【作者】 蒲颖；

【导师】 刘慧霞；

【作者基本信息】 中南大学 ， 老年医学， 2011， 博士

【摘要】 第一部分建立针对肝细胞的指数级富集的配体系统进化技术目的：建立针对肝细胞的指数级富集的配体系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment, SELEX),并获得能识别肝细胞表面膜蛋白的核酸适体(Aptamer)。方法：设计DNA文库,两端各一段长20bp的引物序列,引物1用异硫氰酸荧光素(fluorescein isothiocyanate, FITC)标记,引物2用生物素biotin标记,中间20bp为随机序列。待培养皿(100mm)中人肝细胞癌细胞株HepG2生长丰度达到95%左右时,弃去细胞培养基,用4℃洗涤缓冲液洗涤。取20nmole DNA文库,溶于1ml结合缓冲液中,与细胞在4℃孵育1h后弃去上清液,洗涤缓冲液洗涤两次后,用细胞刮子收集细胞,用1ml蒸馏水洗涤,并将细胞悬液收集在1.5ml离心管中,在95℃加热15min后离心,取上清液,此即首轮筛选的母液,其内包含与HepG2结合的序列。经过聚合酶链式反应(PCR)扩增所得序列,扩增后的产物为双链DNA,包含目标链及其互补链。将PCR产物与链亲素(Streptavidin)覆盖的琼脂糖珠孵育后滤过上清液,用2%NaOH打开PCR双链结构,目标链则溶解于NaOH溶液中,将该溶液通过NAP-5脱盐柱脱去NaOH,测定单链DNA (ssDNA)浓度以确定该轮筛选终产物总量,并将其甩干。从上一轮筛选终产物中取100pmole,并将其溶于500ul含有10%胎牛血清的结合缓冲液中,将其与培养皿(60mm)中生长丰度达到95%左右的HepG2细胞孵育,按照上述方法完成每一轮筛选。每3-5轮筛选后需要检查每轮产物与细胞的结合情况。待细胞生长丰度达95%左右时,用非酶消化液消化细胞,将贴壁生长的细胞配成单个细胞悬液,用洗涤缓冲液洗涤后将细胞溶于结合缓冲液中,每5×105个细胞与25pmole的筛选产物于4℃孵育30min,筛选产物的浓度为250nM。同时将细胞与同等浓度的DNA文库孵育,作为阴性对照。孵育结束后洗涤细胞,用流式细胞仪分析筛选产物与细胞的结合程度。随着筛选轮数的增加,产物与细胞间的结合程度越来越高,直至达到平台期。选取结合程度最高的三轮产物进行测序,所得序列即针对靶细胞的核酸适体。用DNA合成仪合成所得序列并用流式细胞仪检测各条序列与靶细胞的结合程度,从而确定本筛选最终所得的核酸适体。分别用胰酶以及蛋白酶K处理细胞,处理时间从5min-30min不等,观察酶处理前后各核酸适体与细胞结合的情况。同时,将各核酸适体与不同种类的细胞孵育(包括LH86、Huh7、WT、IRS/KO、M7617、Ramos、CEM、H23、H69、A549、HBE、H661、TOV-21G、CAOV3等细胞),观察各序列的结合情况。结果：本文通过应用SELEX对HepG2细胞展开筛选,经过测序分析发现了4条针对HepG2细胞的核酸适体：IR01、IR03、IR04以及IR06。其中IR01、IR04与靶细胞的亲和性最佳,其解离常数分别为11.287±3.786nM和88.849±22.339nM; IR03虽然与HepG2细胞结合程度较低,解离常数较高(129.513±47.924nM),但其特异性最好,能特异性与HepG2细胞结合,而与其它种类细胞几乎无交叉反应；IR01、IR04、IR06对大部分肝脏来源的细胞株均有较好结合；IR01是本实验所得的核酸适体当中与靶细胞结合程度最强的一条序列,其仅与肝脏来源的细胞有结合,而与其他组织来源的细胞无交叉反应,并且该条核酸适体与细胞的结合不容易被蛋白酶的作用所破坏。相反,IR03, IR04, IR06三条核酸适体与靶细胞的结合均容易被酶的消化而破环,胰酶或者蛋白酶K处理HepG2细胞5min后上述三条核酸适体与靶细胞的结合即被完全破坏。结论：通过SELEX,可以获得与靶细胞高亲和性以及高特异性结合的核酸适体,这些核酸适体通过识别靶细胞膜上所表达的某种生物分子从而达到识别表达相同生物分子的不同细胞株。第二部分筛选针对肝细胞表面与高胰岛素处理相关膜蛋白的核酸适体目的：在第一部分实验的基础上筛选出针对肝细胞表面与高胰岛素处理相关膜蛋白的核酸适体(Aptamer)。方法：待HepG2细胞生长丰度达70%左右后血清饥饿12h,然后分成四组,第一组继续予以不含胰岛素的培养基培养,第二组予以加入生理剂量的胰岛素(0.1nM)的培养基培养,第三组予以加入高胰岛素(100nM)的培养基培养,第四组予以加入超高胰岛素(500nM)的培养基培养。同时,予以高胰岛素(100nM)处理胎鼠肝细胞野生型(Wild type, WT)与胰岛素受体底物2基因(Insulin receptor substrate 2, IRS2)敲除后的胎鼠肝细胞(IRS2/KO),并设立正常培养组(不加入胰岛素处理)作为对照。上述各组细胞的培养基中均不含血清。24h后用非酶消化液消化细胞,将贴壁生长的细胞配成单个细胞悬液,用洗涤缓冲液洗涤后将细胞溶于结合缓冲液中,每5×105个细胞与浓度为250nM的核酸适体25pmole于4℃孵育30min。孵育结束后洗涤细胞,用流式细胞仪分析各核酸适体与上述各细胞的结合程度在高胰岛素处理前后有无变化。结果：IR04与HepG2细胞的结合明显受到100nM胰岛素的抑制；进一步加大胰岛素的剂量(500nM)则抑制作用更加明显,经过500nM胰岛素处理后IR04与HepG2的结合几乎被完全消减；但IR04与HepG2细胞的结合不受生理剂量胰岛素的影响。同样,高胰岛素处理也能减弱IR04与WT细胞的结合；但IR04与IRS2/KO细胞的结合程度不受高胰岛素处理的影响。而IR01、IR03、IR06与细胞的结合均不受高胰岛素处理的影响。结论：IR04与靶细胞的结合被高胰岛素处理所减弱,且其减弱的程度与高胰岛素的浓度呈正相关；但生理浓度范围内的胰岛素不影响IR04与靶细胞的结合；IR04可能的靶标为肝细胞表面某种与胰岛素作用相关的膜蛋白,此种膜蛋白在高胰岛素处理过程中被下调,但具体机制有待进一步研究；IR04所针对的靶物质在人、鼠肝细胞的表达具有高度同源性,且其受高胰岛素影响而发生下调的机理可能与人类相似。第三部分基于核酸适体修饰石墨烯的胰岛素检测目的：应用核酸适体修饰后的氧化石墨烯(GO)来进行胰岛素的检测,并通过加入催化剂(DNA酶)来实现其检测信号的放大。方法：将天然石墨粉和氯化钠结晶研磨从而减小石墨颗粒的体积。去掉氯化钠后,将研磨过的石墨粉加入到浓硫酸中,强力搅拌下加入高锰酸钾,并用体积分数3%的双氧水还原剩余的高锰酸钾和二氧化锰,使其变为无色可溶的硫酸锰。在双氧水的处理下,悬浮液变成亮黄色。最后,过滤、洗涤3次,然后超声处理4h,离心后取上层溶液用于下一步实验。异硫氰酸荧光素(FITC)标记的胰岛素核酸适体(insulin binding aptamer, IBA)稀释成100nM的胰岛素缓冲液。将IBA与GO按照摩尔浓度1：1于室温条件下混匀,避光孵育30min。此即本实验的工作溶液。由于GO能淬灭吸附在其表面的IBA所携带的荧光,此时溶液中仅存在一个较弱的荧光信号。