高低分化的胃癌细胞内高尔基体的蛋白质组学研究
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摘要
背景:胃癌是世界上第二种致死性肿瘤,也是我国主要恶性肿瘤之一,其预后也差,中晚期病人的五年生存率仅有20-30%左右,严重威胁着人民的身体健康。研究发现高中分化腺癌的凋亡指数明显高于低分化腺癌,而分化差的胃癌细胞却具有逃避细胞凋亡的机制。细胞凋亡受抑制可能在胃癌的浸润和转移中发挥着重要的作用。有报道称胃癌的预后不仅和肿瘤自身的增殖活性有关,还和肿瘤自身的凋亡活性有关,那些具有较高的细胞凋亡指数的胃癌患者,其预后较好。并且在前期研究工作中也发现,具有侵袭转移的胃癌细胞中高尔基体囊泡扩大,而分泌功能无或低的胃癌细胞内高尔基体含量极少。可见高尔基体与胃癌细胞的发生发展存在着紧密联系。此外Kellokumpu等报道结肠癌糖基化异常与高尔基体结构的改变有关。这些均提示高尔基体在胃肠癌的发生发展及其生物学行为中起着重要作用。
高尔基体(golgi apparatus)是一个分泌通道的主要细胞器,除了进行蛋白加工,运输外,还参与蛋白质的糖基化、细胞的分泌和溶酶体的形成,最近有报道称高尔基体还参与细胞的凋亡,高尔基体本身体内就有一套凋亡信号网络。此外该细胞器还与细胞的增值分化,细胞分裂等诸多细胞活动有关,这些均表明此细胞器的功能复杂,目前对该细胞器功能的认识在许多方面的机制仍不清楚。此外据人类基因组推测,高尔基体上至少有一千多种蛋白,而目前已知的蛋白却仅有五,六百个。这些问题的存在均说明该细胞器亟待深入研究。
蛋白质组学技术的迅猛发展,特别是双向电泳和生物质谱技术的突破性发展,为组织或细胞的全蛋白质谱的研究提供了强大的技术支撑。其中发展出来的一个分支——亚细胞器蛋白质组学,主要针对细胞内一些特定细胞器的蛋白质进行分析,具有明确的亚细胞定位性,不仅可以明显提高蛋白质的分辨率与鉴定效率,还能大大提高某些低丰度蛋白质的检出。因此,这一技术的应用特别有利于帮助认识细胞器的结构和功能,提供细胞器上的蛋白组成情况,并提供关于蛋白质在细胞间转移和相互作用这些方面的信息。故亚细胞器蛋白质组学技术的提出和应用对高尔基体的功能研究也是非常适用的,在于一方面可以发现高尔基体上的新蛋白,完善高尔基体的蛋白组成情况;另一方面,可以找出在病理条件或细胞功能状态发生改变时,高尔基体的蛋白变化,为高尔基体的功能研究提供有力的实验证据。
基于以上背景,本实验主要是以分化不同的胃癌细胞的高尔基体为研究对象,运用亚细胞器蛋白质组学的技术路线研究高尔基体在胃癌细胞分化增殖中的作用。首先将胃癌细胞内的高尔基体进行分离纯化,提出纯度较高的高尔基体。然后运用双向电泳技术进行蛋白分离,建立高低分化的胃癌细胞内高尔基体的蛋白图谱,并找出两者中的差异蛋白。最后运用飞行时间质谱对差异蛋白进行鉴定,western blotting进行验证。从而对高尔基体在胃癌细胞的发生发展中的作用进行探讨。
本研究主要包括以下三部分:
第一部分:胃癌细胞内高尔基体的分离与鉴定
目的:从胃癌细胞内分离出纯度较高的高尔基体,并对所分离纯化的高尔基体进行形态学及酶学鉴定,以了解其纯度。为亚细胞器蛋白质组学的开展奠定基础。
方法:运用体外大量胃癌细胞的分批分次培养,差速离心结合蔗糖密度梯度离心法纯化分离出高尔基体后,电镜及中性红超活染色进行形态学鉴定,高尔基体标志酶半乳糖基转移酶及硫胺素焦磷酸酶活性测定进行纯度分析。
结果:通过蔗糖密度梯度离心法分离出高尔基体,电镜显示为一些囊泡性结构,中性红超活染色显示所得分离物被染成玫瑰红色,说明分离物为高尔基体。高尔基体标志酶半乳糖基转移酶及硫胺素焦磷酸酶与粗匀浆液相比,分别富集了107,98倍。而其他细胞器的标志酶的含量均大大降低或检测不出。
结论:首次从胃癌细胞内成功分离出纯度较高的高尔基体,为对高尔基体功能的深入研究及后续试验的开展奠定基础。
第二部分:高低分化的胃癌细胞内高尔基体的双向电泳的建立
目的:分别建立高,低分化胃癌细胞内高尔基体的高分辨率,稳定的双向电泳图,了解在两种细胞中高尔基体蛋白的组成情况,对比分析找出两者中的主要差异蛋白。
方法:采用蔗糖密度梯度的超速离心方法分离纯化高尔基体,通过对样品制备,蛋白上样量,聚焦条件及聚焦仪器的选择等方面参数的选择,优化双向电泳条件。在优化条件下,运用双向凝胶电泳(2-DE)技术分离高尔基体蛋白质,用ImageMaster 2D软件分析所得图谱。
结果:通过一系列条件优化,分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱。采用17cm, pH 3~10非线性胶条和12%SDS-PAGE所获得的高分化及低分化胃癌细胞高尔基体的双向电泳图谱背景干净,蛋白点的分布均匀。通过图象扫描和软件分析可以明显检测到800余个蛋白点,高分化胃癌细胞的高尔基体可检测到的蛋白点有769±22个,而低分化胃癌细胞的高尔基体的蛋白点有837±43个,对两者进行了比较蛋白质组学研究,共发现主要有差异蛋白100余个,其中低分化胃癌细胞的高尔基体主要有80多个蛋白表达上调,而高分化胃癌细胞内主要有30多个蛋白表达上调。
