本文選題：精胺化普魯蘭　切入點(diǎn)：siRNA　出處：《北京協(xié)和醫(yī)學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：RNA干擾(RNAi)技術(shù)在多種蛋白表達(dá)相關(guān)的疾病或病毒感染的治療中具有應(yīng)用前景。實(shí)現(xiàn)RNAi的關(guān)鍵條件之一是獲得具有RNA藥物高裝載量和高轉(zhuǎn)染效率、無(wú)毒且無(wú)免疫原性、并易于商業(yè)化生產(chǎn)的載體。非病毒型載體具有易化學(xué)合成、低免疫原性、保護(hù)siRNA免于降解等優(yōu)勢(shì)。在眾多非病毒載體中,陽(yáng)離子多糖具有良好的生物相容性并且易修飾,一直是核酸載體的研發(fā)重點(diǎn)之一。但是,在遞送siRNA過(guò)程中,陽(yáng)離子多糖會(huì)與血清蛋白發(fā)生非特異性結(jié)合,從而影響RNA干擾效率。為了減少血清蛋白的影響,通常需要使用無(wú)血清條件培養(yǎng)細(xì)胞。然而,在無(wú)血清環(huán)境中,陽(yáng)離子多糖又會(huì)通過(guò)靜電相互作用而與細(xì)胞膜發(fā)生非特異性結(jié)合,由此而導(dǎo)致嚴(yán)重的細(xì)胞毒性；此外,當(dāng)在體內(nèi)應(yīng)用時(shí),無(wú)血清環(huán)境的要求也無(wú)法中得到滿足。慢性髓系白血病(CML)是由造血干細(xì)胞染色體移位而產(chǎn)生融合基因BCR-ABL所導(dǎo)致的血液惡性腫瘤。CML的主要治療方法為激酶抑制劑和異體造血干細(xì)胞移植,但患者會(huì)逐漸對(duì)激酶抑制劑產(chǎn)生耐藥,同時(shí)還有部分患者對(duì)藥物不耐受。越來(lái)越多的研究致力于將RNA干擾技術(shù)應(yīng)用于CML的治療,但是,面臨著CML細(xì)胞難以轉(zhuǎn)染的挑戰(zhàn)。目前對(duì)CML細(xì)胞轉(zhuǎn)染的方法主要包括電穿孔和病毒載體兩種。但是,電穿孔轉(zhuǎn)染不能用于體內(nèi)實(shí)驗(yàn),而病毒載體具有引起免疫反應(yīng)的風(fēng)險(xiǎn),并且易引起宿主基因突變,因此,迫切需要研發(fā)針對(duì)CML細(xì)胞的新型載體。精胺化普魯蘭多糖(Pullulan-spermine, Ps)是一種典型的陽(yáng)離子多糖,由于其特有的化學(xué)成分而在肝臟細(xì)胞的RNAi中得到廣泛研究。實(shí)驗(yàn)室之前的研究結(jié)果顯示,培養(yǎng)基中的血清濃度會(huì)影響Ps與siRNA所形成的復(fù)合物(Ps/siRNA)被乳腺癌細(xì)胞攝取的數(shù)量及其溶酶體逃逸能力,由此而影響RNA干擾效率,但是關(guān)于血清蛋白(Pr)與Ps的相互作用及Pr與Ps的質(zhì)量比(Pr/Ps)與RNAi之間的關(guān)系尚需要深入研究。此外,尚未有研究報(bào)道Ps在血液惡性腫瘤RNAi中的應(yīng)用。本研究用Ps作為模型陽(yáng)離子多糖載體,開展以下兩方面的研究：(1)Ps對(duì)蛋白分子的吸附作用以及Pr/Ps對(duì)Ps/siRNA復(fù)合物的粒徑和RNA干擾效率的調(diào)控作用；(2)以Ps為siRNA載體,沉默CML細(xì)胞功能基因BCR-ABL,并比較Ps在CML細(xì)胞、RAW264.7和Jurkat細(xì)胞上引起的RNA干擾效率的差異。方法：采用等溫滴定量熱法研究Ps與牛血清白蛋白(BSA)之間的吸附作用,通過(guò)瓊脂糖凝膠電泳檢測(cè)Ps/siRNA復(fù)合物的形成,以及BSA對(duì)Ps與siRNA結(jié)合穩(wěn)定性的影響；通過(guò)動(dòng)態(tài)光散射實(shí)驗(yàn)(DLS)研究溶液體系中Pr/Ps對(duì)Ps/siRNA復(fù)合物顆粒尺寸的影響。以MDA-MB-231和MDA-MB-231-EGFP細(xì)胞為模型,用流式細(xì)胞術(shù)和激光共聚焦顯微術(shù)檢測(cè)Pr/Ps對(duì)復(fù)合物進(jìn)入細(xì)胞的影響和對(duì)RNA干擾效率的影響。應(yīng)用RT-PCR技術(shù)研究血清對(duì)Ps、Lipo 3000介導(dǎo)β-actin/BCR-ABL siRNA對(duì)K562、KU812、MEG-01、RAW、Jurkat細(xì)胞的RNA干擾效率的影響；以Ps/PGL4.51轉(zhuǎn)染K562、RAW、Jurkat細(xì)胞,并用微孔板塊熒光檢測(cè)儀檢測(cè)Luc的表達(dá)量。結(jié)果：Ps與siRNA完全結(jié)合的質(zhì)量比為0.75：1。在溶液中Ps會(huì)吸附BSA分子,但BSA的吸附不影響Ps/siRNA的穩(wěn)定性。在一定siRNA質(zhì)量范圍內(nèi),隨著Pr/Ps的增大,Ps/siRNA的粒徑減小,同時(shí),復(fù)合物Ps/siRNA進(jìn)入細(xì)胞的能力、對(duì)細(xì)胞的毒性和介導(dǎo)基因沉默的效率也逐漸降低；在Pr/Ps固定的條件下,隨siRNA量的增加,RNA干擾效率逐漸提高,當(dāng)siRNA濃度達(dá)到1 μg/mL后,RNA干擾效率不再增加。在Ps/siRNA與細(xì)胞共孵育后,使用氯喹(CQ)處理細(xì)胞,可使RNA干擾效率進(jìn)一步提高。當(dāng)N/P為2.5時(shí),復(fù)合物Ps/siRNA的粒徑不受培養(yǎng)基中血清濃度的影響。同時(shí),無(wú)論在無(wú)血清培養(yǎng)基中還是在有血清培養(yǎng)的條件下,K562的細(xì)胞活性都在80%以上;Ps/FAM-siRNA在3種CML細(xì)胞上的陽(yáng)性率均大于95%,而在RAW和Jurkat兩種細(xì)胞上的陽(yáng)性率在85%左右。對(duì)5種細(xì)胞的β-actin基因進(jìn)行RNA干擾,在Opti-MEM培養(yǎng)基條件下,Ps在3種CML細(xì)胞上介導(dǎo)的干擾效率高于Lipo3000,在RAW、Jurkat細(xì)胞上低于Lipo 3000;在10%的血清濃度條件下,Lipo3000和Ps介導(dǎo)的干擾效率均很低。對(duì)3種CML細(xì)胞的BCR-ABL基因進(jìn)行RNA干擾,在上述兩種培養(yǎng)基條件下,載體Ps在K562細(xì)胞上介導(dǎo)的干擾效率明顯高于Lipo3000；對(duì)于KU812細(xì)胞,Ps與Lipo3000介導(dǎo)的干擾效率沒(méi)有差異；對(duì)于MEG-01細(xì)胞,在Opti-MEM培養(yǎng)基條件下,Ps介導(dǎo)的干擾效率高于Lipo3000,而在10%血清濃度條件下,低于Lipo 3000。當(dāng)N/P為2.5時(shí),Ps在K562及其耐藥株上介導(dǎo)的RNA干擾效率不受血清濃度影響,但在HELA細(xì)胞上介導(dǎo)的干擾效率則隨血清濃度的增加而降低。此外,Ps/PGL4.51在K562細(xì)胞上介導(dǎo)的熒光素酶的表達(dá)量高于在RAW和Jurkat細(xì)胞上的表達(dá)量。結(jié)論：溶液中血清蛋白與陽(yáng)離子多糖的質(zhì)量比Pr/Ps是調(diào)控陽(yáng)離子多糖Ps與siRNA所形成復(fù)合物的顆粒尺寸的關(guān)鍵參數(shù),對(duì)復(fù)合物進(jìn)入細(xì)胞的能力及其所介導(dǎo)的RNA干擾效率具有重要的調(diào)控作用。此外,Ps在CML類細(xì)胞上表現(xiàn)出優(yōu)于Lipo3000的沉默白血病相關(guān)功能基因的性能,有望成為介導(dǎo)CML細(xì)胞RNA干擾的新載體。
