中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
47期
9393-9397
,共5页
蔡寨%厉周%黄宗海%方素珍%崔大祥%高峰
蔡寨%厲週%黃宗海%方素珍%崔大祥%高峰
채채%려주%황종해%방소진%최대상%고봉
聚酰胺-胺型树枝状高聚合物%结直肠癌%反义寡核苷酸%Survivin
聚酰胺-胺型樹枝狀高聚閤物%結直腸癌%反義寡覈苷痠%Survivin
취선알-알형수지상고취합물%결직장암%반의과핵감산%Survivin
背景:近年来纳米载体已被视为突破基因转移瓶颈最有前途的技术之一.聚酰胺-胺型树枝状高聚合物(polyamidoamine,PAMAM)是一种新型的纳米材料,体内外实验表明,PAMAM比其他阳离子载体的基因转染效率高、细胞毒性低.目的:探讨PAMAM介导Survivin反义寡核苷酸(Survivin antisense oligonucleotide,Survivin-asODN)对结直肠癌裸鼠移植瘤的抑制作用.设计、时间及地点:肿瘤基因治疗体内实验,于2008-02/08在上海交通大学微纳米科学技术研究院生物纳米工程研究室及南方医科大学珠江医院中心实验室完成.材料:人结直肠癌细胞株SW620购自中科院上海细胞所,PAMAM树形分子由上海交通大学微纳米研究院生物纳米工程研究室提供,脂质体Lipolifectmain~(TM)2000购自美国Invitrogen公司.Survivin-asODN 由上海生工公司合成.方法:将PAMAM和阳离子脂质体分别与Survivin-asODN混合得到载反义基因转染复合物.透射电镜观察复合物的形态,激光散射粒径分析仪测定粒径,zeta电位分析仪测定复合物的zeta电位,离心法和紫外分光分度仪测定复合物的包封率和体外DNA释放速度.将对数生长期的人结直肠癌细胞SW620细胞接种于18只Balb/C裸鼠皮下建立结直肠癌裸鼠皮下移植瘤模型.随机均分为脂质体组、PAMAM组和空白对照组.分别注射脂质体-survivin-asODN转染复合物、PAMAM-survivin-asODN转染复合物、血清培养液,观察两组移植瘤体积,Western bloting方法检测移植瘤组织中survivin基因的表达.主要观察指标:阳离脂质体-survivin-asODN复合物及PAMAM-survivin-asODN复合物的粒经,zeta电位,基因载药率,包封率,释放率,转染后survivin表达率及对凋亡的诱导率,种植肿瘤生长抑制率,移植瘤细胞Survivin蛋白的表达及活性.结果:PAMAM-survivin-asODN复合物的粒径小于脂质体-survivin-asODN复合物的粒径(P<0.01),而zeta电位高于PAMAM-survivin-asODN复合物zeta电位(P<0.05),基因包封率两组差异无显著性意义.PAMAM对DNA持续释放达14 d,但脂质体复合物只持续5 d.PAMAM-survivin-asODN复合物治疗组裸鼠移植瘤survivin蛋白表达低于脂质体-survivin-asODN复合物组(P<0.05).PAMAM-survivin-asODN复合物治疗组移植瘤体积低于脂质体-survivin-asODN复合物组(P<0.05).结论:PAMAM能将Survivin-asODN高效递送到结直肠癌移植瘤细胞,降低survivin蛋白的表达,抑制移植瘤生长.
