【摘要】：p53蛋白,也被認(rèn)為是腫瘤的抑制蛋白。自從發(fā)現(xiàn)以來,被證實(shí)參與細(xì)胞周期調(diào)控,激活DNA修復(fù)、維持基因組穩(wěn)定及促進(jìn)細(xì)胞凋亡。在超過50%的人類的癌癥中都出現(xiàn)p53功能缺失,其中就包括單位點(diǎn)和多位點(diǎn)的突變及結(jié)構(gòu)的扭曲。同樣也有越來越多的研究證實(shí),p53蛋白的突變,使得其空間構(gòu)象發(fā)生變化,從而失去對細(xì)胞周期調(diào)控與促進(jìn)細(xì)胞凋亡的重要生物學(xué)功能,與此同時(shí),還會(huì)得到新的功能(Gain-of-Function, GOF),如促進(jìn)細(xì)胞轉(zhuǎn)移、侵染等,由抑癌基因變?yōu)榘┗颉Ｄ壳霸絹碓蕉嗟难芯拷M開始關(guān)注p53蛋白突變體的研究,其中就包括促進(jìn)突變體的回復(fù)突變及抑制其生物學(xué)功能。此項(xiàng)研究對于癌癥的靶點(diǎn)治療具有重要的生物學(xué)研究與醫(yī)學(xué)價(jià)值。 核酸適配體(aptamer)是一段短的單鏈核酸分子,其特點(diǎn)是能夠根據(jù)結(jié)構(gòu)特異性針對性地地結(jié)合多種靶標(biāo)分子,如蛋白質(zhì)、小分子、糖、脂肪等。它是由一個(gè)叫SELEX (Systematic Evolution of Ligand Exponential enrichment)的體外篩選進(jìn)程得到的。SELEX技術(shù)自從1990年由三個(gè)研究組同時(shí)發(fā)明并已廣泛地運(yùn)用到諸如核糖體開關(guān)、靶向治療等生物及醫(yī)學(xué)應(yīng)用。于是我們有理由相信,通過此篩選方法,我們也可以篩選到對諸如p53突變體有高親和力和特異性的核酸適配體。 目前,還未見研究組篩選到p53突變體適配體的報(bào)導(dǎo)。在本論文中,我們的首要研究目的就是能夠篩選到p53R175H的適配體。由于此突變型與野生型的唯一區(qū)別只在于第175位的氨基酸突變,所以我們對傳統(tǒng)的SELEX篩選過程進(jìn)行了改進(jìn),名為差別競爭SELEX篩選,將p53野生型與突變型蛋白質(zhì)分別經(jīng)過固相偶聯(lián),同時(shí)投入體系中,競爭性地結(jié)合庫分子,最大限度地增加篩選特異性與差異性。經(jīng)過5輪的差別競爭SELEX篩選,我們得到了21個(gè)p53R175H蛋白的候選適配體。通過親和力檢測,適配體p53R175H-APT與p53R175H蛋白的親和力大大超過對于野生型p53的親和力,并且在凝膠遷移實(shí)驗(yàn)中我們最終確定p53R175H-APT能夠在體外和p53R175結(jié)合。所以我們最終確定的p53R175H-APT作為我們后續(xù)研究的對象。 盡管在親和力檢測中,p53R175H-APT對于p53突變體有著相對于野生型p53較強(qiáng)的親和力,但其細(xì)胞學(xué)效應(yīng)還有待研究。在本論文中,我們試圖闡述篩選到的適配體P53R175H-APT在細(xì)胞中的功能。首先,我們必須確定P53R175H-APT在其靶標(biāo)的選擇上具有嚴(yán)格的特異性。在含有p53野生型的HEK293T和HeLa細(xì)胞中,通過將p53R175H-APT轉(zhuǎn)染入上述兩種細(xì)胞中,無論從表型還是從細(xì)胞生長于凋亡的情況看,均沒有發(fā)現(xiàn)與對照組有顯著性差異。同時(shí),在只含有p53的另一個(gè)突變體p53R273H的H1299細(xì)胞中,經(jīng)過轉(zhuǎn)染p53R175H-APT,相對于對照組我們依然沒有觀察到明顯的差別。在完成了以上實(shí)驗(yàn)證實(shí)p53R175H-APT對于含有p53野生型與p53其他突變體的細(xì)胞沒有顯著性作用之后,我們將此適配體構(gòu)建到質(zhì)粒并轉(zhuǎn)染入H1299-p53R175H穩(wěn)轉(zhuǎn)細(xì)胞系中,我們發(fā)現(xiàn),經(jīng)過p53R175H-APT處理的細(xì)胞,細(xì)胞增殖得到明顯的抑制,細(xì)胞的死亡明顯增多。而對于含有p53野生型的細(xì)胞,則沒有此現(xiàn)象。側(cè)面說明此RNA適配體可以特異性地識(shí)別p53R175H并顯著影響細(xì)胞增殖與細(xì)胞凋亡。為了更加確定產(chǎn)生如此明顯的生物學(xué)差別與p53突變體相關(guān),我們通過p53的免疫沉淀證實(shí)了在細(xì)胞中,p53R175H-APT與p53突變體結(jié)合的確大于IgG和scramble序列對p53突變體的結(jié)合能力。與此同時(shí),通過克隆形成實(shí)驗(yàn)與soft agar實(shí)驗(yàn)我們證明了通過此適配體處理的細(xì)胞,其惡性程度與生長能力也有明顯降低。由于很多p53的突變屬于功能獲得型突變,腫瘤細(xì)胞的遷移能力會(huì)不同程度地提高。為此我們設(shè)計(jì)了Transwell遷移實(shí)驗(yàn)和wound healing實(shí)驗(yàn),證實(shí)經(jīng)過此適配體處理過的細(xì)胞其遷移能力得到明顯降低。進(jìn)一步,在分子水平上,p53R175H-APT處理的細(xì)胞表現(xiàn)出更像p53野生型的調(diào)控方式,多個(gè)p53相互作用基因均有激活,一定程度上說明此適配體可使p53突變體的功能回復(fù)。同時(shí),我們通過免疫熒光更加直觀地證明了,經(jīng)過p53R175H-APT處理過的p53突變體,能夠從構(gòu)象上使其回復(fù)成p53野生型。 為了從更高水平驗(yàn)證此效應(yīng)可以運(yùn)用到實(shí)體瘤的治療中,我們建立了裸鼠腫瘤模型,并將適配體通過實(shí)體瘤注射與靜脈注射兩種方法進(jìn)行治療。相對于對照組,適配體組的裸鼠腫瘤明顯減小并維持較低水平,甚至消失。通過對實(shí)體瘤切片的TUNEL凋亡檢測染色,我們發(fā)現(xiàn)在適配體治療組中的凋亡細(xì)胞明顯多于對照組。更加證實(shí)了p53R175H-APT可以通過p53R175H作用于腫瘤細(xì)胞,促進(jìn)腫瘤細(xì)胞的凋亡、降低細(xì)胞生長能力、限制腫瘤細(xì)胞的遷移及具有一定程度的臨床潛力。 綜上所述,本論文的研究成果闡述了一個(gè)新的SELEX篩選方法,并通過此方法成功篩選出與野生型p53只有一個(gè)堿基差異的p53R175H的適配體p53R175H-APT。體外實(shí)驗(yàn)證實(shí)其可以抑制腫瘤細(xì)胞生長并促進(jìn)其凋亡,體內(nèi)實(shí)驗(yàn)進(jìn)一步證實(shí)了其潛在的臨床價(jià)值,對于腫瘤靶向治療提供新的思路。
