胶质瘤研究两种常用细胞系的蛋白质组学研究

发布时间：2018-05-02 16:16

本文选题：胶质母细胞瘤 + U87　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景神经胶质瘤,简称胶质瘤,由于发生于神经外胚层,故又称作神经外胚层肿瘤或神经上皮肿瘤。神经胶质瘤是颅内原发恶性肿瘤中最常见的一种,根据WTO分级,可将神经胶质瘤分为4个等级,多形胶质母细胞瘤(glioblastoma multiforme GBM)属于Ⅳ级胶质瘤,在现有的治疗手段下,患者确诊GBM后的生存期平均为1-2年。目前对它的治疗主要是以手术治疗为主的综合治疗。GBM本身病死率高,又极易复发,癌细胞存在一定的化疗耐药性及放射抵抗,预后欠佳,为社会和家庭带来了巨大的经济和精神负担。开展胶质瘤相关的基础与临床研究对于寻找对其更为有效的治疗方法意义重大。 U251细胞和U87细胞为胶质瘤体外实验两种常用的工具细胞,两种细胞系从GBM中分离建立而来,具有很强的代表性。U251细胞来源于一位老年男性GBM患者,而U87来源于一位女性GBM患者,均由Ponten.J教授及其同事建立。自两细胞系建立以来,以该两种细胞为对象的大量研究使得GBM的增殖,侵袭,迁移和凋亡等生物学习性越来越清晰。从目前的研究观察到,尽管两者均来源于GBM患者,但U251和U87细胞系在细胞生物学特征并不相同,表现为在正常培养的情况下,显微镜下观察到的U251细胞跟U87细胞的细胞形态并不一致,U251细胞胞体成呈裙摆形,树突轴突等突起较为短小,U87细胞的胞体成梭形,有多个较长轴突,相对于U251而言,U87细胞具有更强的增殖,迁移和侵袭能力,而对TMZ的敏感性比U251强,与实验中观察到的结果为相同,由此可见,U251细胞和U87细胞虽同来源于GBM,但生物学性状并不完全一致,尤其是侵袭能力、成瘤能力差异显著。既然U251和U87侵袭性、成瘤能力差异显著,那么,以二者为工具,探讨其分子基础,将有助于揭示GBM侵袭的分子机制,为GBM治疗提供潜在分子靶点,但是,经文献检索检索发现,U251细胞和U87细胞究竟存在哪些蛋白表达差异,并不清楚。因此,本研究以U251细胞和U87细胞为研究对象,用蛋白组学的方法蛋白双向电泳及同位素相对标记与绝对定量技术(isobaric tags forrelative and absolute quantitation, iTRAQ)方法对比分析两细胞蛋白表达谱表达情况,探讨这两种细胞系来源是否一致,并检测两细胞差异蛋白的表达,通过对差异蛋白进行GO及pathway富集分析等生物信息学分析,解析两细胞细胞形态,增殖,迁移和侵袭能力及药敏性等细胞生物学差异的原因,从筛选得到的差异蛋白中选出部分蛋白质,验证其蛋白表达水平,干扰其U251细胞和U87细胞中表达,观察干扰后U251细胞和U87细胞生物学特征的变化。研究方法一、U251细胞和U87细胞培养,使用MTT检测两细胞系的增殖能力及对不同浓度TMZ的药敏性,使用划痕实验检测其迁移能力。二、蛋白双向凝胶电泳(two-dimensional gel electrophoresis2-DE)检测U251和U87细胞系蛋白表达谱,验证其组织学来源是否一致。三、iTRAQ比较U251细胞和U87细胞系差异蛋白表达谱,结果使用行基质辅助激光解析电离-飞行时间质谱分析(MALDI-TOF-MS)找差异蛋白点,并使用G0富集分析及Pathway富集分析等对两细胞差异蛋白进行分析。四、用qPCR技术验证U251细胞和U87细胞系10对基因TPM4, FLNC, C10orf58, PDLIM1, MYH10, PSIP1, YA61, SYNM, SLC9A3R2and BCAM mRNAs表达差异。五、使用siRNA干扰两种细胞系TPM4,PDLIM1, PSIP13种基因表达,qPCR技术检测干扰效率。划痕实验分别检测siRNA干扰后U251和U87细胞各处理组细胞的迁移能力。六、统计学处理：采用SPSS19.0软件进行统计学处理。计量资料用x±s表示,组间比较采用单因素方差分析,P0.05定义为有统计学意义。研究结果一、同等培养条件下,U87比U251细胞增殖更快,侵袭能力更强,同时,前者比后者对TMZ更为敏感。二、Image Master检测U251和U87细胞系的蛋白质表达谱之间的差异点,发现两者蛋白表达谱基本一致,提示U251和U87细胞系组织学来源一致,,与ATCC数据库描述一样,而存在着少数差异蛋白,提示其生物学性质存在差异。三、同位素相对标记与绝对定量技术分析两细胞系蛋白组,总蛋白数量3660种,根据蛋白质丰度水平,当差异倍数达到1.5倍以上,且经统计检验其p-value值小于0.05时,视为差异蛋白,507种蛋白在表达量上存在明显的差异,其中U251细胞相对U87细胞上调蛋白数量为244种,下调蛋白数量为263种。四、qPCR检测结果提示,在10个基因当中,C10orf58, FLNC, PDLIM1,YA61,TPM4在U251细胞中的表达比在U87细胞中表达的高,MYH10, PSIP1, SYNM, SLC9A3R2and BCAM,在U251细胞中的表达比在U87细胞中表达的低,差异均超过了4倍,比较iTRAQ检测结果,提示以上10种差异蛋白的基因表达水平在两细胞上下调方向一致。五、siRNA干扰TPM4,PDLIM1,PSIP1表达后划痕试验提示U251中干扰TPM4表达组细胞(以下简称T组)迁移最快,干扰PSIP1表达组细胞次之(以下简称PS组),干扰PDLIM1表达组细胞(以下简称PD组)与阴性对照组(NC组)细胞迁移最慢,后两组细胞迁移速度并没有明显的差异。研究结论 U251细胞和U87细胞系来源于胶质母细胞瘤,它们的组织学来源是一致的,两者蛋白表达谱有差异,差异表达蛋白总共达到了507种,其中PDLIM1, TPM4基因和相对应蛋白表达在U251细胞中的表达比在U87细胞中表达的高,PSIP1基因和相对应蛋白表达在U251细胞中的表达比在U87细胞中表达的低,PDLIM1,TPM4对U87迁移有负向调节作用,即可抑制U87细胞的迁移,PSIP1基因表达量在两细胞中较低,对两者迁移能力影响不明显。本研究意义一、为基于U251细胞和U87细胞系的研究提供蛋白水平差异的数据资料。二、由于两种GBM生物学特性并不完全一致,进一步阐明差异蛋白的作用,有助于GBM特定功能的解析。