当溶液中存在胰岛素时,与GO结合的IBA则从GO表面解离,与胰岛素结合,FITC标记的IBA游离到溶液中,GO对FITC的淬灭作用大大减弱,从而使得溶液中的荧光信号增加；进一步在工作溶液中加入DNA酶,加入不同浓度的胰岛素(胰岛素的加入量从5nM到50μM)后孵育2h。此时与胰岛素结合的IBA将被DNA酶消化成片段,从而失去与胰岛素结合的能力,胰岛素重新游离于工作液中；而与GO结合的IBA由于被GO保护,不被DNA酶消化,但将会从GO表面解离,并与游离的胰岛素结合,从而被DNA酶消化成片段。理论上该反应可以无限循环至IBA耗尽。终止反应后检测溶液中荧光浓度。同时设立阴性对照组(生物素、链霉亲和素、牛血清蛋白)以明确上述胰岛素检测体系的特异性。结果：本实验通过将胰岛素核酸适体修饰在氧化石墨烯单层表面,成功构建了胰岛素的生物感应器,实现了胰岛素的便捷检测,检测下限为500nM；通过进一步加入DNA酶来实现信号放大后,胰岛素的检测下限降低到5nM。结论：胰岛素核酸适体修饰后的氧化石墨烯可以作为胰岛素检测的良好工具,通过加入DNA酶实现检测信号的放大,将检测下限降低了100倍。更多还原

【Abstract】 Chapter 1 Investigation on the construction of Cell-based Systematic Evolution of Ligands by Exponential EnrichmentObjective:To constract the Cell-based systematic evolution of ligands by exponential enrichment(SELEX)and to obtain the aptamers for the membrane proteins on the liver cells.Methods:The DNA library is consisted by two primers which length are 20bp. Primer1 is labeled with fluorescein isothiocyanate(FITC), while Primer2 is labeled with biotin. There is 20bp random sequence in the middle. When the HepG2 in the cell culture dish(100mm)grows to 95% confluence, get rid of the cell culture medium, and wash it by 4℃washing buffer. Dillute 20nmole library by 1ml binding buffer, and incubate it with HepG2 in 4℃for 1h. Get rid of the suspension and wash the cell by washing buffer twice. Use cell brush collect the cells in 1.5ml centrifuge tube by 1ml DI water, and heat the tube in the heat block in 95℃for 15min. Then centrifuge the tube and get the supernatant. This is our first pool for selection. Our binding sequences was inside. Using PCR to amplify these sequences. After PCR, we have double strain DNA(dsDNA), which contain the target strain and the complemental strain. Incubate these dsDNA with streatavidin coated beads, and get rid of the suspension. The dsDNA is opened into ssDNA by 2%NaOH, then our target strain was dissolved in the NaOH solution. Desalt the solution by NAP-5 column and meansure the concentration of these ssDNA to get the gross.At last dry the final sollution by DNA dryer. That is our first pool. Culture our target cells in 60mm cell culture dish. Dissovle 100pmole pooll by 500ul binding buffer which contain 10%FBS. And then do the next round of SELEX follow the way hereinbefore. Test the bound between each pool and our target cells every 3 to 5 rounds. When HepG2 grows to 95% confluence, using non-enzymatic buffer digest the cells, and make the cells into single one. Wash the cells, and put dillute the cells by binding buffer. Every 5×105 cells incubate with 25pmole pool in 4℃for 30min, the concentration of the pool is 250nM. The cells incubate with DNA library is the negative control. Using flow cytometry to analyse the binding. When the binding signal stop increasing, take the best 3 binding pools to do the 454 sequencing. After we get the sequences, synthesize these sequences and test the binding one by one with target cells to determine our aptamers. Using trypsin and proteinase K digesting HepG2 cells respectively, the treating time range from 5min to 30min, and compare the