[Abstract]:Objective: RNA interference (RNAi) technology has the application prospect of protein expression in a variety of disease treatment or virus infection related in. One of the key conditions for the realization of RNAi is obtained with RNA high drug loading and high transfection efficiency, non-toxic and non immunogenic, and easy to support the commercial production of non viral vectors have. Easy chemical synthesis, low immunogenicity, protect siRNA from degradation and other advantages. Among the non viral vector, cationic polysaccharide has good biocompatibility and easy functionalization, has been one of the key research of nucleic acid vectors. However, in the delivery process of siRNA, cationic polysaccharide combined with non serum protein the opposite sex, thus affecting the RNA interference efficiency. In order to reduce the influence of serum protein, usually need to use serum-free cultured cells. However, in serum-free environment, cationic polysaccharide and through electrostatic interaction With the cell membrane of nonspecific binding, which leads to severe cytotoxicity; in addition, when applied in vivo, serum free environmental requirements cannot be met. In chronic myeloid leukemia (CML) is the main method of treatment by hematopoietic stem cell translocation generated blood malignant tumor induced by BCR-ABL.CML fusion gene the kinase inhibitor and allogeneic hematopoietic stem cell transplantation, but the patient will gradually produce resistance to kinase inhibitors, as well as some patients of drug intolerance. More and more studies focus on the RNA interference technology is applied to CML treatment, however, facing the CML cells are difficult to challenge. The transfection method for CML cell transfection mainly includes two kinds of electroporation and viral vectors. However, electroporation can not be used for in vivo experiments, and viral vectors have risks caused by the immune response, and easy to cause The host gene mutation, therefore, there is an urgent need to develop new carrier for CML cells. Fine amination of pullulan (Pullulan-spermine, Ps) is a kind of typical cationic polysaccharide, due to its unique chemical composition have been studied extensively in liver cells. RNAi research laboratory before results showed that the serum concentration in medium the complex formed by the influence of Ps and siRNA (Ps/siRNA) is the number of lysosomes and uptake of breast cancer cells escape ability, thus affect the efficiency of RNA interference, but the serum protein (Pr) and the quality of the interaction of Ps and Pr and Ps ratio (Pr/Ps) and the relationship between RNAi still need further research. In addition, there is no study reported the application of Ps in hematological malignancies in RNAi. This study model of cationic polysaccharide with Ps as carrier, to carry out research on the following two aspects: (1) the adsorption effect of Ps on protein molecules to Ps/siRNA and Pr/Ps on the composite particle size and the regulation effect of RNA interference efficiency; (2) using Ps as siRNA carrier, silencing CML gene BCR-ABL cell function, and compare the differences in Ps CML cells, RNA interference efficiency by RAW264.7 and on Jurkat cells. Methods: using isothermal titration calorimetry study with Ps bovine serum albumin (BSA) adsorption between, formed by agarose gel electrophoresis of the Ps/siRNA complex, and BSA of Ps and siRNA combined with the stability of influence; through the dynamic light