbits in each group. Except for rabbits in control group (sham scald at 37 ℃ ) , rabbits in the other 3 groups were subjected to 30% TBSA full-thickness scald. Rabbits in SA invasion 2 and 6 hs groups were injected with 2 mL (1.0×108 CFU/mL) SA suspension, which was in the log growth phase, via auricle vein 18 hs and 22 hs after injury. Whole blood samples were collected from carotid artery of rabbits in 4 groups 24 hs after scald. Lymphocytes were isolated and its ex-tracted proteins were analyzed by two-dimensional gel electrophoresis coupled with mass spectroscopy. Re-suits About 1030 protein spots of lymphocytes were detected in each group. Compared with that of control group, 19 protein spots were found to be differentially expressed in the other 3 groups, and 11 spots (10 pro-teins) were identified. Expression levels of cofilin, cyclophilin A, ubiquitin, nucleoside diphosphate ki-nase, glutamate dehydrogenase and annexin 1 were down-regulated, but expression level of peroxiredoxin was up-regulated obviously. Conclusions There is obvious proteornic change in lymphocytes of scalded rabbits or of scalded rabbits invaded by SA, and it may relate to immune suppression and sepsis after injury.     

人股动脉粥样硬化相关蛋白的鉴定及其生物学作用

目的 应用比较蛋白质组学方法探讨人动脉粥样硬化闭塞症(ASO)差异蛋白质的表达及其在ASO发病机制中的作用.方法 选取ASO股动脉8例及正常股动脉5例,提取组织蛋白质,行双向凝胶电泳分离、质谱分析及数据库查询,获得差异蛋白信息.结果　成功建立ASO和正常股动脉双向电泳图谱,含量相差大于2倍以上的蛋白质53个,27个上调,26个下调,质谱鉴定出13种蛋白质,主要与炎症、免疫、氧化应激、脂质代谢等相关.结论 ASO与正常股动脉蛋白质组明显差异,差异蛋白质可能在ASO中起重要作用.

Abstract：

Objective We used proteomic profiling in an attempt to differentiate and identify histological proteins that were associated with atherosclerosis obliterans (ASO) of human femoral artery.Methods We comparatively analyzed the proteome of 8 atherosclerotic and 5 normal femoral arteries. The differentially expressed proteins were visualized by two dimensional electrophoresis (2-DE) and sequenced by matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). The protein identification program was used to search the National Center for Biotechnology Information (NCBI) database and International Protein Index (IPI). Results A total of 53 distinct spots corresponding to 13 different proteins were identified by MALDI-TOF-MS using the NCBI and IPI databases. The function information of these 13 proteins mainly involved the pathogenetic mechanisms such as inflammation, innate immunity, oxidative stress, lipid metabolism, amyloid degeneration and so on. Conclusion ASO is associated with distinct patterns of protein expression in the femoral arteries, and differentially expressed 13 proteins may contribute to the pathogenesis. These findings might provide needed biomarkers for ASO and new insight into its pathophysiology.     

胰腺癌血清相关蛋白质分子的差异表达

目的 利用双向差异凝胶电泳(2D-DIGE)的方法建立胰腺癌、慢性胰腺炎和正常对照人群外周血清的差异蛋白质双向凝胶电泳图谱并分析差异蛋白质点.方法 双向差异凝胶电泳分离20例胰腺癌患者、10例慢性胰腺炎患者和20例正常对照组人群外周血清蛋白,比较不同血清中蛋白质表达的差异.结果 成功建立胰腺癌、慢性胰腺炎和正常人之间的外周血清蛋白质双向差异凝胶电泳图谱,软件分析共发现了168个明显差异表达的蛋白质点,其中在胰腺癌组与正常对照组的比较中有22个蛋白质点在胰腺癌血清中表达上调,29个蛋白质点下调;在胰腺癌组和慢性胰腺炎组的比较中有24个蛋白质点在胰腺癌血清中表达上调,54个表达下调;在慢性胰腺炎组和正常对照组的比较中有20个蛋白质点表达上调,19个蛋白质点表达下调.结论 双向差异凝胶电泳是快速有效的分离蛋白质新的方法,得到的双向差异凝胶电泳图谱以及显著表达差异的蛋白质点为质谱鉴定提供了实验基础.