揹景:近年來納米載體已被視為突破基因轉移瓶頸最有前途的技術之一.聚酰胺-胺型樹枝狀高聚閤物(polyamidoamine,PAMAM)是一種新型的納米材料,體內外實驗錶明,PAMAM比其他暘離子載體的基因轉染效率高、細胞毒性低.目的:探討PAMAM介導Survivin反義寡覈苷痠(Survivin antisense oligonucleotide,Survivin-asODN)對結直腸癌裸鼠移植瘤的抑製作用.設計、時間及地點:腫瘤基因治療體內實驗,于2008-02/08在上海交通大學微納米科學技術研究院生物納米工程研究室及南方醫科大學珠江醫院中心實驗室完成.材料:人結直腸癌細胞株SW620購自中科院上海細胞所,PAMAM樹形分子由上海交通大學微納米研究院生物納米工程研究室提供,脂質體Lipolifectmain~(TM)2000購自美國Invitrogen公司.Survivin-asODN 由上海生工公司閤成.方法:將PAMAM和暘離子脂質體分彆與Survivin-asODN混閤得到載反義基因轉染複閤物.透射電鏡觀察複閤物的形態,激光散射粒徑分析儀測定粒徑,zeta電位分析儀測定複閤物的zeta電位,離心法和紫外分光分度儀測定複閤物的包封率和體外DNA釋放速度.將對數生長期的人結直腸癌細胞SW620細胞接種于18隻Balb/C裸鼠皮下建立結直腸癌裸鼠皮下移植瘤模型.隨機均分為脂質體組、PAMAM組和空白對照組.分彆註射脂質體-survivin-asODN轉染複閤物、PAMAM-survivin-asODN轉染複閤物、血清培養液,觀察兩組移植瘤體積,Western bloting方法檢測移植瘤組織中survivin基因的錶達.主要觀察指標:暘離脂質體-survivin-asODN複閤物及PAMAM-survivin-asODN複閤物的粒經,zeta電位,基因載藥率,包封率,釋放率,轉染後survivin錶達率及對凋亡的誘導率,種植腫瘤生長抑製率,移植瘤細胞Survivin蛋白的錶達及活性.結果:PAMAM-survivin-asODN複閤物的粒徑小于脂質體-survivin-asODN複閤物的粒徑(P<0.01),而zeta電位高于PAMAM-survivin-asODN複閤物zeta電位(P<0.05),基因包封率兩組差異無顯著性意義.PAMAM對DNA持續釋放達14 d,但脂質體複閤物隻持續5 d.PAMAM-survivin-asODN複閤物治療組裸鼠移植瘤survivin蛋白錶達低于脂質體-survivin-asODN複閤物組(P<0.05).PAMAM-survivin-asODN複閤物治療組移植瘤體積低于脂質體-survivin-asODN複閤物組(P<0.05).結論:PAMAM能將Survivin-asODN高效遞送到結直腸癌移植瘤細胞,降低survivin蛋白的錶達,抑製移植瘤生長.
배경:근년래납미재체이피시위돌파기인전이병경최유전도적기술지일.취선알-알형수지상고취합물(polyamidoamine,PAMAM)시일충신형적납미재료,체내외실험표명,PAMAM비기타양리자재체적기인전염효솔고、세포독성저.목적:탐토PAMAM개도Survivin반의과핵감산(Survivin antisense oligonucleotide,Survivin-asODN)대결직장암라서이식류적억제작용.설계、시간급지점:종류기인치료체내실험,우2008-02/08재상해교통대학미납미과학기술연구원생물납미공정연구실급남방의과대학주강의원중심실험실완성.재료:인결직장암세포주SW620구자중과원상해세포소,PAMAM수형분자유상해교통대학미납미연구원생물납미공정연구실제공,지질체Lipolifectmain~(TM)2000구자미국Invitrogen공사.Survivin-asODN 유상해생공공사합성.방법:장PAMAM화양리자지질체분별여Survivin-asODN혼합득도재반의기인전염복합물.투사전경관찰복합물적형태,격광산사립경분석의측정립경,zeta전위분석의측정복합물적zeta전위,리심법화자외분광분도의측정복합물적포봉솔화체외DNA석방속도.장대수생장기적인결직장암세포SW620세포접충우18지Balb/C라서피하건립결직장암라서피하이식류모형.수궤균분위지질체조、PAMAM조화공백대조조.분별주사지질체-survivin-asODN전염복합물、PAMAM-survivin-asODN전염복합물、혈청배양액,관찰량조이식류체적,Western bloting방법검측이식류조직중survivin기인적표체.주요관찰지표:양리지질체-survivin-asODN복합물급PAMAM-survivin-asODN복합물적립경,zeta전위,기인재약솔,포봉솔,석방솔,전염후survivin표체솔급대조망적유도솔,충식종류생장억제솔,이식류세포Survivin단백적표체급활성.결과:PAMAM-survivin-asODN복합물적립경소우지질체-survivin-asODN복합물적립경(P<0.01),이zeta전위고우PAMAM-survivin-asODN복합물zeta전위(P<0.05),기인포봉솔량조차이무현저성의의.PAMAM대DNA지속석방체14 d,단지질체복합물지지속5 d.PAMAM-survivin-asODN복합물치료조라서이식류survivin단백표체저우지질체-survivin-asODN복합물조(P<0.05).PAMAM-survivin-asODN복합물치료조이식류체적저우지질체-survivin-asODN복합물조(P<0.05).결론:PAMAM능장Survivin-asODN고효체송도결직장암이식류세포,강저survivin단백적표체,억제이식류생장.