scattering experiment (DLS) effect of Pr/Ps solution on Ps/siRNA complex particle size. With MDA-MB-231 and MDA-MB-231-EGFP cells as model. Effect of impact on the complex into cells by flow cytometry and confocal laser microscopy for detection of Pr/Ps and RNA interference efficiency. Research on the application of RT-PCR on serum Ps, Lipo mediated -actin/BCR-AB beta 3000 L siRNA of K562, KU812, MEG-01, RAW, RNA interference influence efficiency of Jurkat cells transfected by Ps/PGL4.51; K562, RAW, Jurkat cells, and expression of microporous plate fluorescence detector for detection of Luc. Results: the quality of Ps and siRNA completely with ratio of 0.75:1. Ps in the solution will be adsorbed BSA molecules. But the adsorption of BSA does not affect the stability of the Ps/siRNA. The siRNA quality range, with the increase of Pr/Ps, the grain size of Ps/siRNA decreases, at the same time, the complex Ps/siRNA ability to enter the cell, on the cell toxicity and mediated gene silencing efficiency is gradually reduced; in Pr/Ps fixed conditions, with the increase of siRNA the amount of RNA interference efficiency increased gradually when the concentration of siRNA reached 1 g/mL after RNA interference efficiency did not increase. In Ps/siRNA and cells were incubated, chloroquine (CQ) cells, the RNA interference efficiency further improved. When N/P is 2.5, composite Ps/siRNA particle size will not be affected by the culture medium serum concentration. At the same time, both in serum-free medium or in serum culture conditions, the activity of K562 cells are above 80%; Ps/FAM-siRNA was greater than 95% in 3 positive rate of CML cells, while the positive rate in two and RAW Jurkat cells in about 85%. RNA interference beta -actin gene of the 5 kinds of cells, the medium in the condition of the Opti-MEM, Ps in 3 CML cell mediated interference efficiency is higher than that of Lipo3000, in RAW, Jurkat cells than Lipo 3000; in the serum concentration of 10%, and the interference efficiency of Lipo3000 Ps mediated RNA interference are very low. The BCR-ABL gene of 3 CML cells, in the above two kinds of medium conditions, the efficiency of interference vector Ps in K562 cells mediated by KU812 cells was significantly higher than that of Lipo3000; the interference efficiency of Ps, and Lipo3000 mediated no difference of; In MEG-01 cells cultured under Opti-MEM condition, the interference efficiency mediated by Ps is higher than Lipo3000, while in the 10% serum concentration, less than 3000. Lipo when N/P is 2.5, Ps is not affected by the effects of serum concentration in RNA interference efficiency K562 and drug-resistance mediated, but the interference efficiency in HELA cells mediated the guide is increased with the increase of the concentration of serum decreased. In addition, the expression of Ps/PGL4.51 in K562 cell mediated luciferase expression was higher than that in RAW and Jurkat cells. Conclusion: the quality of serum protein and cationic polysaccharide solution than Pr/Ps is a key parameter of particle size of Ps and regulation of siRNA cationic polysaccharide formed the complex, has an important role in the regulation of RNA interference efficiency ability complex into cells and mediated. In addition, Ps showed the performance of leukemia related genes silence is better than that of Lipo3000 in CML cells, It is expected to be a new vector to mediate RNA interference in CML cells.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R96