结论:首次成功建立了高,低分化胃癌细胞中高尔基体的双向电泳图,直观的展示了胃癌细胞高尔基体的蛋白分布情况,找出了一百多个差异蛋白点,为胃癌细胞内高尔基体功能的深入研究奠定了基础,并首次明确展示了高尔基体在胃癌细胞增值分化发展过程中的蛋白变化情况。
第三部分:高低分化的胃癌细胞内高尔基体中差异蛋白的质谱分析及western blotting验证
目的:通过质谱分析,将高低分化胃癌细胞内的差异蛋白进行鉴定,明确是高尔基体上的哪些蛋白与胃癌细胞的增殖分化有关,从而探讨高尔基体在胃癌发生发展中的作用。
方法:对差异蛋白经过胶内切取,胰酶消化后,运用基质辅助激光解吸离子化飞行时间质谱(MALDI—TOF MS)鉴定蛋白质点,数据库搜索,共鉴定出39个蛋白点。并经western blotting进一步验证了鉴定出的两种蛋白TCP-1和Annexin 2。
结果:在对高低分化胃癌细胞高尔基体中的七十多个较明显蛋白点进行质谱鉴定和数据库检索分析时,发现60%以上的蛋白点的肽质量指纹图谱都有较好的信噪比和质量精度,并成功鉴定出39个蛋白点。这些蛋白,按功能分,包括膜活性相关蛋白,细胞结构蛋白,调节蛋白及代谢酶,癌相关蛋白,囊泡运输蛋白,细胞增殖分化相关蛋白,凋亡相关蛋白,未知蛋白等。其中低分化胃癌细胞的高尔基体中与凋亡相关的蛋白大大减少,而影响细胞增殖分化的蛋白却大大增加。这些蛋白变化,对人们认识高尔基体的功能提供了有效的参考。并且为高尔基体在胃癌发生发展中所起的作用也提供了蛋白信息。
结论:经过前述一系列蛋白质组学研究途径,发现了在高分化胃癌细胞内高尔基体上的抗凋亡蛋白明显高于低分化的胃癌细胞,而低分化胃癌细胞内的高尔基体上与分化增殖有关的蛋白却大大增加。高尔基体上出现的这些蛋白变化,说明高尔基体通过对这类蛋白含量的调节,从而影响胃癌细胞的增值分化,提示高尔基体可能在胃癌发生发展中通过对凋亡蛋白,增值分化蛋白的调节,影响胃癌的发生发展。
Background: gastric cancer is still the second most common cause of death from cancer in Asia and worldwide. It remains a great clinical challenge, due to its very poor prognosis. There are reports indicated that the prognosis of gastric cancer is not only relevant to proliferation, but also apoptosis of tumor itself. The poorly differentiated gastric carcinomas have escape apoptosis mechanism, and apoptosis inhibited may exert important role in invasion and metastasis of gastric carcinomas. Our preceding research suggested that there was a rapid proliferation and expansion of the GA in gastric mucinous adenocarcinomas with high secretion of hydratase and liability to invasion and metastasis, while it’s hard to see GA in gastric tubular adenocarcinomas with no or little secretion of hydratase. Another report shows that the Golgi has altered structure and abnormal glycosylation in colorectal cancer. In addition, the transportation of Golgi appeared functional disturbance in gastric cancer. So we carried out our study using gastric cancer cells from the point of Golgi and hoped to find out the function of Golgi played in gastric cancer from protein level.
