CAJ | 학술논문

目的：制备载血管紧张素转换酶（ACE）短发卡RNA（shRNA）壳聚糖纳米粒，并且给予聚乙二醇（PEG）表面修饰，分析其相关物理学、生物学特性，以期获得一种缓释的非病毒载体介导系统。方法应用本实验室前期实验中筛选出的能显著下调ACE基因的靶序列，采用菌液用碱裂解法大量抽提和纯化重组质粒，随机酶切测序，并检测质粒浓度与纯度，采用离子交联法制备壳聚糖纳米粒， PEG修饰壳聚糖纳米粒，复凝聚法制备不同pH值、体积比以及PEG化的载ACEshRNA壳聚糖纳米。喷金电镜下对各种壳聚糖纳米粒悬液扫描并照相，观察纳米粒与形态。应用凝胶阻滞分析验证载ACEshRNA壳聚糖纳米多聚复合物的形成及电荷性质。结果①壳聚糖纳米粒的粒径随着溶液pH值的升高而增大，当pH值为5．5时的PEG壳聚糖纳米粒平均粒径（125．8±5．6）nm左右，大小均匀，多分散度最小，分布比较集中，zeta电位为正，有利于与带负电荷的质粒结合；PEG化后也对粒径以及zeta电位无明显影响，但是与壳聚糖相同条件相比，分散度均明显减小，可见更适合制备均匀的纳米粒，利于和质粒结合；壳聚糖与质粒体积比（质量比）为1∶1制备的壳聚糖纳米粒平均粒径较小，故通过细胞膜的通透性较好，多分散度较小，分布比较集中， zeta电位也为正值，有利于与带负电荷的质粒结合。②壳聚糖纳米以及PEG化壳聚糖纳米质粒复合物均有效结合质粒，由于中和了质粒所带的负电荷，凝胶电泳时，质粒不出孔，而裸质粒则出孔；pH＜7时壳聚糖分子中大部分的氨基带正电荷能与质粒DNA有效地结合，质粒不出孔；在体积比为1∶1、1∶2、1∶3时，壳聚糖纳米粒能有效地结合质粒。结论所制备的载ACE shRNA-PEG壳聚糖纳米粒大小均匀，粒径分布范围较窄，并且筛选出在pH＝5．5时，与质粒体积比为1∶1时，以PEG壳聚糖纳米粒作为载体有良好的结合力，为后期体外转染培养细胞以及体内转染提供了实验基础。目的：製備載血管緊張素轉換酶（ACE）短髮卡RNA（shRNA）殼聚糖納米粒，併且給予聚乙二醇（PEG）錶麵脩飾，分析其相關物理學、生物學特性，以期穫得一種緩釋的非病毒載體介導繫統。方法應用本實驗室前期實驗中篩選齣的能顯著下調ACE基因的靶序列，採用菌液用堿裂解法大量抽提和純化重組質粒，隨機酶切測序，併檢測質粒濃度與純度，採用離子交聯法製備殼聚糖納米粒， PEG脩飾殼聚糖納米粒，複凝聚法製備不同pH值、體積比以及PEG化的載ACEshRNA殼聚糖納米。噴金電鏡下對各種殼聚糖納米粒懸液掃描併照相，觀察納米粒與形態。應用凝膠阻滯分析驗證載ACEshRNA殼聚糖納米多聚複閤物的形成及電荷性質。結果①殼聚糖納米粒的粒徑隨著溶液pH值的升高而增大，噹pH值為5．5時的PEG殼聚糖納米粒平均粒徑（125．8±5．6）nm左右，大小均勻，多分散度最小，分佈比較集中，zeta電位為正，有利于與帶負電荷的質粒結閤；PEG化後也對粒徑以及zeta電位無明顯影響，但是與殼聚糖相同條件相比，分散度均明顯減小，可見更適閤製備均勻的納米粒，利于和質粒結閤；殼聚糖與質粒體積比（質量比）為1∶1製備的殼聚糖納米粒平均粒徑較小，故通過細胞膜的通透性較好，多分散度較小，分佈比較集中， zeta電位也為正值，有利于與帶負電荷的質粒結閤。②殼聚糖納米以及PEG化殼聚糖納米質粒複閤物均有效結閤質粒，由于中和瞭質粒所帶的負電荷，凝膠電泳時，質粒不齣孔，而裸質粒則齣孔；pH＜7時殼聚糖分子中大部分的氨基帶正電荷能與質粒DNA有效地結閤，質粒不齣孔；在體積比為1∶1、1∶2、1∶3時，殼聚糖納米粒能有效地結閤質粒。結論所製備的載ACE shRNA-PEG殼聚糖納米粒大小均勻，粒徑分佈範圍較窄，併且篩選齣在pH＝5．5時，與質粒體積比為1∶1時，以PEG殼聚糖納米粒作為載體有良好的結閤力，為後期體外轉染培養細胞以及體內轉染提供瞭實驗基礎。
목적：제비재혈관긴장소전환매（ACE）단발잡RNA（shRNA）각취당납미립，병차급여취을이순（PEG）표면수식，분석기상관물이학、생물학특성，이기획득일충완석적비병독재체개도계통。방법응용본실험실전기실험중사선출적능현저하조ACE기인적파서렬，채용균액용감렬해법대량추제화순화중조질립，수궤매절측서，병검측질립농도여순도，채용리자교련법제비각취당납미립， PEG수식각취당납미립，복응취법제비불동pH치、체적비이급PEG화적재ACEshRNA각취당납미。분금전경하대각충각취당납미립현액소묘병조상，관찰납미립여형태。응용응효조체분석험증재ACEshRNA각취당납미다취복합물적형성급전하성질。결과①각취당납미립적립경수착용액pH치적승고이증대，당pH치위5．5시적PEG각취당납미립평균립경（125．8±5．6）nm좌우，대소균균，다분산도최소，분포비교집중，zeta전위위정，유리우여대부전하적질립결합；PEG화후야대립경이급zeta전위무명현영향，단시여각취당상동조건상비，분산도균명현감소，가견경괄합제비균균적납미립，리우화질립결합；각취당여질립체적비（질량비）위1∶1제비적각취당납미립평균립경교소，고통과세포막적통투성교호，다분산도교소，분포비교집중， zeta전위야위정치，유리우여대부전하적질립결합。②각취당납미이급PEG화각취당납미질립복합물균유효결합질립，유우중화료질립소대적부전하，응효전영시，질립불출공，이라질립칙출공；pH＜7시각취당분자중대부분적안기대정전하능여질립DNA유효지결합，질립불출공；재체적비위1∶1、1∶2、1∶3시，각취당납미립능유효지결합질립。결론소제비적재ACE shRNA-PEG각취당납미립대소균균，립경분포범위교착，병차사선출재pH＝5．5시，여질립체적비위1∶1시，이PEG각취당납미립작위재체유량호적결합력，위후기체외전염배양세포이급체내전염제공료실험기출。