[Abstract]:Research background
Glioma, called glioma, is also called neuroectodermal tumor or neuroepithelial tumor because it occurs in the neuroectoderm. Glioma is the most common type of primary intracranial malignant tumor. According to WTO classification, glioma can be divided into 4 grades, and the glioblastoma multiforme GBM belongs to IV. The average survival time of patients with grade glioma is 1-2 years after the diagnosis of GBM. At present, the treatment of.GBM is mainly a comprehensive treatment based on surgical treatment, which has high mortality and easy recurrence. The cancer cells have a certain chemotherapeutic resistance and radiation resistance, and the prognosis is not good. The basic and clinical research of glioma is of great significance in finding a more effective treatment for glioma.
U251 cells and U87 cells are two common tool cells in vitro experiment of glioma. The two cell lines are isolated from GBM. The strong representative.U251 cells are derived from an elderly male GBM patient, and U87 originated from a female GBM patient. All of them were established by Professor Ponten.J and his colleagues. Since the establishment of the two cell line, it has been established. A large number of studies on two kinds of cells have made the biological learning of GBM proliferation, invasion, migration and apoptosis more and more clear. From the present study, although both of them are derived from GBM patients, the U251 and U87 cell lines are different in cell biological characteristics, showing the U251 observed under the microscope under normal conditions. The cell morphology of the cells is not consistent with the cell morphology of the U87 cells. The cell body of the U251 cell is a skirt shape, the protuberance of the dendritic axon is relatively short, the cell body of the U87 cell is spindle shaped, and there are many longer axons. Compared with the U251, U87 cells have stronger proliferation, migration and invasion ability, and the sensitivity to TMZ is stronger than that of U251, and the results observed in the experiment. In the same way, it can be seen that although U251 and U87 cells are from GBM, the biological characters are not exactly the same, especially the invasive ability, and the difference of the ability of tumor formation is significant. Since U251 and U87 are invasive and the difference of the ability of tumor formation is significant, then the two groups are used as a tool to explore the molecular mechanism of GBM invasion, and it will be used to treat GBM. Treatment provides potential molecular targets. However, a literature search shows that the difference in protein expression between U251 cells and U87 cells is not clear.