The Golgi complex is a major organelle of the secretory pathway. The key function of it is to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. Nevertheless the Golgi complex has many other complex functions, such as cell polarization and differentiation, mitosis and intracellular signaling, as well as cell division. Recent datas show that the Golgi also plays an important role in apoptosis. These unresolved questions regarding nonclassical functions of the Golgi have stimulated us to do more research on this organelle with the goal of comprehensively identifying its protein components.
Proteome studies have so far been developed with the technology. It includes two main technology approaches, one of which is using 1- or 2-DE for protein separation and MS for protein identification, and the other is the multidimensional protein identification technology (MudPIT) or‘shotgun’proteomics approach. The second one enabled more comprehensive high-throughput profiling strategies of enriched organellar fractions, resulting in hundreds of proteins identifications. It is very feasible to build up a holistic picture of research material and discover new proteins. However the traditional and the most used method is the first one, because the reproducible two-dimensional (2D) gel systems can give the direct picture about the protein complements. Highly reproducible 2D gels allow the one-time identification of a protein spot from a complex or organelle without the requirement of any prior knowledge about the protein being examined. Furthermore, it is possible to begin to address functional questions by comparative analyses of proteomes obtained for organelles during the change of functional states.
In the present study,we used the organellar proteomic analysis to study gastric cancer cells with different differentiation for the first time. The protein complements of purified golgi fractions isolated from well-differentiated gastric cancer cell line MKN-28 and poorly differentiated gastric cancer cell line SGC-7901 respectively were separated in 2-DE gels first, then about 100 proteins different expressed in both pictures were found, and 39 proteins were indentified using MS at last. These proteins have different functions, involving in cell division, transportation, apoptosis, and play a role in the differentiation and development of gastric cancer and so on. It is the first time to open the door which would guide us to understand the relationship between Golgi and gastric cancer and it would help us to do more research for better knowing the function of Golgi in gastric cancer.
The research includes three parts:
Part 1: The Isolation and Assessment of Golgi Apparatus from Gastric Cancer Cells
Objective: To isolate and purify the Golgi apparatus from Gastric Cancer Cells.
Methods: A large number of gastric cancer cells SGC7901 were cultivated in vitro, then Golgi apparatus were isolated from the cells by differential centrifugation combined with sucrose density gradient ultra-centrifugation. Its purity was characterized biochemically by enzymatic assays, morphologically by electron microscopy (EM) and neutral red supravital staining.
Results: Electron microscopy detection showed that the majorities of product were Golgi apparatus. The fraction was stained red by neutral red. Marker enzyme analysis demonstrated that TPP activity increased to 97-fold, and the relative specific activity of galactosyltransferase was 107-fold enriched over the homogenate.
Conclusion: The first successful isolation of Golgi apparatus from gastric cancer cells by using ultra- centrifugation will lead to do more research into the function of GA.
Part 2: Two-dimensional gel electrophoresis of Golgi apparatus isolated from the well-, and poor-differentiated gastric cancer cells respectively.
Objective: To establish a relatively perfect, reproducible and high-resolution technique of two-dimensional electrophoresis (2-DE) for Golgi apparatus. Compare these two pictures to find out the main proteins expressed differently between these two cells.
Methods: To Optimize the condition of two-dimensional electrophoresis(2-DE) by choosing the best parameters of sample preparation,the amount of sample, the focal condition, and the focal setting instrument, and so on. The proteins of GA were seperated by 2-DE. Electrophoresis patterns were analyzed by imagemaster software.