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 魯慧英;Detection of hepatitis C virus RNA sequences in cholangiocarcinomas in Chinese and American patients[J];Chinese Medical Journal;2000年12期

2 ;Detection the HCV RNA by PCR-microplate hybridization method from clinical specimens[J];中國(guó)輸血雜志;2001年S1期

3 付漢江,鄭曉飛;類mRNA RNA研究進(jìn)展[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));2002年04期

4 Verheyden B ,徐其武;口服脊髓灰質(zhì)炎疫苗核殼和RNA的穩(wěn)定性[J];國(guó)外醫(yī)學(xué).預(yù)防.診斷.治療用生物制品分冊(cè);2002年01期

5 CMBE譯文組;探索小RNA的功能[J];現(xiàn)代臨床醫(yī)學(xué)生物工程學(xué)雜志;2004年03期

6 沈維干;RNA interference and its current application in mammals[J];Chinese Medical Journal;2004年07期

7 ;Potent and specific inhibition of SARS-CoV antigen expression by RNA interference[J];Chinese Medical Journal;2005年09期

8 孫娣;汪洋;張麗娟;閆玉清;;一種簡(jiǎn)捷提取植物總RNA的方法[J];黑龍江醫(yī)藥;2005年06期

9 熊慧華;于世英;胡廣原;莊亮;;Effects of Survivin Expression Suppressed by Short Hairpin RNA on MCF-7 Cells[J];華中科技大學(xué)學(xué)報(bào)(醫(yī)學(xué)英德文版);2006年03期