Objective To prepare angiotensin converting enzyme (ACE) short hairpin RNA (shRNA) chitosan nanoparticles which are subsequently surface-modified with polyethylene glycol (PEG), and to analyze the relevant physical and biological characteristics of this complex in order to obtain a sustained release non-viral vector-mediated system. Methods The significantly down-regulated ACE gene target sequence screened out from our pre-experiment was applied in the study. The substantial extraction and purification of recombinant plasmid were completed by alka-line lysis of bacteria. The sequencing was detected by random enzyme digestion, and the concentration and purity of plasmid were measured. The chitosan nanoparticles were prepared by ionic crosslinking method and modified by PEG. Then, PEG-chitosan nanoparticles containing ACEshRNA of different pH values and the volume ratios were prepared by complex coacervation. Different suspensions of chitosan nanoparticles were studied and photographed under scan-ning electron microscopy with gold-coating to observe their morphology. The formation and charge characteristics of ACE-shRNA-containing chitosan nanoparticles complex were analyzed and verified by gel retardation assay. Results①The size of chitosan nanoparticles increased with the rise of solution pH value. At the pH value of 5.5, the PEG chitosan nanoparticles were even in size [mean(125.8±5.6)nm], with minimum polydispersity, concentrated distribu-tion and positive zeta potential which facilitated binding to the negatively charged plasmid. After PEG modification, there were no significant change in particle size and zeta potential. However, comparing with non-PEG chitosan under the same conditions, the dispersity of PEG modified chitosan nanoparticles was even lowered, suggesting that PEG modification may be more suitably used to prepare uniform-sized nanoparticles which readily binds to with plasmids. A smaller mean size of chitosan nanoparticles obtained with 1∶1 volume ratio (mass ratio) of chitosan nanoparticles to plasmid, presented with better membrane permeability, smaller dispersity, more concentrated distribution and positive zeta potential, which favored their binding to the negatively charged plasmid. ②Both chitosan nano-and PEG-chitosan nano-plasmid complexes may effectively bound to plasmid. As the negatively charged plasmid was neutralized, the plasmid retained inside the spores in contrast to the opposite as did the naked plasmid during gel electrophoresis. At volume ratio of 1∶1, 1∶2 and 1∶3, the chitosan nanoparticles can effectively combine with plasmid. Conclusion The prepared PEG-chitosan nanoparticles containing ACE-shRNA has uniform size and narrower particle size distribution. At a PH of 5.5 and the volume ratio to plasmid of 1∶1, PEG chitosan nanoparticles as a vector have good binding ca-pacity. This study provide a foundation for the future in vitro transfection of cultured cells and in-vivo transfection ex-periments.