Results: High-resolution electrophoregrams were obtained and were repeatable. Using pH 3-10 non-liner strip and 12% SDS-PAGE, almost 800 spots can be obtained from 2-DE map of GA. After silver stained, 769±22 spots are detected in the GA of well-differentiated gastric cancer cells, while 837±43 spots are identified in the GA of poorly-differentiated gastric cancer cells. Compare these two pictures, we can see that the spot distribution patterns are similar between them, but the differences are obvious between them, ans the differentiated spots are numbered.
Conclusion: Protein patterns of GA from well-, and poor-differentiated gastric cancer cells were obtained by using ultra-centrifugation and 2-DE. The first installment of the proteins' profile of GA from gastric cancer cells will benefit further investigation on Golgi function in gastric cancer.
Part 3: The main differentiated spots of GA between the well-, and poor-differentiated gastric cancer cells were identified by MALDI—TOF MS and the results were verified by Western bolt.
Objective: To identify the differential expression proteins by using mass spectrum. These identified proteins have close relationship with proliferation and differentiation of gastric cancer cells, which will help explaining the initiation and development mechanism of gastric cancer at Golgi apparatus level and providing another way to do more reaserch on GA in gastric cancer.
Methods: The differentiated spots are excised from the gels, digested by trypsin, and analyzed by MALDI-TOF MS. The peptide mass fingerprint (PMF) of the differentiated spots was obtained, and these datas were searched in MASCOT on line. At last 39 proteins differentially expressed were identified by tandem mass spectrometry. The TCP-1 and Annexin 2 were verified by Western bolt.
Results: 39 proteins differentially expressed in GF between SGC-7901 and MKN-28 were identified by tandem mass spectrometry. Based on the main functions of these proteins, they were classified as protein synthesis- and folding-related proteins, membrane fusion protein, cytoskeleton proteins, metabolic enzymes, apoptosis/tumorgenesis-related proteins, transcription-and translation-related proteins, angiogenesis/metastasis-related proteins, and cell communication/signal transduction-related proteins, and et cl. To affirm the result of identification, TCP-1 and Annexin 2 were verified by Western bolt. And the results of Western bolt were perfect matched with the results of 2-DE.
Conclusion: by a serize of proteomic methods, we found that the contant of apoptosis proteins was higer in GA of well-differentiated gastric cancer cells compared with poor-differentiated gastric cancer cells, while the proteins relate to cell differentiation and proliferation were expressed highly in GA of poor-differentiated gastric cancer cells. Such proteins change on the GA, suggested that regulation of these proteins by GA can influence the differentiation and proliferation of gastric cancer. So this fist report on proteomic analysis of GA isolated between well-, and poor-differentiated gastric cancer cells will benefit further investigation on Golgi function in gastric cancer.