Abstract：

Objective To analyze and compare the proteomes expressed by human pancreatic carcinoma, chronic pancreatitis and normal control groups. Methods Differencial expression of the proteomes in peripheral serum was analyzed by fluorescence 2-D difference gel electrophoresis (2D-DIGE). Results 2D-DIGE protein maps in 20 patients with pancreatic cancer, 10 patients with chronic pancreatitis and 20 normal controls were analyzed and 168 spots were identified by gel-analysis software. Between pancreatic cancer group and normal control group 22 protein spots were up-regulated and 29 spots down-regulated; 24 spots were up-regulated and 54 spots down-expressed between pancreatic cancer group and chronic pancreatitis group; 20 spots were up-regulated and 19 spots down-regulated between chronic pancreatitis group and normal control group. Conclusion 2D-DIGE was a rapid and efficient method to separate proteins. 2D-DIGE protein maps and different protein spots would provide an exprimental basis for the phase of mass spectrometry.     

特发性高草酸尿症大鼠肝脏差异蛋白质的筛选

目的 寻找特发性高草酸尿症大鼠肝脏组织差异表达的蛋白质.方法 取3只雄性特发性高革酸尿症大鼠和3只正常对照雄性大鼠,分别取其肝脏组织约300 mg后提取其中的总蛋白.以荧光差异显示凝胶电泳(DIGE)技术分离肝脏中的全部蛋白质,用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)和DeCyderTM v6.5软件进行蛋白质的分析和鉴定.结果 总共筛选出21个差异表达蛋白质点,其中11个蛋白质点在特发性高草酸尿症组表达上调,10个蛋白质点下调.最终有18个差异蛋白质点被鉴定确认.结论 筛选出的这些差异蛋白质可能与特发性高草酸尿症有关.     

胆管癌肿瘤标志物的蛋白质组学初步研究

目的:蛋白质组学分析被认为是一种寻找潜在生物标志物,尤其是肿瘤标志物的有效方法。二维电泳蛋白质组学分析是一种流行和行之有效的技术。使用二维微分凝胶电泳(2-D DIGE)的研究是为了解癌症病人和对照组不同的蛋白质表达,并从蛋白质在二维图像上分布模式发现潜在的肿瘤标志物。方法:选取5例胆管癌病人和5例对照组胆管标本作为实验样本。然后提取DIGE裂解的蛋白质。随后提取的蛋白质以3种不同CyDyes进行标记,并通过2-D DIGE分离。结果:2-D DIGE从肿瘤和对照组标本中分离出多种蛋白质,点状分布在长13 cm、pH值在3.0～10.0的固相pH梯度胶条上。经过DeCyder软件的生物变异分析模块分析,所有胆管癌病人有20处蛋白质点被发现显着升高,5处蛋白质点下调(配对t检验,P     

特发性高草酸尿大鼠肝脏蛋白质组的电泳分析

目的：建立稳定的特发性高草酸尿大鼠肝脏蛋白质组的双向电泳图谱.探讨其差异蛋白质组与特发性高草酸尿的关系.方法：取特发性高草酸尿大鼠及正常对照大鼠的肝组织300 mg.匀浆提取肝组织总蛋白,分别用Cy3或Cy5标记,每一对Cy3和Cy5标记样品均与一个Cy2标记的内标等量混合,采用凝胶内差异显示电泳（DIGE）技术进行电泳分离,经过不同激光下扫描得到不同样品的蛋白质组图谱.获得的图谱经DeCyderTM v6.5软件进行分析.结果：在特发性高草酸尿大鼠肝组织中共筛选出21个差异表达蛋白质点,有11个蛋白质表达水平显著增加,另外10个蛋白质表达水平显著下降.结论：利用DIGE技术可以作胶内对比分析,并可根据内标消除胶与胶之间的差异,从而提高统计可信度.分析出的21个差异蛋白质可能与特发性高草酸尿的发生有密切关系.     

