多功能核酸納米載藥系統(tǒng)對(duì)腫瘤的療效檢測(cè)
發(fā)布時(shí)間：2018-08-25 19:58
【摘要】：癌癥的診斷和治療在生化分析領(lǐng)域研究中備受關(guān)注,涉及的方法也越來(lái)越具有豐富性和創(chuàng)新性,其中關(guān)于功能核酸納米體系已呈現(xiàn)出較好的研究和應(yīng)用前景。本文設(shè)計(jì)了一種多功能核酸納米體系,一方面,利用DNA自組裝原理,通過(guò)滾環(huán)擴(kuò)增(Rolling Circle Amplification,RCA)技術(shù)和末端轉(zhuǎn)移反應(yīng),形成了多功能DNA納米球體系,作為抗癌藥物阿霉素(DOX)的載體,用于對(duì)不同種癌細(xì)胞的特異性識(shí)別和靶向給藥;同時(shí)還可用于細(xì)胞中還原性巰基化合物的含量測(cè)定。實(shí)驗(yàn)結(jié)果表明本DNA納米球材料具有良好的生物相容性和較強(qiáng)的靶向選擇性,降低正常細(xì)胞損傷的同時(shí)實(shí)現(xiàn)了對(duì)癌細(xì)胞的殺滅作用,對(duì)谷胱甘肽等還原性巰基化合物的檢測(cè)具有靈敏度高、重復(fù)性好的良好特性;另一方面,本文還建立在滾環(huán)擴(kuò)增方法的基礎(chǔ)之上,利用鄰近連接效應(yīng)(proximity ligation,PLA),進(jìn)一步與氧化石墨烯和介孔二氧化硅這兩種無(wú)機(jī)納米材料相結(jié)合,實(shí)現(xiàn)了細(xì)胞內(nèi)小分子物質(zhì)的檢測(cè)和胞內(nèi)對(duì)藥物的釋放這兩個(gè)過(guò)程的進(jìn)行。該設(shè)計(jì)有效降低了生物體內(nèi)除檢測(cè)樣本外無(wú)關(guān)物質(zhì)的消化作用影響,使檢測(cè)更具可行性和高靈敏性。其主要內(nèi)容如下:1.基于DNA自組裝技術(shù)和末端轉(zhuǎn)移反應(yīng)的多功能磁性DNA納米球?qū)入赘孰牡臋z測(cè)和抗癌藥物DOX的靶向輸送:首先,利用DNA自組裝技術(shù)原理,通過(guò)滾環(huán)擴(kuò)增方法,形成DNA納米球。其次,在合成DNA納米球的基礎(chǔ)上,引入磁性微球,形成磁性DNA納米球,便于分離。通過(guò)對(duì)參與組裝的DNA引物鏈進(jìn)行改造,可使其與含有雙硫鍵的DNA單鏈進(jìn)行雜交反應(yīng),從而形成了含有雙硫鍵的DNA納米球,該納米球可實(shí)現(xiàn)對(duì)細(xì)胞內(nèi)谷胱甘肽等還原性巰基化合物含量的檢測(cè),檢測(cè)靈敏度高。再者,通過(guò)對(duì)合成DNA納米球過(guò)程中參與滾環(huán)擴(kuò)增過(guò)程的模板鏈進(jìn)行改造,使其含有sgc8核酸適配體序列,該序列可特異性識(shí)別CEM細(xì)胞上過(guò)度表達(dá)的PTK7蛋白,由此合成的含sgc8核酸適配體序列的DNA納米球可實(shí)現(xiàn)對(duì)CEM細(xì)胞的選擇性藥物輸送。最后,建立在DNA納米球的基礎(chǔ)上,我們還通過(guò)末端轉(zhuǎn)移酶的作用,在滾環(huán)擴(kuò)增形成的線性長(zhǎng)鏈末端轉(zhuǎn)移上多個(gè)腺嘌呤(A)堿基,此末端可與修飾有葉酸的多聚胸腺嘧啶(T)單鏈進(jìn)行雜交反應(yīng),從而形成了含有葉酸的DNA納米球,該納米球可對(duì)那些會(huì)在表面過(guò)度表達(dá)葉酸受體的癌細(xì)胞,如HeLa細(xì)胞等進(jìn)行特異性識(shí)別,實(shí)現(xiàn)藥物的有效輸送。2.基于滾環(huán)擴(kuò)增方法和鄰近連接效應(yīng)的信號(hào)放大檢測(cè)ATP并誘導(dǎo)介孔二氧化硅開孔實(shí)現(xiàn)藥物釋放的體系:鄰近連接(proximity ligation,PLA)可將兩條不同的單鏈DNA分子分別與同一個(gè)蛋白質(zhì)識(shí)別分子相結(jié)合,使得兩條DNA單鏈的尾部可在空間上緊密靠近,在DNA連接酶的作用下,游離的5’端和3’端發(fā)生連接反應(yīng),從而形成滾環(huán)擴(kuò)增方法所必需的環(huán)狀結(jié)構(gòu)。因此我們?cè)O(shè)計(jì)了一條含有ATP適體序列的DNA單鏈作為連接鏈,一條可與連接鏈進(jìn)行末端互補(bǔ)的模板鏈,一條可與模板鏈的序列部分相似的信號(hào)鏈。三種DNA單鏈同時(shí)吸附到氧化石墨烯納米材料上,具有良好生物相容性和納米級(jí)粒徑的氧化石墨烯可通過(guò)胞吞作用進(jìn)入癌細(xì)胞。該體系一旦與ATP分子相遇,即可發(fā)生ATP適體鏈部分與ATP分子的結(jié)合作用,從而發(fā)生鄰近連接效應(yīng),并在DNA連接酶的作用下形成環(huán)狀DNA復(fù)合物,進(jìn)而完成滾環(huán)擴(kuò)增過(guò)程,所形成的長(zhǎng)線性DNA單鏈含有成千上萬(wàn)的堿基,可結(jié)合大量附著在氧化石墨烯上的信號(hào)鏈,釋放出較強(qiáng)的熒光。通過(guò)對(duì)釋放的熒光進(jìn)行檢測(cè)來(lái)實(shí)現(xiàn)對(duì)ATP濃度的測(cè)量,檢測(cè)信號(hào)強(qiáng)、靈敏度高。同時(shí),為了提高該系統(tǒng)的選擇性,我們?cè)谘趸┑谋砻嫘揎椓巳~酸分子,可與過(guò)度表達(dá)葉酸受體的癌細(xì)胞,如HeLa細(xì)胞進(jìn)行特異性結(jié)合,在與缺乏葉酸受體的MCF-7細(xì)胞的對(duì)比實(shí)驗(yàn)中,表現(xiàn)出明顯的較高選擇性特點(diǎn),實(shí)驗(yàn)結(jié)果進(jìn)一步說(shuō)明了本系統(tǒng)在癌癥診斷中的應(yīng)用潛能;另一方面,我們又設(shè)計(jì)了一條與上述系統(tǒng)中所提及的信號(hào)鏈堿基序列一致但不含熒光基團(tuán)的DNA單鏈,將該DNA單鏈通過(guò)靜電吸引作用吸附到介孔硅的微孔道口,作為門DNA單鏈,封鎖微孔道口,封裝介孔二氧化硅內(nèi)部預(yù)先裝備好的藥物DOX。該系統(tǒng)所形成封閉的藥物運(yùn)載及控釋結(jié)構(gòu),只有在ATP的作用下,發(fā)生上述滾環(huán)擴(kuò)增過(guò)程后所形成的線性長(zhǎng)鏈出現(xiàn)時(shí),可能與門DNA單鏈雜交,使其脫離介孔二氧化硅表面,將微孔上的“門”打開,從而釋放出藥物。這一設(shè)計(jì)可實(shí)現(xiàn)對(duì)藥物的可控釋放,有效降低阿霉素對(duì)正常細(xì)胞的強(qiáng)毒副作用,具有很強(qiáng)的實(shí)用性。同樣地,為了提高該系統(tǒng)的特異性選擇能力,對(duì)介孔二氧化硅表面進(jìn)行葉酸的修飾,實(shí)現(xiàn)對(duì)過(guò)度表達(dá)葉酸受體的癌細(xì)胞的特異性識(shí)別,進(jìn)而靶向給藥。該體系有效降低了目標(biāo)分子外其他物質(zhì)的消化影響,并降低了抗癌藥物的毒副作用,使得檢測(cè)方法更具高靈敏性、選擇性和可行性。