高尔基体(golgi apparatus)是一个分泌通道的主要细胞器,除了进行蛋白加工,运输外,还参与蛋白质的糖基化、细胞的分泌和溶酶体的形成,最近有报道称高尔基体还参与细胞的凋亡,高尔基体本身体内就有一套凋亡信号网络。此外该细胞器还与细胞的增值分化,细胞分裂等诸多细胞活动有关,这些均表明此细胞器的功能复杂,目前对该细胞器功能的认识在许多方面的机制仍不清楚。此外据人类基因组推测,高尔基体上至少有一千多种蛋白,而目前已知的蛋白却仅有五,六百个。这些问题的存在均说明该细胞器亟待深入研究。
蛋白质组学技术的迅猛发展,特别是双向电泳和生物质谱技术的突破性发展,为组织或细胞的全蛋白质谱的研究提供了强大的技术支撑。其中发展出来的一个分支——亚细胞器蛋白质组学,主要针对细胞内一些特定细胞器的蛋白质进行分析,具有明确的亚细胞定位性,不仅可以明显提高蛋白质的分辨率与鉴定效率,还能大大提高某些低丰度蛋白质的检出。因此,这一技术的应用特别有利于帮助认识细胞器的结构和功能,提供细胞器上的蛋白组成情况,并提供关于蛋白质在细胞间转移和相互作用这些方面的信息。故亚细胞器蛋白质组学技术的提出和应用对高尔基体的功能研究也是非常适用的,在于一方面可以发现高尔基体上的新蛋白,完善高尔基体的蛋白组成情况;另一方面,可以找出在病理条件或细胞功能状态发生改变时,高尔基体的蛋白变化,为高尔基体的功能研究提供有力的实验证据。
基于以上背景,本实验主要是以分化不同的胃癌细胞的高尔基体为研究对象,运用亚细胞器蛋白质组学的技术路线研究高尔基体在胃癌细胞分化增殖中的作用。首先将胃癌细胞内的高尔基体进行分离纯化,提出纯度较高的高尔基体。然后运用双向电泳技术进行蛋白分离,建立高低分化的胃癌细胞内高尔基体的蛋白图谱,并找出两者中的差异蛋白。最后运用飞行时间质谱对差异蛋白进行鉴定,western blotting进行验证。从而对高尔基体在胃癌细胞的发生发展中的作用进行探讨。
本研究主要包括以下三部分:
第一部分:胃癌细胞内高尔基体的分离与鉴定
目的:从胃癌细胞内分离出纯度较高的高尔基体,并对所分离纯化的高尔基体进行形态学及酶学鉴定,以了解其纯度。为亚细胞器蛋白质组学的开展奠定基础。
方法:运用体外大量胃癌细胞的分批分次培养,差速离心结合蔗糖密度梯度离心法纯化分离出高尔基体后,电镜及中性红超活染色进行形态学鉴定,高尔基体标志酶半乳糖基转移酶及硫胺素焦磷酸酶活性测定进行纯度分析。
结果:通过蔗糖密度梯度离心法分离出高尔基体,电镜显示为一些囊泡性结构,中性红超活染色显示所得分离物被染成玫瑰红色,说明分离物为高尔基体。高尔基体标志酶半乳糖基转移酶及硫胺素焦磷酸酶与粗匀浆液相比,分别富集了107,98倍。而其他细胞器的标志酶的含量均大大降低或检测不出。
结论:首次从胃癌细胞内成功分离出纯度较高的高尔基体,为对高尔基体功能的深入研究及后续试验的开展奠定基础。
第二部分:高低分化的胃癌细胞内高尔基体的双向电泳的建立
目的:分别建立高,低分化胃癌细胞内高尔基体的高分辨率,稳定的双向电泳图,了解在两种细胞中高尔基体蛋白的组成情况,对比分析找出两者中的主要差异蛋白。
方法:采用蔗糖密度梯度的超速离心方法分离纯化高尔基体,通过对样品制备,蛋白上样量,聚焦条件及聚焦仪器的选择等方面参数的选择,优化双向电泳条件。在优化条件下,运用双向凝胶电泳(2-DE)技术分离高尔基体蛋白质,用ImageMaster 2D软件分析所得图谱。
结果:通过一系列条件优化,分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱。采用17cm, pH 3~10非线性胶条和12%SDS-PAGE所获得的高分化及低分化胃癌细胞高尔基体的双向电泳图谱背景干净,蛋白点的分布均匀。通过图象扫描和软件分析可以明显检测到800余个蛋白点,高分化胃癌细胞的高尔基体可检测到的蛋白点有769±22个,而低分化胃癌细胞的高尔基体的蛋白点有837±43个,对两者进行了比较蛋白质组学研究,共发现主要有差异蛋白100余个,其中低分化胃癌细胞的高尔基体主要有80多个蛋白表达上调,而高分化胃癌细胞内主要有30多个蛋白表达上调。
结论:首次成功建立了高,低分化胃癌细胞中高尔基体的双向电泳图,直观的展示了胃癌细胞高尔基体的蛋白分布情况,找出了一百多个差异蛋白点,为胃癌细胞内高尔基体功能的深入研究奠定了基础,并首次明确展示了高尔基体在胃癌细胞增值分化发展过程中的蛋白变化情况。
第三部分:高低分化的胃癌细胞内高尔基体中差异蛋白的质谱分析及western blotting验证
目的:通过质谱分析,将高低分化胃癌细胞内的差异蛋白进行鉴定,明确是高尔基体上的哪些蛋白与胃癌细胞的增殖分化有关,从而探讨高尔基体在胃癌发生发展中的作用。
方法:对差异蛋白经过胶内切取,胰酶消化后,运用基质辅助激光解吸离子化飞行时间质谱(MALDI—TOF MS)鉴定蛋白质点,数据库搜索,共鉴定出39个蛋白点。并经western blotting进一步验证了鉴定出的两种蛋白TCP-1和Annexin 2。
结果:在对高低分化胃癌细胞高尔基体中的七十多个较明显蛋白点进行质谱鉴定和数据库检索分析时,发现60%以上的蛋白点的肽质量指纹图谱都有较好的信噪比和质量精度,并成功鉴定出39个蛋白点。这些蛋白,按功能分,包括膜活性相关蛋白,细胞结构蛋白,调节蛋白及代谢酶,癌相关蛋白,囊泡运输蛋白,细胞增殖分化相关蛋白,凋亡相关蛋白,未知蛋白等。其中低分化胃癌细胞的高尔基体中与凋亡相关的蛋白大大减少,而影响细胞增殖分化的蛋白却大大增加。这些蛋白变化,对人们认识高尔基体的功能提供了有效的参考。并且为高尔基体在胃癌发生发展中所起的作用也提供了蛋白信息。
结论:经过前述一系列蛋白质组学研究途径,发现了在高分化胃癌细胞内高尔基体上的抗凋亡蛋白明显高于低分化的胃癌细胞,而低分化胃癌细胞内的高尔基体上与分化增殖有关的蛋白却大大增加。高尔基体上出现的这些蛋白变化,说明高尔基体通过对这类蛋白含量的调节,从而影响胃癌细胞的增值分化,提示高尔基体可能在胃癌发生发展中通过对凋亡蛋白,增值分化蛋白的调节,影响胃癌的发生发展。
Background: gastric cancer is still the second most common cause of death from cancer in Asia and worldwide. It remains a great clinical challenge, due to its very poor prognosis. There are reports indicated that the prognosis of gastric cancer is not only relevant to proliferation, but also apoptosis of tumor itself. The poorly differentiated gastric carcinomas have escape apoptosis mechanism, and apoptosis inhibited may exert important role in invasion and metastasis of gastric carcinomas. Our preceding research suggested that there was a rapid proliferation and expansion of the GA in gastric mucinous adenocarcinomas with high