胃癌基因表达谱和蛋白质表达谱的分析研究

目的从转录组水平和蛋白质组水平寻找胃癌组织与正常胃组织间的差异表达基因和蛋白。方法应用含有14592个已知基因及表达序列标签的cDNA表达谱芯片获取基因表达谱信息．50％以上样本中Cy3／Cy5荧光强度比大于或等于2和小于或等于0．5的基因分别为在胃癌中表达上调或下调的基因。采用双向凝胶电泳（2-DE）分离32例经手术切除的胃癌患者的胃癌组织和正常胃组织总蛋白．对差异表达蛋白质点利用基质辅助激光解析电离飞行时间质谱（MALDI—TOF—MS）进行分析．获取肽质量指纹图谱（PMF）。同时搜索SWISS-PROT数据库鉴定蛋白质。结果与正常组织相比．胃癌组织中差异表达基因共有387个,其中表达上调的有149个基因．上调超过3倍的有29个基因：表达下调的有238个基因．其中下调超过5倍的有21个基因。在蛋白质水平鉴定了15个差异表达蛋白．在肿瘤组织中高表达的有10个．其余5个在肿瘤组织中低表达。胃癌中过表达的基凶与蛋白产物主要与细胞骨架运动、细胞增殖及信号传导有关．表达下调的则主要与细胞免疫防御、毒理代谢有关。结论从转录组水平和蛋白质组水平对胃癌基因表达谱进行分析．不仅有助于从分子水平全方位理解胃癌的发病机制及生物学特性．同时也有助于进一步发现新的胃癌相关标志物和基因治疗的靶点。     

血清CEA阴性结直肠癌患者血清差异蛋白质的初步筛选

目的分析血清CEA阴性结直肠癌患者、血清CEA阳性结直肠癌患者与正常人血清中差异表达蛋白,筛选潜在的与血清CEA阴性结直肠癌患者诊断及预后密切相关的血清蛋白质。方法血清CEA阴性结直肠癌患者、血清CEA阳性结直肠癌患者与正常人空腹血清标本各11例分别等量混合成3组样本,利用双向凝胶电泳（2-DE）及基质辅助激光解吸电离飞行时间质谱（MALDI_TOF-MS／MS）技术筛选及鉴定差异表达的蛋白点,并对其部分蛋白进行生物学分析。结果筛选出3组间表达量差异2倍以上差异蛋白点共13个,进行MALDI-TOF-MS／MS分析,鉴定出：①血清CEA阴性结直肠癌患者与正常人血清比较,表达下调的蛋白有纤维胶凝蛋白2前体,纤维胶凝蛋白3,载脂蛋白L1,间a胰蛋白酶抑制剂轻链H4,转甲状腺素蛋白,表达上调的蛋白为免疫球蛋白lambda轻链。②血清CEA阳性结直肠癌患者与正常人血清比较,表达下调的蛋白有纤维胶凝蛋白2前体,纤维胶凝蛋白3,载脂蛋白L1,间a胰蛋白酶抑制剂轻链H4,转甲状腺素蛋白,表达上调的蛋白为：载脂蛋白E,结合珠蛋白。③血清CEA阴性结直肠癌患者与血清CEA阳性结直肠癌患者的1个差异点,经鉴定为免疫球蛋白lambda轻链。结论血清CEA阴性结直肠癌患者与血清CEA阳性结直肠癌患者、正常人血清的蛋白质表达谱表现出一定差异,这些差异蛋白有可能成为血清CEA阴性结直肠癌患者诊断与判定预后的血清标志物。     