[Abstract]:The p53 protein is also thought to be a tumor suppressor protein. Since the discovery, it has been demonstrated to be involved in cell cycle regulation, to activate DNA repair, to maintain genomic stability, and to promote cell apoptosis. P53 function deletions occur in more than 50% of human cancers, including mutation and structural distortion of the unit and multi-site points. In the same way, more and more studies have confirmed that the mutation of p53 protein causes the spatial conformation of the p53 protein to change, thus losing the important biological function of regulating and regulating the cell cycle and promoting the cell apoptosis, and at the same time, a new function (Gain-of-Function, GOF) can be obtained, such as promoting cell transfer, Infeiting and the like, the tumor suppressor gene becomes an oncogene. At present, more and more research groups have started to focus on the study of p53 protein mutants, including the promotion of the reverse mutation of the mutant and the inhibition of its biological function. The study has important biological and medical value for the treatment of cancer. The nucleic acid aptamer is a short-stranded nucleic acid molecule, which is characterized in that a plurality of target molecules, such as proteins, small molecules, sugar, fat, and so on. It is obtained by an in vitro screening process called SELEX The. SELEX technology has been invented by the three research groups in 1990 and has been widely used in the fields of biology and medicine, such as the ribosome switch, targeted therapy, etc. With this, we have reason to believe that by this screening method, we can also screen for nucleic acid adaptation, such as the high affinity and specificity of the p53 mutant in addition, it has not been found in that study group to screen the variant of the p53 mutant In this paper, our primary purpose is to be able to filter to p53R175H As the only difference between this mutant and the wild type is the amino acid mutation at position 175, we have improved the traditional SELEX screening process, called the differential competitive SELEX screening, and the p53 wild-type and the mutant protein are coupled through a solid phase, respectively, at the same time In the system, the library molecules are bound in a competitive way, and the screening specificity is increased to the maximum extent. The difference. After a 5-wheel differential competitive SELEX screening, we got a candidate for 21 p53R175H proteins The affinity of the aptamer p53R175H-APT with the p53R175H protein greatly exceeded the affinity for wild-type p53 by affinity detection, and in the gel migration experiment we finally determined that the p53R175H-APT was capable of in vitro and p53R17 5. So our final p53R175H-APT is our follow-up study The p53R175H-APT has a strong affinity for p53 mutants with respect to wild-type p53, but its cytologic effect Also to study. In this paper, we try to set out the filter to which the aptamer P53R175H-APT is fine The function in the cell. First, we must determine that the P53R175H-APT has a strict selection of its target The specificity of the cells. In HEK293T and HeLa cells containing the p53 wild type, none of the two cells was found by transfecting p53R175H-APT into the two cells, either from the phenotype or from the cell growth to the apoptosis. At the same time, in the H1299 cells of another mutant p53R273H containing only p53, the p53R175H-APT was transfected and we