10 楊益超;;RNA干擾及其在醫(yī)學(xué)中的應(yīng)用[J];廣西醫(yī)學(xué);2007年10期

相關(guān)會(huì)議論文 前10條

1 金由辛;;面向21世紀(jì)的RNA研究[A];面向21世紀(jì)的科技進(jìn)步與社會(huì)經(jīng)濟(jì)發(fā)展（下冊(cè)）[C];1999年

2 ;第四屆RNA全國(guó)研討會(huì)大會(huì)報(bào)告日程安排[A];第四屆全國(guó)RNA進(jìn)展研討會(huì)論文集[C];2005年

3 ;Function of Transfer RNA Modifications in Plant Development[A];植物分子生物學(xué)與現(xiàn)代農(nóng)業(yè)——全國(guó)植物生物學(xué)研討會(huì)論文摘要集[C];2010年

4 王峰;張秋平;陳金湘;;棉花總RNA的快速提取方法[A];中國(guó)棉花學(xué)會(huì)2011年年會(huì)論文匯編[C];2011年

5 關(guān)力;陳本iY;iJ云虹;郭培芝;魏重琴;邱蘇吾;苗健;;關(guān)于動(dòng)物}D~T中RNAn,定方法的研究[A];中國(guó)生理科學(xué)會(huì)學(xué)術(shù)會(huì)議論文摘要匯編（生物化學(xué)）[C];1964年

6 夏海濱;;小RNA在免疫學(xué)領(lǐng)域中的應(yīng)用研究進(jìn)展[A];中國(guó)免疫學(xué)會(huì)第五屆全國(guó)代表大會(huì)暨學(xué)術(shù)會(huì)議論文摘要[C];2006年

7 ;The stability of hepatitis C virus RNA in various handling and storage conditions[A];中國(guó)輸血協(xié)會(huì)第四屆輸血大會(huì)論文集[C];2006年

8 郭德銀;;RNA干擾在病毒研究和控制中的應(yīng)用[A];2006中國(guó)微生物學(xué)會(huì)第九次全國(guó)會(huì)員代表大會(huì)暨學(xué)術(shù)年會(huì)論文摘要集[C];2006年

9 甘儀梅;楊業(yè)華;王學(xué)奎;曹燕;楊特武;;棉花總RNA快速提取[A];中國(guó)棉花學(xué)會(huì)2007年年會(huì)論文匯編[C];2007年

10 ;Identification and characterization of novel interactive partner proteins for PCBP1 that is a RNA-binding protein[A];中國(guó)優(yōu)生優(yōu)育協(xié)會(huì)第四屆全國(guó)學(xué)術(shù)論文報(bào)告會(huì)暨基因科學(xué)高峰論壇論文專輯[C];2008年

相關(guān)重要報(bào)紙文章 前10條

1 記者 馮衛(wèi)東;研究人員發(fā)現(xiàn)可破壞腫瘤抑制基因的小RNA[N];科技日?qǐng)?bào);2009年

2 記者 儲(chǔ)笑抒 通訊員 盛偉;人體微小RNA有望提前發(fā)出癌癥預(yù)警[N];南京日?qǐng)?bào);2011年

3 瀘州醫(yī)學(xué)院副教授、科普作家 周志遠(yuǎn);“大頭兒子”與環(huán)狀RNA[N];第一財(cái)經(jīng)日?qǐng)?bào);2014年

