褪黑素对人脐血来源EPC作用及应用蛋白质芯片检测蛋白质表达差异研究

发布时间：2018-06-01 14:58

本文选题：内皮祖细胞 + SELDI　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 目的内皮祖细胞(endothelial progenitor cells, EPCs)是血管内皮细胞的前体细胞,其在组织缺血后的血管发生和血管新生中起着重要作用。本研究旨在建立体外培养脐血来源内皮祖细胞的培养体系及褪黑素对内皮祖细胞的干预影响；探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选褪黑素干预后EPC蛋白表达差异；并通过Tricine-SDS-PAGE电泳分离差异蛋白,FT-MS分析鉴定差异蛋白。从而探讨褪黑素对EPC的作用及其机制,建立从细胞培养及药物干预到蛋白质组学研究的方法体系,并为血管新生及肿瘤抑制调控提供研究依据。方法脐带血经密度梯度离心获得单个核细胞,建立本实验室的EPCs培养体系培养,以免疫细胞化学和流式细胞术对经本体系培养7天后的单个核细胞进行内皮祖细胞表型CD34.CD133. vWF.CD146, CD144鉴定。对培养至7天的EPC进行低中高三个浓度组(1μM,5u M,10μM)褪黑素作用24h。后收集药物干预组、同期非药物干预对照组细胞,对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和本研究建立的细胞裂解液法提取蛋白,以BCA法测定蛋白浓度；分别以磁珠活化后点样和生物芯片处理器使蛋白样品与芯片结合；并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2, SAX2, IMAC-Cu,H50不同类型芯片捕获蛋白差异,最后以WCX2芯片筛选蛋白差异表达。对差异蛋白峰采用Tricine-SDS-PAGE体系分离差异蛋白,根据SELDI差异蛋白表达结果通过选取53kDa左右差异峰蛋白做FT-MS分析鉴定,探讨差异蛋白点的鉴定。结果细胞接种后前5天为生长的潜伏期,细胞开始贴壁,无明显扩增。第6天平均每个视野下细胞数目为287±45；第9天细胞数为282±46；第12天开始,细胞进入对数生长期,细胞数为805±67(P0.05)；第19天细胞继续增殖,细胞数为1115±182(P0.05)；第23天时,细胞进入凋亡期,数量明显减少,为265±61(P0.05)。vWF,CD146,CD144表达阳性。流式细胞术结果表明,梭形样细胞群体中,CD34阳性率为88.98%±5.15%(P0.05),CD133阳性率为1.20%±1.44%(P0.05),VEGFR2阳性率为41.83%±3.23%(P0.05)。药物干预24h后,光学显微镜下观察,低浓度(1uM)褪黑素能促进细胞生长,高浓度(10μM)褪黑素则对EPC有较强的抑制和促凋亡作用。相同培养条件细胞以上述三种蛋白提取方法获得的蛋白浓度分别为：0.25±0.034μg／μL,0.6±0.06μg/μL,1.02±0.077μg/μL；生物芯片处理器点样法操作简单,要求样本量较少,点样时间短；SELDI芯片蛋白质峰图谱能反映蛋白浓度情况；WCX2, SAX2, H50, IMAC-Cu芯片捕获的蛋白质种类有较大区别；在分子量1000-300000Da范围内,以WCX2芯片共检测到87个药物干预差异蛋白峰,其中17个差异呈趋势变化。通过Tricine-SDS-PAGE系统可获得分离蛋白条带,通过FT-MS分析鉴定,方法已建立,初步测试的分子量为53kDa左右的差异峰蛋白条带为α-微管蛋白。结论利用本实验室的培养体系成功培养出内皮祖细胞。镜下观察,低浓度褪黑素能促进EPC生长和增殖,高浓度抑制其生长。三种蛋白提取方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究；生物芯片处理器能较好的使蛋白与芯片结合；SELDI芯片不仅能准确的定位蛋白,也能较好的反应蛋白浓度关系；SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。SELDI芯片检测到17个药物干预趋势差异表达蛋白峰,通过Tricine-SDS-PAGE,能够分离差异蛋白,并可通过FT-MS分析进行准确鉴定。
[Abstract]:Objective endothelial progenitor cells (EPCs) is a precursor cell of vascular endothelial cells, which plays an important role in angiogenesis and angiogenesis after tissue ischemia. The aim of this study was to establish the culture system of endothelial progenitor cells derived from umbilical cord blood in vitro and the effect of melatonin on endothelial progenitor cells. SELDI chip technology is the most suitable method and condition for the analysis of cell specimen protein. The difference of EPC protein expression is screened by the screening of melatonin, and the differential protein is separated by Tricine-SDS-PAGE electrophoresis, and the differential protein is identified by FT-MS analysis. The effect of melatonin on EPC and its mechanism are discussed, and the cell culture and drug intervention to protein are established. Methodological system of omics research, and provide evidence for angiogenesis and tumor suppressor regulation.
