RNAi技术沉默PTHrP基因的表达诱导软骨肉瘤细胞凋亡的实验研究  

Experimental study on RNA interference mediated inhibition of PTHrP expression in proliferating chondrosarcoma cells

作　　者：韩会峰[1] 许铁[1] 裴冬生[2] 秦宏敏[1] 

机构地区：[1]徐州医学院附属医院急救中心,江苏徐州221002 [2]徐州医学院生物化学与分子生物学研究中心,江苏徐州221002

出　　处：《徐州医学院学报》2008年第1期30-34,共5页Acta Academiae Medicinae Xuzhou

基　　金：江苏省自然科学基金(05KJB320135);江苏省高校自然科学基金(BK2005015)

摘　　要：目的研究甲状旁腺相关蛋白(parathyroid hormone-related protein,PTHrP)对软骨肉瘤细胞增殖的影响。方法本实验设置空白对照组、空质粒组和siPTHrP转染组,每组样本数均为6。空白对照组不做处理;分别将空质粒pSilencer3.1H1neo(空质粒组)和重组质粒pSilencer3.1H1neo-siPTHrP(siPTHrP转染组)转染至SW1353软骨肉瘤细胞株,通过MTT法检测各组细胞活力并绘制细胞生长曲线,利用流式细胞技术测定细胞凋亡率,半定量RT-PCR和Western blotting法检测PTHrP基因在mRNA水平及蛋白水平表达上的变化。结果siPTHrP转染组的细胞生长较空白对照组软骨肉瘤细胞明显缓慢;siPTHrP转染组细胞凋亡率明显升高;重组质粒pSilencer3.1H1neo-siPTHrP能明显抑制软骨肉瘤细胞的PTHrP基因在mRNA转录水平和蛋白水平上的表达。结论重组质粒pSilencer3.1H1neo-siPTHrP可明显抑制软骨肉瘤细胞增殖及其内源基因PTHrP在mRNA转录水平和蛋白水平的表达,为PTHrP介导的软骨肉瘤基因沉默疗法提供了理论基础。Objective To investigate the influence of small interfering RNAs targeting parathyroid hormone - related protein （PTHrP） on the proliferation of chondrosarcoma cells. Methods The experiment was carried out in three groups of cell - culture wells （ n =- 6 each） ： blank control group, empty plasmid group and siPTHrP transfection group. The empty plasmid （pSilencer3.1H1 neo） and recombinant plasmid （pSilencer3.1H1 neo -siPTHrP） were transfected into the SW1353 chondrosarcoma cell line. The cell growth curve was drawn in terms of MTT data. Flow cytometry was performed to determine the cell apoptotic rate. Western blotting and immunohistochemical staining were made to show the changes in PTHrP expression. Results The introduction of pSilencer3.1 H1 neo - siPTHrP caused effective downregulation of the growth of chondrosarcoma cells, elevated the rate of apoptosis and evidently inhibited the expression of PTHrP in the process of RNA transcription and protein expression. Conclusions The small interfering RNA targeting PTHrP gene can dramatically inhibit the proliferation of chondrosarcoma cells and the expression of endogenic PTHrP gene, providing a theoretical basis for PTHrP - mediated gene silencing therapy for chondrosarcoma.

关 键 词：甲状旁腺相关蛋白(PTHrP) RNA干扰 基因沉默 软骨肉瘤 细胞凋亡 

分 类 号：R738.3[医药卫生—肿瘤]