binding before and after the enzyme treatment. On the other hand, we incubate each aptamer with other kinds of cells (Including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3) and find the binding specificity.Results:In this paper, we do SELEX on HepG2 cells, and finally got 4 binding aptamers. There are IR01, IR03, IR04 and IR06. The best binding aptamers are IR01 and IR04, whose binding dissociated constant (Kd) are 11.287±3.786nM and 88.849±22.339nM respectively; Although our IR03 only have weak binding with HepG2 cells (Kd=129.513±47.924nM), but seems it only binds with HepG2, and almost no cross link with other kinds of cells; IR01, IR04 and IR06 can not only bind with HepG2 but also can bind with other kinds of liver cells. IR01 is the strongest binding sequence we got. And the most interesting thing is the bound of IR01 can not be eliminated by the digestion of proteinase.Conclusion:By doing SELEX, we can get the aptamers for the target cells with high specificity and high affinity. These aptamers can bind with their target biomolecules which are on the surface of cells membrane. In that way, they can bind different kinds of cells which express same biomolecules. Chapter 2 Select one aptamer for high insulin treatment related membrane protein on the surface of liver cellObjective:Based on the first part experiment, to select one aptamer for high insulin treatment related membrane protein on the surface of liver cell from the aptamers we got from the Cell-based SELEX on HepG2.Methods:Let the HepG2 cells grow to 70% confluence, starve from FBS for 12h, then divide these cells into four groups. The first group is treated by OnM insulin. The second group incubate with 0.1 nM insulin. The third group was treated with 100nM insulin. The last group was treated by 500nM insulin. All the four groups were incubated 24h. We also treat the fetal mice hepatocyte wild type(WT) and insulin receptor substrate 2 gene knock out fetal mice hepatocytes(IRS/KO) with 100nM insulin in the same way as HepG2 cells. At the same time, we also treat the WT and IRS/KO cells with OnM insulinfor control. And all the above cells are incubated in the FBS free medium. After 24h, using non-enzymatic buffer digest the cells into single one. Wash them by washing buffer and dissolve the cells into binding buffer. Every 5×105 cells incubate with 25pmole aptamers for 30min in 4℃. The concentration of aptamers is 250nM. After incubation, washing the cells by washing buffer, and using the flow cytometry to analyse if the high insulin treatment will change the binding aptamers and cells.Results:The binding between IR04 and HepG2 cells was inhibited by 100nM insulin obviously. When the concentration of insulin increased to 500nM, the binding between IR04 and HepG2 almost disappeared completely. But those binding won’t affect by physiological dose insulin. Also the high insulin treatment can decrease the binding between IR04 and fetal mice hepatocyte (WT), but won’t affect the binding between IR04 and the insulin receptor substrate 2 gene knock