were still not observed with respect to the control group There is a clear difference. After the above experiments have been completed to confirm that p53R175H-APT has no significant effect on the cells containing the p53 wild-type and p53 other mutants, we construct the aptamer into the plasmid and transfect into the H1299-p53R175H stable-transfer cell line, and we have found that the cell proliferation obtained through the p53R175H-APT treatment Obvious inhibition, cell death There is a significant increase in death, whereas for cells containing wild-type p53, There is no such phenomenon. The side shows that this RNA aptamer can specifically recognize p53R175H and significantly affect cell proliferation In order to make it more determined that such a significant biological difference is associated with a p53 mutant, the immunoprecipitation of p53 confirmed that the combination of p53R175H-APT with the p53 mutant is indeed greater than that of the IgG and scabble sequence to the p53 mutant At the same time, by cloning and forming an experiment with soft agar, we demonstrated that the cells treated with this aptamer, the degree of malignancy and the ability to grow, There is a significant reduction in the ability of the tumor cells to migrate due to a variety of p53 mutations that are functional to type mutations. To this end, we designed the Transwell Migration Experiment and the Sound Rheing experiment to confirm that the cells treated by this aptamer have the ability to migrate. Further, at the molecular level, the cells treated with p53R175H-APT exhibited more p53 wild-type control, and many of the p53-interacting genes were activated to a certain extent that the aptamer could be used to make the p53 mutant The functional response of the p53R175H-APT, which was treated by p53R175H-APT, was more intuitively demonstrated by immunofluorescence. 53 Wild-type. In order to verify this effect from a higher level that can be applied to the treatment of solid tumors, we established a nude mouse tumor model and injected the adapter through solid tumor injection and intravenous injection. In contrast to the control group, the tumor of the nude mice of the aptamer group was significantly reduced and kept low The level, or even the disappearance, was detected by the TUNEL apoptosis test of the solid tumor section, and we found that the apoptotic cells in the aptamer treatment group It is more evident that p53R175H-APT can act on the tumor cells through p53R175H, promote the apoptosis of the tumor cells, reduce the cell growth ability, limit the migration of the tumor cells, In conclusion, the results of this paper describe a new SELEX screening method and successfully screened the adaptor p53 of p53R175H with only one base difference with the wild-type p53 by this method. R175H-APT. In vitro experiments confirm that it can inhibit the growth of tumor cells and promote its apoptosis. In vivo, the potential clinical value is further confirmed, and for tumor target
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R73-36

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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8 陳宏健;謝松;李理想;柳峰松;;一種基于干擾肌動(dòng)蛋白基因?qū)е路叫尉W(wǎng)紋n，

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