中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2008年
8期
566-572
,共7页
王瑾%张振中%周天洋%刘宇琼%李惠翔
王瑾%張振中%週天洋%劉宇瓊%李惠翔
왕근%장진중%주천양%류우경%리혜상
聚乙烯亚胺%端粒酶逆转录酶%反义寡核苷酸%NGR%食管癌细胞系EC9706
聚乙烯亞胺%耑粒酶逆轉錄酶%反義寡覈苷痠%NGR%食管癌細胞繫EC9706
취을희아알%단립매역전록매%반의과핵감산%NGR%식관암세포계EC9706
Polyethylenimine%TERT%Antisense oligodeoxynucleotide%NGR%Human esophageal squamous carcinoma cell line EC9706
目的 探讨纳米载体介导的人端粒酶逆转录酶(hTERT)基因反义寡核苷酸(ASODN)在体内外对食管癌EC9706细胞的增殖抑制作用及端粒酶表达的影响.方法 制备线型聚乙烯亚胺(L-PEI)/ASODN纳米级复合物及NGR修饰的L-PEI/ASODN靶向纳米复合物,转染人食管癌细胞系EC9706.采用激光共聚焦显微镜观察细胞对纳米复合物的摄入情况,通过二苯基溴化四氮唑蓝(MTT)法检测细胞抑制率,采用半定量逆转录聚合酶链反应(RT-PCR)和免疫组化法分别检测hTERT mRNA和蛋白的表达情况,Annexin V-FITC/PI双标法检测凋亡细胞.通过体内药物分布实验和抑瘤实验观察NGR的靶向肿瘤细胞作用、裸鼠移植瘤的生长情况、组织形态学和超微形态学改变.结果 共聚焦显微镜观察可见,L-PEI可以保护hTERT ASODN有效进入细胞核内;N/P=10、ASODN终浓度40 μg/ml的L-PEI/ASODN复合物转染细胞后24、48、72和96 h,增殖抑制率分别为45.6%、55.8%、67.0%和83.1%;L-PEI/ASODN转染组hTERT mRNA水平明显降低,hTERT条带与内对照条带灰度值的比值为0.27±0.04,与空白对照组、ASODN转染组和L-PEI/SODN转染组相比,差异均有统计学意义(均P＜0.05),hTERT蛋白不表达;转染48 h后,L-PEI/ASODN转染组细胞凋亡明显,早期凋亡率和晚期凋亡率分别为38.0%和52.2%.NGR修饰的L-PEI/ASODN-FAM纳米复合物注射于荷瘤小鼠,其靶向肿瘤作用明显;给药8 d后,NGR/L-PEI/ASODN静脉给药组和皮下给药组的肿瘤体积均明显小于生理盐水组和NGR/L-PEI/SODN组(均P＜0.05);超微结构观察可见典型凋亡小体.结论 NGR修饰、L-PEI介导下的hTERT ASODN可抑制EC9706细胞的增殖,降低其端粒酶的表达,还可有效到达裸鼠人食管癌移植瘤内,抑制肿瘤的生长.
目的 探討納米載體介導的人耑粒酶逆轉錄酶(hTERT)基因反義寡覈苷痠(ASODN)在體內外對食管癌EC9706細胞的增殖抑製作用及耑粒酶錶達的影響.方法 製備線型聚乙烯亞胺(L-PEI)/ASODN納米級複閤物及NGR脩飾的L-PEI/ASODN靶嚮納米複閤物,轉染人食管癌細胞繫EC9706.採用激光共聚焦顯微鏡觀察細胞對納米複閤物的攝入情況,通過二苯基溴化四氮唑藍(MTT)法檢測細胞抑製率,採用半定量逆轉錄聚閤酶鏈反應(RT-PCR)和免疫組化法分彆檢測hTERT mRNA和蛋白的錶達情況,Annexin V-FITC/PI雙標法檢測凋亡細胞.通過體內藥物分佈實驗和抑瘤實驗觀察NGR的靶嚮腫瘤細胞作用、裸鼠移植瘤的生長情況、組織形態學和超微形態學改變.結果 共聚焦顯微鏡觀察可見,L-PEI可以保護hTERT ASODN有效進入細胞覈內;N/P=10、ASODN終濃度40 μg/ml的L-PEI/ASODN複閤物轉染細胞後24、48、72和96 h,增殖抑製率分彆為45.6%、55.8%、67.0%和83.1%;L-PEI/ASODN轉染組hTERT mRNA水平明顯降低,hTERT條帶與內對照條帶灰度值的比值為0.27±0.04,與空白對照組、ASODN轉染組和L-PEI/SODN轉染組相比,差異均有統計學意義(均P＜0.05),hTERT蛋白不錶達;轉染48 h後,L-PEI/ASODN轉染組細胞凋亡明顯,早期凋亡率和晚期凋亡率分彆為38.0%和52.2%.NGR脩飾的L-PEI/ASODN-FAM納米複閤物註射于荷瘤小鼠,其靶嚮腫瘤作用明顯;給藥8 d後,NGR/L-PEI/ASODN靜脈給藥組和皮下給藥組的腫瘤體積均明顯小于生理鹽水組和NGR/L-PEI/SODN組(均P＜0.05);超微結構觀察可見典型凋亡小體.結論 NGR脩飾、L-PEI介導下的hTERT ASODN可抑製EC9706細胞的增殖,降低其耑粒酶的錶達,還可有效到達裸鼠人食管癌移植瘤內,抑製腫瘤的生長.
