發(fā)布時間：2018-02-02 12:47

  本文關(guān)鍵詞： 納米金剛石 CpG ODN 免疫刺激活性 抗腫瘤效應(yīng)　出處：《中國科學院研究生院(上海應(yīng)用物理研究所)》2016年博士論文　論文類型：學位論文

【摘要】：治療型核酸,包括功能化修飾的寡脫氧核苷酸(ODNs),適配體(aptamers),小干擾RNA(siRNA),在一系列疾病(從遺傳性到后天獲得的疾病以及癌癥)的治療中顯示出很強的應(yīng)用前景。未甲基化的胞嘧啶-磷酸鹽-鳥嘌呤寡脫氧核苷酸(CpG ODNs)就是其中一種具有免疫刺激活性的核酸,最初發(fā)現(xiàn)于病毒及細菌的DNA中。人工合成的CpG ODNs是一種很好的免疫佐劑,在抗感染、癌癥以及過敏性疾病的中有廣泛的應(yīng)用。CpG ODNs的臨床實驗?zāi)壳疤幱冖?Ⅲ期。然而,一些副作用的顯現(xiàn)使其無法繼續(xù)研究。這些副作用主要與高劑量的重復(fù)給藥相關(guān),因為單鏈CpG的細胞攝取率很低且很容易被代謝清除。因此,急需發(fā)展一種新的輸運系統(tǒng)不但可以提高細胞的攝取率而且可以抵抗核酸酶的降解發(fā)揮長時間的免疫刺激效應(yīng)。納米材料作為控釋的藥物輸運載體有廣闊的應(yīng)用前景。各種各樣的納米材料,從無機、有機到生物大分子都被用作CpG的載體刺激細胞產(chǎn)生相應(yīng)的免疫反應(yīng)。然而,高昂的費用及潛在的毒性效應(yīng)限制了其在體內(nèi)的應(yīng)用。納米材料的一系列載體中,納米金剛石(NDs)擁有卓越的載運能力、靈活的表面可修飾性以及良好的生物相容性。特別是NDs在生理條件下可以自發(fā)形成多孔團簇結(jié)構(gòu),對于載運小分子化療藥物及生物大分子顯示出“海綿”效應(yīng)。這篇工作,我們采用多聚賴氨酸(PDL)修飾的功能化的納米金剛石(fNDs)用于細胞內(nèi)及體內(nèi)CpG寡核苷酸的輸運并且探討其免疫刺激活性及潛在的抗腫瘤效應(yīng)。主要研究內(nèi)容和結(jié)果如下:(1)首先設(shè)計CpG ODN載體,用陽離子聚合物PDL修飾原始的NDs,形成fNDs復(fù)合物;隨后通過靜電相互作用吸附帶負電的CpG,形成CpG-fNDs復(fù)合物。原始NDs的平均粒徑為247nm,zeta電位為33.0eV;當包裹了帶正電荷的PDL之后,fNDs的平均粒徑增加到325nm,zeta電位增加到67.3eV;fNDs靜電吸附了CpG之后,平均粒徑進一步增大,達到338nm;然而Zeta電位則降低為40.6eV。(2)然后,我們使用激光共聚焦熒光顯微鏡評估cpg-fnds的細胞攝取。攝取時間為6h,細胞內(nèi)平均熒光強度定量分析顯示:與單鏈cpg相比,cpg-fnds組的細胞攝取率提高了將近3個數(shù)量級;之后研究攝取的cpg-fnds在細胞內(nèi)的清除,結(jié)果顯示在最初的6h攝取孵育結(jié)束后,24,48,72h細胞內(nèi)的平均熒光強度分別是單鏈cpgodn組的900倍,830倍以及400倍。(3)接下來,我們研究了cpg-fnds的體外免疫刺激活性。結(jié)果發(fā)現(xiàn)cpg-fnds孵育raw264.7細胞6h,可以顯著的促進腫瘤壞死因子(tnf-α)以及白介素-6(il-6)的釋放。與單鏈cpg組相比,tnf-α和il-6的釋放量分別提高到59倍及20倍。之后檢測了長時間(24h,48h,72h)免疫刺激因子的釋放,發(fā)現(xiàn)cpg-fnds組72h仍然可以檢測到tnf-α和il-6的釋放,釋放量分別為單鏈cpg組的13倍及26倍。體外實驗數(shù)據(jù)表明,cpg-fnds有長期的免疫刺激活性,在細胞水平可以持續(xù)3天。(4)為了進一步評估cpg-fnds潛在的治療效應(yīng),我們又將免疫刺激研究進行到體內(nèi)。icr小鼠尾靜脈注射cpg-fnds之后測定小鼠血清中釋放的免疫因子的表達量變化。結(jié)果發(fā)現(xiàn)在尾靜脈注射3h后可以顯著地誘發(fā)血清中il-12和il-6的釋放。與單鏈cpg組相比,il-12和il-6的釋放量分別提高了19倍以及61倍。接下來,我們研究了其長期的免疫刺激效果,發(fā)現(xiàn)48h的釋放量仍有單鏈cpg組的4倍。體內(nèi)實驗同樣證明,cpg-fnds有長期的免疫刺激活性,在動物水平可以持續(xù)2天。(5)既然免疫刺激效果如此好,那么在體內(nèi)是否安全呢?我們使用近紅外熒光染料xenolightcf750共價標記nds,監(jiān)測其在體內(nèi)生物分布和清除。結(jié)果發(fā)現(xiàn),分布在各器官的nds在72小時會徹底清除。主要臟器病理切片及肝功能無明顯變化,這些結(jié)果證實cpg-fnds在體內(nèi)非常安全。(6)最后,我們評估了cpg-fnds的在tlr9陽性的腫瘤模型(b16-f0和4t1)的抗腫瘤效應(yīng)。結(jié)果發(fā)現(xiàn)cpg-fnds可以顯著的抑制腫瘤的生長,對于b16-f0和4t1腫瘤模型,抑瘤率分別為73.3%和28.4%�？傊�,我們開發(fā)了一種新穎的治療型核酸輸送系統(tǒng),可以提高cpgodn的攝取,在體內(nèi)外均有緩釋效應(yīng),而且cpg-fnds復(fù)合物在小鼠腫瘤模型的成功應(yīng)用,為癌癥的免疫治療提供了新的思路,更有可能將其設(shè)計并應(yīng)用于納米醫(yī)藥領(lǐng)域。
[Abstract]:Treatment of type of nucleic acid, including oligonucleotide functionalized (ODNs), aptamer (aptamers), small interfering RNA (siRNA), in a series of diseases (to get acquired from hereditary disease and cancer) showed a strong application prospects. In the treatment of unmethylated cytosine phosphate guanine oligodeoxynucleotide (CpG ODNs) is one kind of immunostimulatory nucleic acid, originally found in viruses and bacteria in DNA. CpG synthetic ODNs is a good adjuvant in anti infection, clinical trials have been widely used in.CpG ODNs cancer and allergic diseases in the present in the I - III period. However, some side effects appear so that it can not continue to study. These side effects mainly with high doses of repeated administration, because the cellular uptake of single stranded CpG rate is very low and it is easy to be metabolic clearance. Therefore, it is urgent to develop an The new transport system can not only improve the degradation of cell uptake and resistance to nuclease play a long time. The immune stimulatory effect of nano materials as drug controlled release carrier transport have broad application prospects. All kinds of nano materials, from inorganic to organic and biological macromolecules have been used as the carrier of CpG stimulation the cell has the corresponding immune response. However, the high cost and potential toxicity limits its in vivo application. A series of carrier nano materials, nano diamond (NDs) has excellent carrying capacity, flexible surface modification and good biocompatibility. Especially in physiological NDs under the condition of the spontaneous formation of porous