核酸载体精胺化普鲁兰在抗肿瘤RNA干扰中的作用研究

发布时间：2018-03-13 16:23

本文选题：精胺化普鲁兰　切入点：siRNA　出处：《北京协和医学院》2016年硕士论文　论文类型：学位论文

【摘要】：目的：RNA干扰(RNAi)技术在多种蛋白表达相关的疾病或病毒感染的治疗中具有应用前景。实现RNAi的关键条件之一是获得具有RNA药物高装载量和高转染效率、无毒且无免疫原性、并易于商业化生产的载体。非病毒型载体具有易化学合成、低免疫原性、保护siRNA免于降解等优势。在众多非病毒载体中,阳离子多糖具有良好的生物相容性并且易修饰,一直是核酸载体的研发重点之一。但是,在递送siRNA过程中,阳离子多糖会与血清蛋白发生非特异性结合,从而影响RNA干扰效率。为了减少血清蛋白的影响,通常需要使用无血清条件培养细胞。然而,在无血清环境中,阳离子多糖又会通过静电相互作用而与细胞膜发生非特异性结合,由此而导致严重的细胞毒性；此外,当在体内应用时,无血清环境的要求也无法中得到满足。慢性髓系白血病(CML)是由造血干细胞染色体移位而产生融合基因BCR-ABL所导致的血液恶性肿瘤。CML的主要治疗方法为激酶抑制剂和异体造血干细胞移植,但患者会逐渐对激酶抑制剂产生耐药,同时还有部分患者对药物不耐受。越来越多的研究致力于将RNA干扰技术应用于CML的治疗,但是,面临着CML细胞难以转染的挑战。目前对CML细胞转染的方法主要包括电穿孔和病毒载体两种。但是,电穿孔转染不能用于体内实验,而病毒载体具有引起免疫反应的风险,并且易引起宿主基因突变,因此,迫切需要研发针对CML细胞的新型载体。精胺化普鲁兰多糖(Pullulan-spermine, Ps)是一种典型的阳离子多糖,由于其特有的化学成分而在肝脏细胞的RNAi中得到广泛研究。实验室之前的研究结果显示,培养基中的血清浓度会影响Ps与siRNA所形成的复合物(Ps/siRNA)被乳腺癌细胞摄取的数量及其溶酶体逃逸能力,由此而影响RNA干扰效率,但是关于血清蛋白(Pr)与Ps的相互作用及Pr与Ps的质量比(Pr/Ps)与RNAi之间的关系尚需要深入研究。此外,尚未有研究报道Ps在血液恶性肿瘤RNAi中的应用。本研究用Ps作为模型阳离子多糖载体,开展以下两方面的研究：(1)Ps对蛋白分子的吸附作用以及Pr/Ps对Ps/siRNA复合物的粒径和RNA干扰效率的调控作用；(2)以Ps为siRNA载体,沉默CML细胞功能基因BCR-ABL,并比较Ps在CML细胞、RAW264.7和Jurkat细胞上引起的RNA干扰效率的差异。方法：采用等温滴定量热法研究Ps与牛血清白蛋白(BSA)之间的吸附作用,通过琼脂糖凝胶电泳检测Ps/siRNA复合物的形成,以及BSA对Ps与siRNA结合稳定性的影响；通过动态光散射实验(DLS)研究溶液体系中Pr/Ps对Ps/siRNA复合物颗粒尺寸的影响。以MDA-MB-231和MDA-MB-231-EGFP细胞为模型,用流式细胞术和激光共聚焦显微术检测Pr/Ps对复合物进入细胞的影响和对RNA干扰效率的影响。应用RT-PCR技术研究血清对Ps、Lipo 3000介导β-actin/BCR-ABL siRNA对K562、KU812、MEG-01、RAW、Jurkat细胞的RNA干扰效率的影响；以Ps/PGL4.51转染K562、RAW、Jurkat细胞,并用微孔板块荧光检测仪检测Luc的表达量。结果：Ps与siRNA完全结合的质量比为0.75：1。在溶液中Ps会吸附BSA分子,但BSA的吸附不影响Ps/siRNA的稳定性。在一定siRNA质量范围内,随着Pr/Ps的增大,Ps/siRNA的粒径减小,同时,复合物Ps/siRNA进入细胞的能力、对细胞的毒性和介导基因沉默的效率也逐渐降低；在Pr/Ps固定的条件下,随siRNA量的增加,RNA干扰效率逐渐提高,当siRNA浓度达到1 μg/mL后,RNA干扰效率不再增加。在Ps/siRNA与细胞共孵育后,使用氯喹(CQ)处理细胞,可使RNA干扰效率进一步提高。当N/P为2.5时,复合物Ps/siRNA的粒径不受培养基中血清浓度的影响。同时,无论在无血清培养基中还是在有血清培养的条件下,K562的细胞活性都在80%以上;Ps/FAM-siRNA在3种CML细胞上的阳性率均大于95%,而在RAW和Jurkat两种细胞上的阳性率在85%左右。对5种细胞的β-actin基因进行RNA干扰,在Opti-MEM培养基条件下,Ps在3种CML细胞上介导的干扰效率高于Lipo3000,在RAW、Jurkat细胞上低于Lipo 3000;在10%的血清浓度条件下,Lipo3000和Ps介导的干扰效率均很低。对3种CML细胞的BCR-ABL基因进行RNA干扰,在上述两种培养基条件下,载体Ps在K562细胞上介导的干扰效率明显高于Lipo3000；对于KU812细胞,Ps与Lipo3000介导的干扰效率没有差异；对于MEG-01细胞,在Opti-MEM培养基条件下,Ps介导的干扰效率高于Lipo3000,而在10%血清浓度条件下,低于Lipo 3000。当N/P为2.5时,Ps在K562及其耐药株上介导的RNA干扰效率不受血清浓度影响,但在HELA细胞上介导的干扰效率则随血清浓度的增加而降低。此外,Ps/PGL4.51在K562细胞上介导的荧光素酶的表达量高于在RAW和Jurkat细胞上的表达量。结论：溶液中血清蛋白与阳离子多糖的质量比Pr/Ps是调控阳离子多糖Ps与siRNA所形成复合物的颗粒尺寸的关键参数,对复合物进入细胞的能力及其所介导的RNA干扰效率具有重要的调控作用。此外,Ps在CML类细胞上表现出优于Lipo3000的沉默白血病相关功能基因的性能,有望成为介导CML细胞RNA干扰的新载体。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R96

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