siRNA抑制Smad4或Runx2基因表达治疗大鼠异位骨化效果比较  

Comparative Effectiveness of Silencing Smad4 or Runx2 with Specific siRNAs for Treating Heterotopic Ossification in Rats

作　　者：薛涛[1,2] 林霖[1] 魏学磊[3] 张继英[1] 傅欣[1] 段小宁[1] 于长隆[1] 

机构地区：[1]北京大学第三医院运动医学研究所,北京100191 [2]北京大学首钢医院骨科 [3]天津医院骨科,天津300211

出　　处：《中国运动医学杂志》2011年第7期644-649,共6页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金(ZR09-1-01);国家体育总局科研项目基金(TYZJ07-1-05);北京市科技新星计划(2008B04)

摘　　要：目的:研究siRNA抑制Smad4基因表达治疗异位骨化大鼠模型的效果,并与抑制Runx2的疗效进行比较。方法:1、设计6条分别针对大鼠Smad4和Runx2的mRNA模板,用体外转录合成法分别合成6条siRNA,采用RT-PCR从mRNA水平在培养的原代成骨前体细胞中筛选有明显抑制作用的siRNA。2、设计合成针对Smad4基因和Runx2基因编码的发卡样siRNA的双链DNA,与腺病毒的穿梭质粒连接后,取其启动子和终止子连接于非病毒载体上。在培养的大鼠原代成骨细胞中,采用RT-PCR和western blot测定Smad4特异性siRNA非病毒对Smad4的抑制作用和Runx2特异性siRNA非病毒对Runx2的抑制作用。3、采用体内实验测定Smad4或Runx2特异性siRNA非病毒载体对切断大鼠跟腱诱导异位骨化动物模型的影响:实验组行跟腱切断术和植入100μg的Smad4-siRNA或Runx2-siRNA重组非病毒载体,对照组行跟腱切断术和植入100μg的pcDNA4/HisA非病毒载体。10周后,行CT三位重建检测形成的异位骨大小,HE染色观察其组织形态。结果:1、在合成的3条Smad4-siRNA中,siRNA139-159对Smad4的抑制效果最明显,在合成的3条Runx2-siRNA中,siRNA1057-1077对Runx2的抑制效果最明显。2、成功构建重组非病毒载体,转染原代成骨细胞可明显抑制Smad4和Runx2的表达。3、CT三位重建显示,Smad4-siRNA实验组形成的异位骨体积较对照组减小92%,Runx2-siRNA实验组形成的异位骨体积较对照组减小93%,差异均有统计学意义(P<0.05)。组织学观察显示非病毒载体介导的特异性Smad4-siRNA和Runx2-siRNA在体内可以抑制异位骨形成,两者之间的抑制效果差异无统计学意义(P>0.05)。结论:Smad4和Runx2两种信号通路的特异性siRNA可以明显抑制切断跟腱诱导的异位骨化,但两种不同siRNA的抑制效果无差别。Objective To study the effect of Smad4-specific siRNA and Runx2-specific siRNA on the formation of heterotopic ossification in rats.Methods（1）Ambion＇s siRNA target design online tool was utilized to design three sequences of DNA oligonucleotides for targeting rat Smad4 mRNA,and another three sequences of DNA oligonucleotides for targeting rat Runx2 mRNA.They were synthesized in vitro through transcription.The most optimal siRNA and transfection condition were determined by mRNA levels in cultured primary osteoblastic cells through RT-PCR.（2）Ambion＇s siRNA target design online tool was utilized to design two DNA oligonucleotides which encoded two hairpin siRNA templates for the gene of Smad4 and Runx2,the DNA oligonucleotides were ligated into shuttle vectors.The siRNA cassettes were then copy out with AscI and inserted into the AscI site of pBac（3xP3-EGFPafm）.The non-viral vecotrs were transfected into primary osteoblastic cells and then the silencing effects on Smad4 and Runx2 were tested with RT-PCR and Western blot.（3）The evidence of heterotopic ossification after transfer of gene for inhibition of either Smad4 or Runx2 was observed.Results（1）Of the 3 Smad4-siRNA and 3 Runx2-siRNA,the inhibition of siRNA^139-159 for Smad4,and the inhibition of siRNA^1057-1077 for Runx2 appeared to be most evident.（2）Rat primary osteoblast cells were infected with the Smad4-siRNA,and the expression of Smad4 was reduced evidently.Rat primary osteoblast cells were infected with the Runx2-siRNA,and the expression of Runx2 was reduced evidently.（3）The formation of heterotopic ossification induced by tenotomy of Achilles tendon was significantly inhibited.Heterotopic ossification was inhibited with Smad4-specific siRNA by approximately 92%（P〈0.05）and with Runx2-specific siRNA by 93%（P〈0.05）.The inhibition effects between Smad4-specific siRNA and Runx2-specific siRNA was statistical insignificance（P〈0.05）.The blocking way of Smad4 or Runx2 was identical in vivo.Conclusion Non-virus-me

关 键 词：SIRNA SMAD4 RUNX2 非病毒载体 异位骨化 

分 类 号：R87[医药卫生—运动医学]