[Abstract]:Cancer diagnosis and treatment have attracted much attention in the field of biochemical analysis, and the methods involved are becoming more and more rich and innovative. Among them, functional nucleic acid nanosystems have shown good research and application prospects. Rolling Circle Amplification (RCA) and terminal metastasis reactions have led to the formation of a multifunctional DNA nanosphere system as a carrier of doxorubicin (DOX) for the specific recognition and targeted drug delivery of various cancer cells, and also for the determination of reducing sulfhydryl compounds in cells. Nanospheres have good biocompatibility and strong targeting selectivity, which can reduce the damage of normal cells and kill cancer cells at the same time. They have high sensitivity and good reproducibility for the detection of glutathione and other reductive sulfhydryl compounds. On the other hand, this paper is based on the roller amplification method. Furthermore, proximity ligation (PLA) was used to combine with graphene oxide and mesoporous silica to realize the detection of intracellular small molecules and the release of intracellular drugs. The main contents are as follows: 1. Detection of glutathione and targeted delivery of antitumor drug DOX by multifunctional magnetic DNA nanospheres based on DNA self-assembly technology and end-transfer reaction: Firstly, using the principle of DNA self-assembly technology, DNA nanoparticles were formed by rolling amplification method. Secondly, magnetic DNA nanospheres were introduced to form magnetic DNA nanospheres on the basis of synthesizing DNA nanospheres for easy isolation. DNA primer chains were modified to hybridize DNA single strands containing disulfide bonds, thus forming DNA nanospheres containing disulfide bonds. The nanospheres were able to achieve intracellular glutathione. Furthermore, the template chain involved in the roll-ring amplification process during the synthesis of DNA nanospheres was modified to contain the sgc8 aptamer sequence, which can specifically identify the over-expressed PTK7 protein on CEM cells and thus synthesize the aptamer containing sgc8 nucleic acid. Finally, on the basis of DNA nanospheres, we also transfer a number of adenine (A) bases to the linear long chain end formed by ring-rolling amplification, which can be hybridized with the polythymine (T) single strand modified with folic acid, by the action of terminal transferase. DNA nanospheres containing folic acid were formed by the reaction. These nanospheres can identify the cancer cells which overexpress folic acid receptors on the surface, such as HeLa cells, to achieve effective drug delivery. Drug release system: Proximity ligation (PLA) can bind two different single-stranded DNA molecules to the same protein recognition molecule, so that the tail of the two single strands of DNA can be closely spaced. Under the action of DNA ligase, the free 5'and 3'ends of the DNA chain react to form a ring-rolling amplification. Therefore, we have designed a DNA single strand containing ATP aptamer sequence as a link chain, a template chain which can complement the end of the link chain, and a signal chain which can partly resemble the sequence of the template chain. Once the system meets the ATP molecule, it can bind the aptamer chain of ATP to the ATP molecule, which results in the adjacent junction effect, and forms a circular DNA complex under the action of DNA ligase, thus completing the rolling amplification process. Long linear DNA single strands contain thousands of bases, which can bind to a large number of signal chains attached to graphene oxide and emit strong fluorescence. The ATP concentration can be measured by detecting the fluorescence. The detection signal is strong and the sensitivity is high. At the same time, in order to improve the selectivity of the system, we repair the surface of graphene oxide. Folic acid molecule can specifically