Therefore, in this study, U251 cells and U87 cells were used to compare the expression of two cell protein expression profiles by means of proteomic method protein two-dimensional electrophoresis and isotopic relative labeling and absolute quantitative technique (isobaric tags forrelative and absolute quantitation, iTRAQ). The expression of two cell differential proteins was detected. By bioinformatics analysis of GO and pathway enrichment analysis of differential proteins, the reasons of cell biological differences, such as cell morphology, proliferation, migration and invasion ability and drug sensitivity of two cells, were analyzed, and some proteins were selected from the differential proteins selected to verify the protein expression. The expression level of U251 cells and U87 cells was disturbed, and the biological characteristics of U251 cells and U87 cells after interference were observed.
research method
First, U251 cells and U87 cells were cultured. MTT was used to detect the proliferation ability of two cell lines and their sensitivity to different concentrations of TMZ. The migration ability was detected by scratch test.
Two, two-dimensional gel electrophoresis2-DE was used to detect the protein expression profiles of U251 and U87 cell lines, and to verify whether their histological sources were consistent.
Three, iTRAQ compared the differential protein expression profiles of U251 cells and U87 cell lines. The results were found by using line matrix assisted laser analytical ionization time of flight mass spectrometry (MALDI-TOF-MS) to find differential protein points, and the analysis of two cell differential proteins by G0 enrichment analysis and Pathway enrichment analysis.
Four, qPCR technology was used to verify the difference between U251 cells and U87 cell line 10 in gene TPM4, FLNC, C10orf58, PDLIM1, MYH10, PSIP1, YA61, SYNM, and PSIP1.
Five, the expression of TPM4, PDLIM1, PSIP13 gene was expressed by siRNA interference, and the interference efficiency was detected by qPCR technique. The scratch test was used to detect the cell migration ability of U251 and U87 cells after siRNA interference.
Six, statistical processing: SPSS19.0 software was used for statistical processing. The measurement data were expressed in X + s, and single factor analysis of variance was used in groups. P0.05 was defined as statistical significance.
Research results
1. Under the same culture conditions, U87 has faster proliferation and stronger invasiveness than U251 cells, and the former is more sensitive to TMZ than the latter.
Two, Image Master detected the difference between the protein expression profiles of U251 and U87 cell lines, and found that the protein expression profiles were basically the same, suggesting that the histologic sources of U251 and U87 cells were the same, as described by the ATCC database, and there were a few differential proteins, suggesting that there were differences in their biological properties.
Three, isotopic relative labeling and absolute quantification analysis of the two cell line protein group, the total protein number is 3660. According to the protein abundance level, when the difference multiple reaches more than 1.5 times, and the p-value value is less than 0.05 by statistical test, the difference in the amount of protein is apparent in the amount of the 507 proteins, and the relative U87 of the U251 cells is relative to U87. The number of upregulated proteins was 244 and the number of down regulated proteins was 263.
Four, qPCR results showed that in the 10 genes, the expression of C10orf58, FLNC, PDLIM1, YA61, TPM4 in U251 cells was higher than that in U87 cells, MYH10, PSIP1, SYNM, SLC9A3R2and, and the expression in the cells was lower than that in the U251 cells, and the difference was more than 4 times. The above 10 differences were compared. The level of protein expression was consistent with that in two cells.
Five, siRNA interference TPM4, PDLIM1, PSIP1 expression after the scratch test suggested that U251 interference TPM4 expression group cells (hereinafter referred to as T group) migration is the fastest, interfering with the PSIP1 expression group cells (hereinafter referred to PS group), interfering with PDLIM1 expression group cells (hereinafter referred to PD group) and negative group (NC group) cell migration is the slowest, the latter two groups of cell migration speed did not There are obvious differences.
research conclusion
The U251 and U87 cell lines are derived from glioblastoma, and their histological origin is consistent, and the protein expression profiles are different. The differential expression proteins have reached 507 species, of which the expression of PDLIM1, TPM4 and relative protein expression in U251 cells is higher than that in U87 cells, PSIP1 gene and relative protein table. The expression in U251 cells is lower than that expressed in U87 cells. PDLIM1 and TPM4 have negative regulation on U87 migration, which can inhibit the migration of U87 cells. The expression of PSIP1 gene is low in two cells, and there is no obvious effect on the mobility of U87 cells.
The significance of this study
First, provide data for protein level differences based on U251 cell and U87 cell lines.
Two, because the biological characteristics of the two GBM are not completely consistent, further elucidation of the role of differential proteins is helpful to the analysis of GBM specific functions.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

【参考文献】