목적 탐토납미재체개도적인단립매역전록매(hTERT)기인반의과핵감산(ASODN)재체내외대식관암EC9706세포적증식억제작용급단립매표체적영향.방법 제비선형취을희아알(L-PEI)/ASODN납미급복합물급NGR수식적L-PEI/ASODN파향납미복합물,전염인식관암세포계EC9706.채용격광공취초현미경관찰세포대납미복합물적섭입정황,통과이분기추화사담서람(MTT)법검측세포억제솔,채용반정량역전록취합매련반응(RT-PCR)화면역조화법분별검측hTERT mRNA화단백적표체정황,Annexin V-FITC/PI쌍표법검측조망세포.통과체내약물분포실험화억류실험관찰NGR적파향종류세포작용、라서이식류적생장정황、조직형태학화초미형태학개변.결과 공취초현미경관찰가견,L-PEI가이보호hTERT ASODN유효진입세포핵내;N/P=10、ASODN종농도40 μg/ml적L-PEI/ASODN복합물전염세포후24、48、72화96 h,증식억제솔분별위45.6%、55.8%、67.0%화83.1%;L-PEI/ASODN전염조hTERT mRNA수평명현강저,hTERT조대여내대조조대회도치적비치위0.27±0.04,여공백대조조、ASODN전염조화L-PEI/SODN전염조상비,차이균유통계학의의(균P＜0.05),hTERT단백불표체;전염48 h후,L-PEI/ASODN전염조세포조망명현,조기조망솔화만기조망솔분별위38.0%화52.2%.NGR수식적L-PEI/ASODN-FAM납미복합물주사우하류소서,기파향종류작용명현;급약8 d후,NGR/L-PEI/ASODN정맥급약조화피하급약조적종류체적균명현소우생리염수조화NGR/L-PEI/SODN조(균P＜0.05);초미결구관찰가견전형조망소체.결론 NGR수식、L-PEI개도하적hTERT ASODN가억제EC9706세포적증식,강저기단립매적표체,환가유효도체라서인식관암이식류내,억제종류적생장.
Objective To investigate the inhibitory effect of nanoparticle-mediated antisense oligodeoxynucleotide(ASODN)of human telomerase reverse transcriptase(hTERT)on telomeruse in the esophageal cancer EC9706 cells.Methods Line-polyethylenimine(L-PEI)was used to condence ASODN into nanoparticle and to couple NGR peptides into targeting nanoparticle.and the prepared L-PEL/ASODN cornplexes were transfected into the EC9706 cells.Cellular uptake of L-PEI/ASODN cornplexes was detected by laser confocal scanning microscopy.MTY assay was used to detect the inhibitory rate of EC9706 cell growth.The level of hTERT mRNA and its protein expression were measured by RT-PCR and immunohistochemistry,respectively.Annexin V FITC/PI double labeling was used to detect cell apoptosis.The distribution of drug in nude mice Was observed by laser confocal scanning microscopy,and the growth and morphology of the tumor Was examined.Resuits The L-PEI-reediated ASODN uptake was enhanced.After transfection.the inhibitory rate of EC9706 cells Was time-dependant and there was a significant differenee between controI cell group and L-PEI/ASODN group(P＜0.05).At 48 h acter transfection.the level of hTERT mRNA Was decreased significantly compared with that of control cell group(P＜0.05).andthe expression of hTERT protein was negative.There was apparent apoptosis in EC9706 ceUs after transfection with L-PEI/ASODN complexes.For the two NGR/L-PEI/ASODN groups.fluorescence was observed in the liver.kidney,lung and tumor tissues of nude mice,and their uptake intensity was timedependent.The mean volume of tumors in the two NGR/L-PEI/ASODN groups was significantly smaller than those in blank control group and SODN group(P＜0.05).Apoptotic bodies were detected in the tumors of L-PEI/ASODN group.Conclusion The NGR/L-PEI/ASODN nanoparticles can effectively reach into the human esophageal cancer xenograt and inhibit the tumor growth in nude mice.and this may provide a theoretical and experimental basis for gene therapy for human esophageal squamous cell carcinoma.
