CAJ | 학술논문

背景:转染是基因研究最关键的始动环节,高效安全的基因转染试剂是基因研究的热点问题.纳米级材料表面活性强,易于表面修饰,膜通透性高,如何将物质纳米化应用于基因转染尚在探索之中.目的:观察不同分子质量、结脂度的纳米级阳离子聚合物的转染效率,筛选低毒、高效的优化转染试剂.设计:对照实验.单位:南方医科大学基因研究所.方法:实验于2006-03/2007-06在南方医科大学基因工程研究所实验室完成.以脂质体为阳性对照,将9种不同分子质量、结脂度的纳米级阳离子聚合物聚乳酸聚乙醇酸、壳聚糖-聚己内酯、聚乙烯亚胺-聚乙二醇,包裹FITC标记的以bcl-2基因为靶标的siRNA(0.2 nmol/L),转入无血清培养的白血病细胞株K562,于转染后6 h后补加20%胎牛血清培养基.MTT法测定24,48,72 h后细胞增殖状况,荧光显微镜检测转染48 h后各纳米材料转染效率,流式细胞仪检测白血病细胞株K562细胞Bcl-2蛋白的表达和凋亡率.结果:①荧光显微镜结果:不同材料的纳米级颗粒,其转染效率有统计学差异(P < 0.05),同种材料的纳米结构因其结脂度不同,其转染效率也有统计学差异(P < 0.05).②MTT结果:表明细胞增殖率与转染率正相关.③流式细胞仪结果表明:转染siRNA引起的靶基因表达抑制以及细胞凋亡率与转染效率正相关.结论:分子质量为1 800/2 000,脂结合力为29%的纳米级聚乙烯亚胺-聚乙二醇颗粒,为低毒、高效的转染试剂.
배경:전염시기인연구최관건적시동배절,고효안전적기인전염시제시기인연구적열점문제.납미급재료표면활성강,역우표면수식,막통투성고,여하장물질납미화응용우기인전염상재탐색지중.목적:관찰불동분자질량、결지도적납미급양리자취합물적전염효솔,사선저독、고효적우화전염시제.설계:대조실험.단위:남방의과대학기인연구소.방법:실험우2006-03/2007-06재남방의과대학기인공정연구소실험실완성.이지질체위양성대조,장9충불동분자질량、결지도적납미급양리자취합물취유산취을순산、각취당-취기내지、취을희아알-취을이순,포과FITC표기적이bcl-2기인위파표적siRNA(0.2 nmol/L),전입무혈청배양적백혈병세포주K562,우전염후6 h후보가20%태우혈청배양기.MTT법측정24,48,72 h후세포증식상황,형광현미경검측전염48 h후각납미재료전염효솔,류식세포의검측백혈병세포주K562세포Bcl-2단백적표체화조망솔.결과:①형광현미경결과:불동재료적납미급과립,기전염효솔유통계학차이(P < 0.05),동충재료적납미결구인기결지도불동,기전염효솔야유통계학차이(P < 0.05).②MTT결과:표명세포증식솔여전염솔정상관.③류식세포의결과표명:전염siRNA인기적파기인표체억제이급세포조망솔여전염효솔정상관.결론:분자질량위1 800/2 000,지결합력위29%적납미급취을희아알-취을이순과립,위저독、고효적전염시제.
BACKGROUND: Transfection is the most important beginning component of research about gene function. It is a problem to find a transfection agent with high efficiency and safety. Nanosized materials have high surface activity, are easy to be modified and easier to pass the biomembrane. Researchers are studying how to use nanosized materials as a transfection agent.OBJECTIVE: To study the transfection efficiency of different basic polymers with different molecular mass and degree of substitute, and to find the optimized transfection agent.DESIGN: Controlled study.SETTING: Institute of Genetic Engineering, Southern Medical University.METHODS: This study was performed in the Institute of Genetic Engineering, Southern Medical University from March 2006 to June 2007. Using the lipofectamine reagent as the positive control, we transfected siRNA (0.2 nmol/L), FITC-labeling targeting bcl-2, by nine nanometers (polylactic acid-polyglycolic acid, chitosan-poly-caprolactone, polyethyleneimin-macrogol) into leukemic cell K562 cultured without serum. Six hours post-transfection, 20% FBS serum was added. The cell proliferation was measured at 24, 48 and 72 hours by using the MTT method. After transfecting for 48 hours, the cells were collected to detect transfection ratio by using fluorescent microscopy, apoptosis ratio and expression of K562 and bcl-2 protein by flow cytometer (FCM). RESULTS: ① Fluorescence microscope detection showed there were significant differences of transfection efficiency between different materials (P < 0.05). Moreover there were statistical differences between different degrees of substitute, although they were the same material (P < 0.05). ② MTT method indicated the cell proliferation ratio was positively related to transfection ratio. ③ Flow cytometry results showed the suppression of expression of targeting gene and apoptosis ratio were positive correlated with transfection ratio. CONCLUSION:The nanometer poly-ethylene glycol combined with poly-ethylene imine (PEG-PEI), whose molecular mass is 1 800/2 000 and degree of substitute is 29%, has a high efficiency and low toxicity.