out fetal mice hepatocyte (IRS/KO). But aptamers IR01, IR03 and IR06’s binding will not be influented by high insulin treatment.Conclusion:The binding between IR04 and target cells are inhibited by high insulin treatment. And the degree of the binding has positive correlation with insulin concentration. But the physiologic concentration of the insulin won’t affect IR04’s binding. So we guess the target of IR04 is one kind of insulin related membrane protein, which will be down-regulated by high insulin treatment. But the detail is not very clear. The target of IR04 are highly homologous in both human beings’ and mice hepatocyte. Also the mechanism under the high insulin treatment might be the same. Chapter 3 Aptamer Conjugated Graphene Oxide based Insulin DetectionObjective:Use insulin binding aptamer conjugated graphene oxide (GO) to do insulin detection. And add DNase to gain a output amplification.Methods:Ground the graphite powder with NaCl to reduce the particle size. Removing the salt, then graphite was added to the concentrated H2SO4 and stirring for 2 hours. After, KMnO4 was added slowly gradually under stirring.Finally, we add distilled water and H2O2 solution to end the reaction. At last, the mixture was washed again. Sonicate GO dispersion under ambient condition for 4 hours. The resulted sample was centrifuged, and collect the upper solution for future experiments.IBA will bind with insulin and escaped from the GO surface. In that case the quenching effect of GO to FITC-IBA will be decreased a lot. Then we can detect the fluorescence signal. Further more, we add DNase I into the GO-aptamer solution and allow insulin incubate with them for 2 hours. When the IBA bind with insulin, it escaped from the GO surface, so the GO can not protect the IBA from the digestion of DNAse any more, it will be digested into pieces, and can not bind with insulin anymore. But the FITC release into the solution and won’t quench by GO. The other IBA which still on the GO surface can be protect from the DNAse digestion. Because the binding IBA was destroyed by DNAse I, other IBA can continue release from the GO surface and bind with the insulin, and the above reaction go on and on. So one insulin can bind with more than one IBA, and the output fluorescence signal in the solution has been amplified. At the same time, we test biotin, streptavidin and BSA with the same concentration as insulin to determine the specificity of the above insulin detecion system.Results:The IBA conjugated GO is a good tool to do insulin selection. Depend on that, we can detect insulin in a simple and fast way, and our detection limit is 500nM. While with the amplification strategy, we added DNase I to amplify the output signal, the detection limit was decreased into 5nM. And the detection limit is improved 100 times.Conclusion:The IBA conjugated graphene oxide is a wonderful tool for insulin detection, but without singal amplification, the detection limit is not low enough. By adding DNase I, we can improve the detection limit over 100 times.更多还原