secretion of hydratase and liability to invasion and metastasis, while it’s hard to see GA in gastric tubular adenocarcinomas with no or little secretion of hydratase. Another report shows that the Golgi has altered structure and abnormal glycosylation in colorectal cancer. In addition, the transportation of Golgi appeared functional disturbance in gastric cancer. So we carried out our study using gastric cancer cells from the point of Golgi and hoped to find out the function of Golgi played in gastric cancer from protein level.
The Golgi complex is a major organelle of the secretory pathway. The key function of it is to sort, package, distribute, and post-translationally modify newly synthesized proteins and membrane lipids. Nevertheless the Golgi complex has many other complex functions, such as cell polarization and differentiation, mitosis and intracellular signaling, as well as cell division. Recent datas show that the Golgi also plays an important role in apoptosis. These unresolved questions regarding nonclassical functions of the Golgi have stimulated us to do more research on this organelle with the goal of comprehensively identifying its protein components.
Proteome studies have so far been developed with the technology. It includes two main technology approaches, one of which is using 1- or 2-DE for protein separation and MS for protein identification, and the other is the multidimensional protein identification technology (MudPIT) or‘shotgun’proteomics approach. The second one enabled more comprehensive high-throughput profiling strategies of enriched organellar fractions, resulting in hundreds of proteins identifications. It is very feasible to build up a holistic picture of research material and discover new proteins. However the traditional and the most used method is the first one, because the reproducible two-dimensional (2D) gel systems can give the direct picture about the protein complements. Highly reproducible 2D gels allow the one-time identification of a protein spot from a complex or organelle without the requirement of any prior knowledge about the protein being examined. Furthermore, it is possible to begin to address functional questions by comparative analyses of proteomes obtained for organelles during the change of functional states.
In the present study,we used the organellar proteomic analysis to study gastric cancer cells with different differentiation for the first time. The protein complements of purified golgi fractions isolated from well-differentiated gastric cancer cell line MKN-28 and poorly differentiated gastric cancer cell line SGC-7901 respectively were separated in 2-DE gels first, then about 100 proteins different expressed in both pictures were found, and 39 proteins were indentified using MS at last. These proteins have different functions, involving in cell division, transportation, apoptosis, and play a role in the differentiation and development of gastric cancer and so on. It is the first time to open the door which would guide us to understand the relationship between Golgi and gastric cancer and it would help us to do more research for better knowing the function of Golgi in gastric cancer.