bind to cancer cells with over-expression of folate receptor, such as HeLa cells. Compared with MCF-7 cells lacking folate receptor, it shows obvious high selectivity. The experimental results further illustrate the potential of this system in cancer diagnosis. On the other hand, we designed a new system. A single strand of DNA that is identical to the sequence of the signal chains mentioned in the above system but does not contain fluorescent groups is adsorbed to the microporous portal of mesoporous silicon by electrostatic attraction as a single strand of DNA, blocking the microporous portal and encapsulating the pre-equipped drug DOX inside the mesoporous silicon dioxide. Only when the linear long chain formed by the above-mentioned roll-ring amplification process occurs under the action of ATP, the carrier and controlled-release structure may hybridize with the single strand of gate DNA to break away from the surface of mesoporous silica and open the "door" on the micropore, thus releasing the drug. This design can achieve controlled release of the drug and effectively reduce the release of adriamycin. Similarly, in order to improve the selectivity of the system, the surface of mesoporous silica was modified with folic acid to realize the specific recognition of cancer cells overexpressing folic acid receptors, and then targeted drug delivery. The digestive effect and the side effects of anticancer drugs are reduced, which makes the detection method more sensitive, selective and feasible.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-3;TQ460.1
本文編號(hào)：2203946
[Abstract]:Cancer diagnosis and treatment have attracted much attention in the field of biochemical analysis, and the methods involved are becoming more and more rich and innovative. Among them, functional nucleic acid nanosystems have shown good research and application prospects. Rolling Circle Amplification (RCA) and terminal metastasis reactions have led to the formation of a multifunctional DNA nanosphere system as a carrier of doxorubicin (DOX) for the specific recognition and targeted drug delivery of various cancer cells, and also for the determination of reducing sulfhydryl compounds in cells. Nanospheres have good biocompatibility and strong targeting selectivity, which can reduce the damage of normal cells and kill cancer cells at the same time. They have high sensitivity and good reproducibility for the detection of glutathione and other reductive sulfhydryl compounds. On the other hand, this paper is based on the roller amplification method. Furthermore, proximity ligation (PLA) was used to combine with graphene oxide and mesoporous silica to realize the detection of intracellular small molecules and the release of intracellular drugs. The main contents are as follows: 1. Detection of glutathione and targeted delivery of antitumor drug DOX by multifunctional magnetic DNA nanospheres based on DNA self-assembly technology and end-transfer reaction: Firstly, using the principle of DNA self-assembly technology, DNA nanoparticles were formed by rolling amplification method. Secondly, magnetic DNA nanospheres were introduced to form magnetic DNA nanospheres on the basis of synthesizing DNA nanospheres for easy isolation. DNA primer chains were modified to hybridize DNA single strands containing disulfide bonds, thus forming DNA nanospheres containing disulfide bonds. The nanospheres were able to achieve intracellular glutathione. Furthermore, the template chain involved in the roll-ring amplification process during the synthesis of DNA nanospheres was modified