本文選題：內(nèi)皮祖細(xì)胞 + SELDI��； 參考：《中國協(xié)和醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPCs)是血管內(nèi)皮細(xì)胞的前體細(xì)胞,其在組織缺血后的血管發(fā)生和血管新生中起著重要作用。本研究旨在建立體外培養(yǎng)臍血來源內(nèi)皮祖細(xì)胞的培養(yǎng)體系及褪黑素對(duì)內(nèi)皮祖細(xì)胞的干預(yù)影響；探討以SELDI芯片技術(shù)進(jìn)行細(xì)胞標(biāo)本蛋白分析的最適方法及條件,篩選褪黑素干預(yù)后EPC蛋白表達(dá)差異；并通過Tricine-SDS-PAGE電泳分離差異蛋白,FT-MS分析鑒定差異蛋白。從而探討褪黑素對(duì)EPC的作用及其機(jī)制,建立從細(xì)胞培養(yǎng)及藥物干預(yù)到蛋白質(zhì)組學(xué)研究的方法體系,并為血管新生及腫瘤抑制調(diào)控提供研究依據(jù)。 方法臍帶血經(jīng)密度梯度離心獲得單個(gè)核細(xì)胞,建立本實(shí)驗(yàn)室的EPCs培養(yǎng)體系培養(yǎng),以免疫細(xì)胞化學(xué)和流式細(xì)胞術(shù)對(duì)經(jīng)本體系培養(yǎng)7天后的單個(gè)核細(xì)胞進(jìn)行內(nèi)皮祖細(xì)胞表型CD34.CD133. vWF.CD146, CD144鑒定。對(duì)培養(yǎng)至7天的EPC進(jìn)行低中高三個(gè)濃度組(1μM,5u M,10μM)褪黑素作用24h。后收集藥物干預(yù)組、同期非藥物干預(yù)對(duì)照組細(xì)胞,對(duì)細(xì)胞標(biāo)本分別用超聲裂解法,U9細(xì)胞裂解緩沖液配方和本研究建立的細(xì)胞裂解液法提取蛋白,以BCA法測定蛋白濃度；分別以磁珠活化后點(diǎn)樣和生物芯片處理器使蛋白樣品與芯片結(jié)合；并對(duì)提取蛋白進(jìn)行檢測,比較不同蛋白濃度梯度點(diǎn)樣及WCX2, SAX2, IMAC-Cu,H50不同類型芯片捕獲蛋白差異,最后以WCX2芯片篩選蛋白差異表達(dá)。對(duì)差異蛋白峰采用Tricine-SDS-PAGE體系分離差異蛋白,根據(jù)SELDI差異蛋白表達(dá)結(jié)果通過選取53kDa左右差異峰蛋白做FT-MS分析鑒定,探討差異蛋白點(diǎn)的鑒定。 結(jié)果細(xì)胞接種后前5天為生長的潛伏期,細(xì)胞開始貼壁,無明顯擴(kuò)增。第6天平均每個(gè)視野下細(xì)胞數(shù)目為287±45；第9天細(xì)胞數(shù)為282±46；第12天開始,細(xì)胞進(jìn)入對(duì)數(shù)生長期,細(xì)胞數(shù)為805±67(P0.05)；第19天細(xì)胞繼續(xù)增殖,細(xì)胞數(shù)為1115±182(P0.05)；第23天時(shí),細(xì)胞進(jìn)入凋亡期,數(shù)量明顯減少,為265±61(P0.05)。vWF,CD146,CD144表達(dá)陽性。流式細(xì)胞術(shù)結(jié)果表明,梭形樣細(xì)胞群體中,CD34陽性率為88.98%±5.15%(P0.05),CD133陽性率為1.20%±1.44%(P0.05),VEGFR2陽性率為41.83%±3.23%(P0.05)。藥物干預(yù)24h后,光學(xué)顯微鏡下觀察,低濃度(1uM)褪黑素能促進(jìn)細(xì)胞生長,高濃度(10μM)褪黑素則對(duì)EPC有較強(qiáng)的抑制和促凋亡作用。相同培養(yǎng)條件細(xì)胞以上述三種蛋白提取方法獲得的蛋白濃度分別為：0.25±0.034μg／μL,0.6±0.06μg/μL,1.02±0.077μg/μL；生物芯片處理器點(diǎn)樣法操作簡單,要求樣本量較少,點(diǎn)樣時(shí)間短；SELDI芯片蛋白質(zhì)峰圖譜能反映蛋白濃度情況；WCX2, SAX2, H50, IMAC-Cu芯片捕獲的蛋白質(zhì)種類有較大區(qū)別；在分子量1000-300000Da范圍內(nèi),以WCX2芯片共檢測到87個(gè)藥物干預(yù)差異蛋白峰,其中17個(gè)差異呈趨勢變化。通過Tricine-SDS-PAGE系統(tǒng)可獲得分離蛋白條帶,通過FT-MS分析鑒定,方法已建立,初步測試的分子量為53kDa左右的差異峰蛋白條帶為α-微管蛋白。 結(jié)論利用本實(shí)驗(yàn)室的培養(yǎng)體系成功培養(yǎng)出內(nèi)皮祖細(xì)胞。鏡下觀察,低濃度褪黑素能促進(jìn)EPC生長和增殖,高濃度抑制其生長。三種蛋白提取方法比較,選用自配的細(xì)胞裂解液提取蛋白的濃度較高且更適于芯片研究；生物芯片處理器能較好的使蛋白與芯片結(jié)合；SELDI芯片不僅能準(zhǔn)確的定位蛋白,也能較好的反應(yīng)蛋白濃度關(guān)系；SELDI各芯片捕獲蛋白類型不同,選擇適宜芯片或聯(lián)合運(yùn)用芯片檢測更易獲得較理想蛋白差異表達(dá)結(jié)果。SELDI芯片檢測到17個(gè)藥物干預(yù)趨勢差異表達(dá)蛋白峰,通過Tricine-SDS-PAGE,能夠分離差異蛋白,并可通過FT-MS分析進(jìn)行準(zhǔn)確鑒定。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

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