高糖培养下人肾小球系膜细胞蛋白表达谱变化的研究

目的 利用比较蛋白质组学双向电泳技术研究体系,观察高糖培养下人肾小球系膜细胞（HMC）蛋白表达谱的变化。 方法 人肾小球系膜细胞分为高糖培养组（30 mmol/L）和正常糖组（5 mmol/L）,培养48 h后收集细胞,提取总蛋白,以DIGE饱和荧光标记,双向荧光差异凝胶电泳。应用Typhoon多功能成像系统扫描凝胶,DeCyder 2-D差异分析软件进行图像分析,寻找差异表达蛋白质。采用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）和蛋白质数据库检索鉴定差异蛋白。 结果 通过DeCyder 2-D差异分析软件,发现正常糖组和高糖培养组之间差异大于1.5倍的蛋白质斑点147个,对其中96个差异蛋白质斑点进行了肽质指纹图分析,鉴定出37种蛋白质。其中磷脂酰乙醇胺结合蛋白 1（PEBP-1）、粒溶素、ATP合成酶H+转运线粒体F0复合体亚基F2仅在高糖组表达。高糖刺激后表达上调的蛋白质有24个,包括嗜酸细胞阳离子蛋白、RGS膜相互作用蛋白16（MIR16）、肽酰-脯氨酰-顺反式异构酶、disks large homolog DLG2、早发性乳腺癌2（BRCA2）、儿茶酚-邻-甲基转移酶等;表达下调的蛋白有5个,包括O-GlcNAc transferase-interacting protein 106 000 isoform 1、proteasome beta 6 subunit precursor、NEFA-interacting nuclear protein NIP30等。 结论 高糖培养下HMC内147种蛋白质的表达发生变化。这些蛋白质广泛参与高糖对HMC的细胞骨架、糖代谢、细胞分裂、基因转录、信号转导、磷酸化、细胞增殖、凋亡等的调节。深入分析这些差异表达蛋白的功能与调控,有望为阐明糖尿病肾病的发病机制提供重要的实验依据。     

人胆汁蛋白质组双向荧光差异凝胶电泳图谱的建立与差异分析

目的 建立人胆汁蛋白质组双向荧光差异凝胶电泳分析流程用于胆汁比较蛋白质组学研究.方法 实验组与对照组样本各6例,分别取自于胆管癌及胆总管结石病例.样本经纯化处理及定量后,分别标记不同的荧光染料后进行双向电泳与差异分析.结果 两组样本均获得高分辨力的双向电泳图谱;各标记蛋白质点的荧光强度与蛋白表达量呈线性关系;实验组与对照组间共筛选出55个差异表达蛋白,其表达量两组间相差1.5倍以上,t检验有统计学意义(P＜0.05).结论 建立了基于双向荧光差异凝胶电泳基础上的胆汁比较蛋白质组学的分析流程.     

蛋白质组技术在胃癌相关标志物筛查中的应用

目的 建立胃癌组织和正常胃组织双向凝胶电泳图谱，鉴定差异表达蛋白，从中发现有意义的胃癌相关标志物。方法 采用双向凝胶电泳（2-DE）分离胃癌组织和正常胃组织总蛋白，银染显色，PDQuest软件分析，从中选取差异表达蛋白质点，通过基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）或MALDI-TOF-TOF-MS鉴定差异表达的蛋白质。结果 获得重复性和分辨率较好的胃组织双向凝胶电泳图谱；发现23个差异表达蛋白质，其中15个得到鉴定，在肿瘤组织中高表达的有10个，其余5个在肿瘤组织中低表达。结论 建立了胃癌组织和正常胃组织双向凝胶电泳图谱，并应用质谱技术鉴定了15个差异表达蛋白质。     

大肠癌发生和肝转移的蛋白质组学研究

目的 利用蛋白质组研究技术分离、鉴定大肠癌肝转移相关蛋白。方法 用等电聚焦／SDS聚丙烯酰胺双向凝胶电泳对比分析12例大肠癌患者原发灶、癌旁肠黏膜和肝转移灶中疏水蛋白表达差异,经肽质量指纹谱分析、鉴定差异蛋白点。结果 双向电泳图像对比分析表明,正常结直肠黏膜组织、癌原发灶和肝转移灶中蛋白质表达有显著差异。对9个差异表达蛋白点分析鉴定,钙调蛋白联合体、核糖核酸酶-6-前体蛋白以及XP-040720蛋白在正常肠黏膜表达,但在癌原发灶和肝转移组织中表达缺失。前阿朴脂蛋白在正常肠黏膜、癌原发灶及肝转移灶中呈递增表达。β-珠蛋白在肝转移灶和正常肠黏膜中表达,而在癌原发灶中表达缺失。Cdc42蛋白在肝转移灶中特异表达。差异蛋白点C4、N7和N9肽指纹谱与已知蛋白同源性低,为候选大肠癌发生和肝转移相关新蛋白。结论 钙调蛋白联合体、核糖核酸酶-6-前体蛋白、α-甘露糖苷酶Ⅰ表达缺失与大肠癌发生和肝转移有关;前阿朴脂蛋白增强表达与大肠癌发生和肝转移有关;Cdc42蛋白、β-珠蛋白表达与肝转移有关。