to contain the sgc8 aptamer sequence, which can specifically identify the over-expressed PTK7 protein on CEM cells and thus synthesize the aptamer containing sgc8 nucleic acid. Finally, on the basis of DNA nanospheres, we also transfer a number of adenine (A) bases to the linear long chain end formed by ring-rolling amplification, which can be hybridized with the polythymine (T) single strand modified with folic acid, by the action of terminal transferase. DNA nanospheres containing folic acid were formed by the reaction. These nanospheres can identify the cancer cells which overexpress folic acid receptors on the surface, such as HeLa cells, to achieve effective drug delivery. Drug release system: Proximity ligation (PLA) can bind two different single-stranded DNA molecules to the same protein recognition molecule, so that the tail of the two single strands of DNA can be closely spaced. Under the action of DNA ligase, the free 5'and 3'ends of the DNA chain react to form a ring-rolling amplification. Therefore, we have designed a DNA single strand containing ATP aptamer sequence as a link chain, a template chain which can complement the end of the link chain, and a signal chain which can partly resemble the sequence of the template chain. Once the system meets the ATP molecule, it can bind the aptamer chain of ATP to the ATP molecule, which results in the adjacent junction effect, and forms a circular DNA complex under the action of DNA ligase, thus completing the rolling amplification process. Long linear DNA single strands contain thousands of bases, which can bind to a large number of signal chains attached to graphene oxide and emit strong fluorescence. The ATP concentration can be measured by detecting the fluorescence. The detection signal is strong and the sensitivity is high. At the same time, in order to improve the selectivity of the system, we repair the surface of graphene oxide. Folic acid molecule can specifically bind to cancer cells with over-expression of folate receptor, such as HeLa cells. Compared with MCF-7 cells lacking folate receptor, it shows obvious high selectivity. The experimental results further illustrate the potential of this system in cancer diagnosis. On the other hand, we designed a new system. A single strand of DNA that is identical to the sequence of the signal chains mentioned in the above system but does not contain fluorescent groups is adsorbed to the microporous portal of mesoporous silicon by electrostatic attraction as a single strand of DNA, blocking the microporous portal and encapsulating the pre-equipped drug DOX inside the mesoporous silicon dioxide. Only when the linear long chain formed by the above-mentioned roll-ring amplification process occurs under the action of ATP, the carrier and controlled-release structure may hybridize with the single strand of gate DNA to break away from the surface of mesoporous silica and open the "door" on the micropore, thus releasing the drug. This design can achieve controlled release of the drug and effectively reduce the release of adriamycin. Similarly, in order to improve the selectivity of the system, the surface of mesoporous silica was modified with folic acid to realize the specific recognition of cancer cells overexpressing folic acid receptors, and then targeted drug delivery. The digestive effect and the side effects of anticancer drugs are reduced, which makes the detection method more sensitive, selective and feasible.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R73-3;TQ460.1
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