BACKGROUND: In recent years, nano-carriers have been regarded as the most promising technologies for breakthrough the bottleneck of gene transfer. Polyamidoamine dendrimer (PAMAM) is a kind of new nanometer material. PAMAM can transfer target gene to the cell with high efficiency and lower toxic both in vivo and in vitro.OBJECTIVE: To evaluate the antitumour effects of survivin antisense oligonucleotide (Survivin-asODN) carried by PAMAM on colorectal cancer transplanted subcutaneously in nude mice.DESIGN, TIME AND SETTING: An in vivo experiment regarding tumor gene therapy was performed from February to August in 2008 at the Laboratory of Bionanometer Engineering, Research Institute of Micro/nanometer Science & Technology of Shanghai Jiao Tong University and Central Laboratory of Zhujiang Hospital of Southern Medical University.MATERIALS: Human colorectal cancer cells SW620 were from Shanghai Cell Institute of Chinese Academy of Sciences.PAMAM dendrimer was offered by the Bionanometer Engineering Laboratory, Research Institute of Micro/nanometer Science & Technology, Shanghai Jiao Tong University. Lipofectamine ~(TM)2000 was purchased from Invitrogen, USA. Survivin-asODN was synthesized by Shanghai Bioengineering Company.METHODS: The PAMAM and cation liposome were respectively mixed with Survivin-asODN to generate the transfection complex carrying antisense gene. The shape of the complex was observed by transmission electron microscope, the particle size was determined by laser particle size analysator and the zeta potential was measured by an analytical tool. The encapsulating efficiency and release progress in vitro were determined by ultraviolet spectrophotometer in centrifuging method. Human colorectal cancer cells SW620 at logarithmic phase were inoculated into the abdominal region of 18 Blab/C nude mice subcutaneously to produce transplanted tumor models in colorectal cancer nude mice, which were randomly divided into 3 groups: liposome, PAMAM and blank control groups. They were injected respectively with Hposome-survivin-asODN complex,PAMAM-survivin-as ODN transfection complex and seroculture liquid. The volumes of tumor were surveyed in the 2 groups.Western blotting method was used to determine the survivin gene expression in the transplanted tumor tissue.MAIN OUTCOME MEASURES: Particle size, zeta potential, gene loading level, encapsulation efficiency, release rate of cationic liposome-survivin-asODN complex and PAMAM-survivin-asODN complex, as well as survivin expression rate and apoptosis rate after transfection, inhibition rate of the transplanted tumor growth, Survivin protein expression and activity in the transplanted tumor cells.RESULTS: The particle size of PAMAM-survivin-asODN complex was smaller (P < 0.01), but the zeta potential was greater (P < 0.05), compared with liposome-survivin-asODN. There was no significant difference between PAMAM and liposome groups in terms of gene loading rate and transfection efficiency. DNA release lasted for 14 days for PAMAM, but only 5 days for liposome.After colorectal cancer cell transfection, survivin protein expression was lower, but apoptosis rate was higher, in the PAMAM-survivin-asODN complex than in the liposome-survivin-asODN complex (P < 0.05).CONCLUSION: PAMAM facilitates delivery of Survivin-asODN into transplanted colorectal cancer cells SW620. As a result,survivin protein expression was decreased, and apoptosis rate was increased in vivo which inhibited transplanied tumour growth.
