短发夹rna沉默htert基因抑制裸鼠皮下人脑胶质瘤生长的实验研究

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1、短发夹RNA沉默hTERT基因抑制裸鼠皮下人脑胶质瘤生长的实验研究作者：孙卫东于如同马衍刚【摘要】目的研究靶向人端粒酶逆转录酶(hTERT)的短发夹RNA(shRNA)对裸鼠人脑胶质瘤中hTERT基因表达的抑制作用，以及影响肿瘤增殖并诱导细胞凋亡的作用，为脑胶质瘤的基因治疗探讨新的依据。方法体外培养人脑胶质瘤U251细胞株，建立人脑胶质瘤裸鼠皮下接种模型。将成功造模的36只裸鼠随机分为3组(每组12只)。hTERTshRNA组肿瘤体内原位注射hTERT质粒shRNA(pshRNA)20ug+脂质体60ul+PBS；PBS组肿瘤体

2、内原位注射PBS150ul；空质粒组肿瘤体内原位注射空质粒20ug+脂质体60ul+PBS。观察肿瘤生长情况。苏木精-伊红染色观察肿瘤组织的病理改变，Westernblot检测hTERT蛋白的表达，钙依赖性磷脂结合蛋白V+碘化丙噪双染流式细胞仪测凋亡率。结果成功建立稳定的裸鼠U251细胞皮下移植瘤模型。治疗5周后hTERTshRNA组肿瘤体积较PBS组和空质粒组明显缩小，抑瘤率为58.2%；病理学检查hTERTshRNA组肿瘤生长受到抑制，肿瘤细胞散在稀疏，可见大量肿瘤细胞坏死及凋亡；Westernblot示hTERTshRNA

3、组hTERT蛋白表达受抑制；流式细胞仪检测hTERTshRNA组肿瘤细胞凋亡率明显高于PBS组和空质粒组(P<；0.01)o结论hTERTshRNA可在裸鼠移植瘤体内有效发挥作用，抑制人端粒酶逆转录酶蛋白在体内表达，从而能有效抑制裸鼠胶质瘤增殖生长,促进肿瘤细胞凋亡。【关键词】徐州医学院附属医院神经外科，江苏徐州221002Abstract：ObjectiveToexploretheinhibitoryeffectofsilencinghumantelomerasereversetranscriptase(hTERT)gen

4、e'sshorthairpinRNAs(shRNAs)onthegrowthofhumangliomainnudemicebyRNAinterferencetechniquetoaffordanexperimentalbasisforconcernedgenetherapyofglioma・MethodsHumang1iomaU251cells,culturedinvitro,wereusedtoestablishmodelsofhumang1iomabysubcutaneousinjectionintonudemice.The

5、36mousemodelsformedweredividedinto3groupsforvariedpurposes(n=12each):thehTERTshRNAtherapygroupwasgiveninsituinjectionofhTERTshRNADplamid(pshRNA)20ug+lipid60u1+PBS；thePBSgroup,giventhatofPBS150u1；theblankplasmidgroup,giventhatofblankplasmid20ug+lipid60u1+PBS.H~Estaine

6、dspecimensofthexenografttumorwerepreparedforpathohistologicalstudy-aftertheplasmidtherapy,Westernblotmethodwasusedtodetermoinetheprotein1evelofhTERT,f1owcytometrywasforassessingtheapoptosisoftumorcells・ResultsSatisfactorysubcutaneousxenograftgliomamodelofnudemicecoul

7、dbeconstructedwithin15days,withanaveragesizeof132mm3.ComparedwiththatintheblankplasmidandPBSgroups,thetumorgrowthinthetherapygroupwasevidentlydepresseddownto58.2%.Throughlightmicroscopy,itwasfoundinthehTERTshRNAgroupthatthetumorgrowthwasinhibitedobviously,therewerere

8、lativelyfewtumorcells,buttherewerenumbersofnecroticandapoptotictumorcellseverywhere.WesternblotshowedtheexpressionofhTERTproteinwas