4 麥迪信;小分子RNA可能有大作用[N];醫(yī)藥經(jīng)濟(jì)報(bào);2003年

5 董映璧;美發(fā)現(xiàn)基因調(diào)控可回應(yīng)“RNA世界”[N];科技日?qǐng)?bào);2006年

6 張忠霞;特制RNA輕推一下,就能“喚醒”基因[N];新華每日電訊;2007年

7 聶翠蓉;RNA：縱是配角也精彩[N];科技日?qǐng)?bào);2009年

8 馮衛(wèi)東;RNA干擾機(jī)制首次在人體中獲得證實(shí)[N];科技日?qǐng)?bào);2010年

9 馮衛(wèi)東　王小龍;英在地球早期環(huán)境模擬條件下合成類RNA[N];科技日?qǐng)?bào);2009年

10 記者 常麗君;新技術(shù)讓研究進(jìn)入單細(xì)胞內(nèi)RNA的世界[N];科技日?qǐng)?bào);2011年

相關(guān)博士學(xué)位論文 前10條

1 王趙瑋;昆蟲RNA病毒復(fù)制及昆蟲抗病毒天然免疫機(jī)制研究[D];武漢大學(xué);2014年

2 包純;一類新非編碼RNA的發(fā)現(xiàn)以及產(chǎn)生和功能的初探[D];華中師范大學(xué);2015年

3 李語(yǔ)麗;基于MeRIP-seq的水稻RNA m6A甲基化修飾的研究[D];中國(guó)科學(xué)院北京基因組研究所;2015年

4 熊瑜琳;miR-122靶位基因STAT3調(diào)控長(zhǎng)鏈非編碼 RNA Lethe促進(jìn)HCV復(fù)制的機(jī)制研究[D];第三軍醫(yī)大學(xué);2015年

5 范春節(jié);高通量測(cè)序鑒定毛竹小RNA及其功能分析[D];中國(guó)林業(yè)科學(xué)研究院;2012年

6 王加強(qiáng);小鼠著床前胚胎特異ERV相關(guān)長(zhǎng)非編碼RNA的定向篩選及功能研究[D];東北農(nóng)業(yè)大學(xué);2015年

7 王業(yè)偉;非編碼RNA SPIU的結(jié)構(gòu)和功能研究和p19INK4D在APL發(fā)病中的作用[D];上海交通大學(xué);2013年

8 鄒艷芬;子癇前期中非編碼RNA對(duì)滋養(yǎng)細(xì)胞功能的調(diào)控及機(jī)制探索[D];南京醫(yī)科大學(xué);2015年

9 朱喬;miR-10b在人肝細(xì)胞肝癌發(fā)生中的作用及其機(jī)制的初步探索[D];第四軍醫(yī)大學(xué);2015年

10 蔣俊鋒;長(zhǎng)鏈非編碼RNA BACE1-AS促進(jìn)Aβ聚集及其調(diào)節(jié)BACE1和SERF1a的ceRNA機(jī)制研究[D];第二軍醫(yī)大學(xué);2015年

相關(guān)碩士學(xué)位論文 前10條

1 陸聰兒;條紋斑竹鯊再生肝臟中差異RNA的研究[D];浙江理工大學(xué);2015年

2 張曉輝;小鼠幾種長(zhǎng)鏈非編碼RNA基因功能的初步研究[D];昆明理工大學(xué);2015年

3 全弘揚(yáng);長(zhǎng)鏈非編碼RNA在細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)中的相關(guān)作用及機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2015年

4 胡亮;DDX19A識(shí)別PRRSV基因組RNA并激活NLRP3炎癥小體[D];中國(guó)農(nóng)業(yè)科學(xué)院;2015年

5 張帥兵;應(yīng)用RNA干擾技術(shù)對(duì)旋毛蟲Nudix水解酶基因功能的研究[D];鄭州大學(xué);2015年

6 鄭芳芳;肺癌RNA-Seq數(shù)據(jù)中RNA編輯事件的識(shí)別算法和系統(tǒng)分析[D];蘇州大學(xué);2015年

7 陳瑞東;長(zhǎng)鏈非編碼RNA MEG3在胰腺癌發(fā)生發(fā)展中作用的實(shí)驗(yàn)研究[D];蘇州大學(xué);2015年

8 劉駿武;線蟲和水稻中環(huán)狀RNA的預(yù)測(cè)及分析[D];華中農(nóng)業(yè)大學(xué);2015年

9 李猷;利用RNA干擾技術(shù)提高番茄抗TMV侵染能力的研究[D];牡丹江師范學(xué)院;2015年

10 吳術(shù)盛;SVCV感染EPC細(xì)胞microRNA表達(dá)譜的初步研究[D];華中農(nóng)業(yè)大學(xué);2015年

，

本文編號(hào)：1607177