cluster structure for carrying small molecule agents and biological macromolecules show "sponge" effect. This work, we use poly lysine (PDL) functionalized The nano diamond (fNDs) for cells and in vivo CpG oligonucleotide transport and to investigate its immune activity and antitumor effect of potential stimulation. The main research contents and results are as follows: (1) the design of CpG ODN vector, the original NDs modified with cationic polymer PDL, the formation of the fNDs complex; followed by electrostatic interaction. The adsorption of charged CpG, the formation of the CpG-fNDs complex. The original NDs average particle size was 247nm, the zeta potential is 33.0eV; when the package with the positive charge of PDL, the average particle size of the fNDs increased to 325nm, the zeta potential increased to 67.3eV; after fNDs electrostatic adsorption of CpG, the average particle size increase, reach 338nm; however, the Zeta potential is reduced to 40.6eV. (2) and then, cellular uptake we use laser confocal fluorescence microscope to evaluate the cpg-fnds. The uptake time is 6h, the mean fluorescence intensity of cells in quantitative analysis showed that with single stranded CPG Compared with the cellular uptake rate in cpg-fnds group was increased by nearly 3 orders of magnitude; after study the uptake of cpg-fnds in cells was clear, results show that at the end of the 6h uptake after the initial incubation, the average fluorescence intensity of 24,48,72h cells was 900 times of single stranded cpgodn group, 830 times and 400 times (3). Next, we studied the in vitro immune stimulating activity of cpg-fnds. The results showed that cpg-fnds of RAW264.7 cells incubated with 6h could significantly promote tumor necrosis factor alpha (tnf- alpha) and interleukin -6 (IL-6) release. Compared with the single CpG group, the release amount of tnf- alpha and IL-6 were increased to 59 times and 20 times. After the detection of long time (24h, 48h, 72h) immune stimulating factor release, cpg-fnds group 72h still can be detected by tnf- alpha and IL-6 release, the release amount of single stranded CpG group respectively 13 times and 26 times in vitro. The experimental data show that cpg-fnds has the long-term immunity thorn Activated, can last for 3 days at the cellular level. (4) in order to further evaluate the therapeutic effect of cpg-fnds potential, we will study the immune stimulation in.Icr mice after intravenous injection of cpg-fnds for determination of expression of release of immune factors in serum of mice. The results found in the tail vein after injection of 3H significantly IL-12 the IL-6 induced serum release. Compared with the single stranded CpG group. The release amount of IL-12 and IL-6 were increased 19 times and 61 times. Next, we investigated the long-term immune stimulating effect, found that the release amount of 48h is 4 times of single stranded CpG group. In vivo experiments also showed that cpg-fnds has long immunostimulatory activity, in animal level can be continued for 2 days. (5) since the immune stimulating effect is so good, so in the body is safe? We use the near infrared fluorescent dye xenolightcf750 NDS monitoring in the covalent labeling. Biodistribution and clearance. The results showed that the distribution in various organs of the NDS will be completely cleared in 72 hours. The main pathological sections and liver function had no obvious change, these results suggest that cpg-fnds is safe in vivo. (6) finally, we evaluated cpg-fnds in TLR9 positive tumor models (B16-F0 and 4T1). Anti tumor effect. The results showed that cpg-fnds can significantly inhibit tumor growth for B16-F0 and 4T1 tumor model, the inhibition rate was 73.3% and 28.4%. respectively. In conclusion, we developed a novel therapeutic nucleic acid delivery system, can improve the uptake of cpgodn, both in vitro and in vivo release effect, and the cpg-fnds complex in success application of mouse tumor model, provides a new way for cancer immunotherapy, are more likely to be designed and used in nano medicine.

