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_医学论文.docVIP

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_医学论文 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_医学论文 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）。hTERT shRNA组肿瘤体内原位注射hTERT 质粒shRNA（pshRNA）20 μg+脂质体60 μl+PBS；PBS组肿瘤体内原位注射PBS 150 μl；空质粒组肿瘤体内原位注射空质粒20 μg+脂质体60 μl+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，Western blot检测hTERT蛋白的表达，钙依赖性磷脂结合蛋白V+碘化丙啶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hTERT shRNA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hTERT shRNA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；Western blot示hTERT shRNA组hTERT蛋白表达受抑制；流式细胞仪检测hTERT shRNA组肿瘤细胞凋亡率明显高于PBS组和空质粒组（0.01）。结论 hTERT shRNA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 221002 Abstract： Objective To explore the inhibitory effect of silencing human telomerase reverse transcriptase (hTERT) gene′s short hairpin RNAs (shRNAs) on the growth of human glioma in nude mice by RNA interference technique to afford an experimental basis for concerned gene therapy of glioma. Methods Human glioma U251 cells, cultured in vitro, were used to establish models of human glioma by subcutaneous injection into nude mice. The 36 mouse models formed were divided into 3 groups for varied purposes (n=12 each): the hTERT shRNA therapy group was given in situ injection of hTERT shRNAD plamid (pshRNA) 20 μg + lipid 60 μl + PBS the PBS group, given that of PBS 150 μl the blank plasmid group, given that of blank plasmid 20 μg + lipid 60 μl + PBS. H-E stained specimens of the xenograft tumor were prepared for pathohistological study after the plasmid therapy, Western blot method was used to determoine the protein level of hTERT, flow cytometry was for assessing the apoptosis of tumor cells. Results Satisfactory subcutaneous xenograft glioma model of nude mice could be constructed within 15 days, with an average size of 132 mm3. Compared with that in the blank plasmid and PBS groups, the tumor growth in the ther