Methods mononuclear cells were obtained by density gradient centrifugation in umbilical cord blood, and the EPCs culture system was established in this laboratory. The phenotype of endothelial progenitor cells (CD34.CD133. vWF.CD146, CD144) was identified by immunocytochemistry and flow cytometry for 7 days after culture, and CD144 was identified for the low middle elevation of EPC in 7 days. Group (1 M, 5u M, 10 M) melatonin was used to collect drug intervention group after 24h., and non drug intervention control group cells. The cell specimens were extracted by ultrasonic lysis method, U9 cell lysate buffer solution and cell lysate method to extract protein by this study. The protein concentration was determined by BCA method, and the point sample and biochip were activated by magnetic beads respectively. The processor combined the protein sample with the chip, and tested the extracted protein, compared the difference of the different protein concentration points and WCX2, SAX2, IMAC-Cu, H50, and finally screened the protein differential expression with the WCX2 chip. The differential protein peaks were separated by Tricine-SDS-PAGE system, according to the difference of SELDI. The results of protein expression were analyzed by identification of differentially expressed peaks of 53kDa and FT-MS.
Results the cells began to grow in the incubation period 5 days after the cell inoculation, and the cells began to stick to the wall. The average number of cells in sixth days was 287 + 45, and the number of cells in ninth days was 282 + 46. At the beginning of twelfth days, the cells entered the logarithmic growth period, the number of cells was 805 + 67 (P0.05), and the cells continued to proliferate on the nineteenth day, and the number of cells was 1115 + 182 (P0.05). At twenty-third days, the cells entered the apoptotic stage and decreased significantly. The expression of the cells was 265 + 61 (P0.05).VWF, CD146 and CD144 was positive. The results of flow cytometry showed that the positive rate of CD34 was 88.98% + 5.15% (P0.05), the positive rate of CD133 was 1.20% + 1.44% (P0.05), and the positive rate of VEGFR2 was 41.83% + 3.23% (P0.05). Low concentration (1uM) melatonin could promote cell growth, and high concentration (10 mu M) melatonin had a strong inhibition and apoptosis effect on EPC. The protein concentration obtained by the three protein extraction methods of the same culture conditions were 0.25 + 0.034 mu g / mu L, 0.6 + 0.06 u g/ mu L, 1.02 + 0.077 mu g/ mu L, and the biochip processor points The sample method is simple to operate, requires less sample size and short time. The protein peak Atlas of SELDI chip can reflect the protein concentration; WCX2, SAX2, H50, IMAC-Cu chips have a large difference in protein species. In the range of molecular weight 1000-300000Da, a total of 87 drug intervention differential protein peaks are detected by WCX2 chip, of which 17 differences are found. The separation of protein bands can be obtained through the Tricine-SDS-PAGE system. The method has been established by FT-MS analysis. The preliminary test of the difference peak protein band with a molecular weight of about 53kDa is alpha microtubulin.
Conclusion the endothelial progenitor cells were successfully cultured with the culture system in our laboratory. Under the microscope, the low concentration melatonin can promote the growth and proliferation of EPC, and the high concentration inhibits its growth. Compared with the three protein extraction methods, the concentration of the extracted protein extracted from the self matched cell lysate is higher and is more suitable for the chip study; the biochip processor can be better. The SELDI chip not only can accurately locate the protein, but also can reflect the relationship of protein concentration. The type of protein capture in the SELDI chips is different, the selection of the suitable chip or the combined use of the chip is more easy to obtain the difference expression of the ideal protein, and the.SELDI chip can detect the difference expression of the 17 drug intervention trends. Protein peaks can be separated by Tricine-SDS-PAGE and can be identified accurately by FT-MS analysis.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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