【學位授予單位】：中國科學院研究生院(上海應(yīng)用物理研究所)
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R943;TB383.1

【相似文獻】

相關(guān)期刊論文 前10條

1 華麗;從情報價值分析納米金剛石在表面工程中的應(yīng)用[J];金剛石與磨料磨具工程;2004年06期

2 ;科學家揭開“納米金剛石”的秘密[J];超硬材料工程;2005年06期

3 華麗;;納米金剛石的開發(fā)與應(yīng)用概述[J];金剛石與磨料磨具工程;2006年03期

4 孫景;江雷;杜希文;雷貽文;翟琪;楊星;;激光轟擊法制備納米金剛石的提純研究[J];炭素技術(shù);2006年04期

5 靳洪允;侯書恩;楊曉光;;納米金剛石拋光液制備及應(yīng)用[J];金剛石與磨料磨具工程;2007年03期

6 秦宇;王光祖;;發(fā)展中的中國納米金剛石應(yīng)用技術(shù)[J];超硬材料工程;2007年04期

7 陸靜;王艷輝;臧建兵;;納米金剛石表面準原子層沉積硅鍍層[J];超硬材料工程;2007年06期

8 崔仲鳴;楊予勇;王光祖;;納米金剛石作為拋光材料的應(yīng)用[J];機械工程與自動化;2008年04期

9 王光祖;崔仲嗚;;納米金剛石作為拋光材料的應(yīng)用[J];石材;2011年03期

10 ;俄研制出制備納米金剛石的新方法[J];延邊大學學報(自然科學版);2011年04期

相關(guān)會議論文 前10條

1 李靜;諸穎;張小勇;李文新;黃慶;;納米金剛石作為中藥活性成分胞內(nèi)運輸載體的研究[A];第二屆中草藥提取關(guān)鍵技術(shù)與提取物產(chǎn)業(yè)應(yīng)用研討會論文集[C];2009年

2 許向陽;朱永偉;王柏春;沈湘黔;;納米金剛石在水介質(zhì)中的分散與穩(wěn)定[A];2002年材料科學與工程新進展（上）——2002年中國材料研討會論文集[C];2002年

3 李汝R，

本文編號：1484546