The research includes three parts:
Part 1: The Isolation and Assessment of Golgi Apparatus from Gastric Cancer Cells
Objective: To isolate and purify the Golgi apparatus from Gastric Cancer Cells.
Methods: A large number of gastric cancer cells SGC7901 were cultivated in vitro, then Golgi apparatus were isolated from the cells by differential centrifugation combined with sucrose density gradient ultra-centrifugation. Its purity was characterized biochemically by enzymatic assays, morphologically by electron microscopy (EM) and neutral red supravital staining.
Results: Electron microscopy detection showed that the majorities of product were Golgi apparatus. The fraction was stained red by neutral red. Marker enzyme analysis demonstrated that TPP activity increased to 97-fold, and the relative specific activity of galactosyltransferase was 107-fold enriched over the homogenate.
Conclusion: The first successful isolation of Golgi apparatus from gastric cancer cells by using ultra- centrifugation will lead to do more research into the function of GA.
Part 2: Two-dimensional gel electrophoresis of Golgi apparatus isolated from the well-, and poor-differentiated gastric cancer cells respectively.
Objective: To establish a relatively perfect, reproducible and high-resolution technique of two-dimensional electrophoresis (2-DE) for Golgi apparatus. Compare these two pictures to find out the main proteins expressed differently between these two cells.
Methods: To Optimize the condition of two-dimensional electrophoresis(2-DE) by choosing the best parameters of sample preparation,the amount of sample, the focal condition, and the focal setting instrument, and so on. The proteins of GA were seperated by 2-DE. Electrophoresis patterns were analyzed by imagemaster software.
Results: High-resolution electrophoregrams were obtained and were repeatable. Using pH 3-10 non-liner strip and 12% SDS-PAGE, almost 800 spots can be obtained from 2-DE map of GA. After silver stained, 769±22 spots are detected in the GA of well-differentiated gastric cancer cells, while 837±43 spots are identified in the GA of poorly-differentiated gastric cancer cells. Compare these two pictures, we can see that the spot distribution patterns are similar between them, but the differences are obvious between them, ans the differentiated spots are numbered.
Conclusion: Protein patterns of GA from well-, and poor-differentiated gastric cancer cells were obtained by using ultra-centrifugation and 2-DE. The first installment of the proteins' profile of GA from gastric cancer cells will benefit further investigation on Golgi function in gastric cancer.
Part 3: The main differentiated spots of GA between the well-, and poor-differentiated gastric cancer cells were identified by MALDI—TOF MS and the results were verified by Western bolt.
Objective: To identify the differential expression proteins by using mass spectrum. These identified proteins have close relationship with proliferation and differentiation of gastric cancer cells, which will help explaining the initiation and development mechanism of gastric cancer at Golgi apparatus level and providing another way to do more reaserch on GA in gastric cancer.
Methods: The differentiated spots are excised from the gels, digested by trypsin, and analyzed by MALDI-TOF MS. The peptide mass fingerprint (PMF) of the differentiated spots was obtained, and these datas were searched in MASCOT on line. At last 39 proteins differentially expressed were identified by tandem mass spectrometry. The TCP-1 and Annexin 2 were verified by Western bolt.
Results: 39 proteins differentially expressed in GF between SGC-7901 and MKN-28 were identified by tandem mass spectrometry. Based on the main functions of these proteins, they were classified as protein synthesis- and folding-related proteins, membrane fusion protein, cytoskeleton proteins, metabolic enzymes, apoptosis/tumorgenesis-related proteins, transcription-and translation-related proteins, angiogenesis/metastasis-related proteins, and cell communication/signal transduction-related proteins, and et cl. To affirm the result of identification, TCP-1 and Annexin 2 were verified by Western bolt. And the results of Western bolt were perfect matched with the results of 2-DE.
Conclusion: by a serize of proteomic methods, we found that the contant of apoptosis proteins was higer in GA of well-differentiated gastric cancer cells compared with poor-differentiated gastric cancer cells, while the proteins relate to cell differentiation and proliferation were expressed highly in GA of poor-differentiated gastric cancer cells. Such proteins change on the GA, suggested that regulation of these proteins by GA can influence the differentiation and proliferation of gastric cancer. So this fist report on proteomic analysis of GA isolated between well-, and poor-differentiated gastric cancer cells will benefit further investigation on Golgi function in gastric cancer.
引文
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