【作者】 李岚；

【导师】 刘江华；

【作者基本信息】 南华大学 ， 内科学（专业学位）， 2015， 硕士

【摘要】 目的:比较分析血管钙化过程中呈差异性表达的mi RNA谱,寻找并验证可能调控动脉钙化的关键mi RNA。方法:对动脉钙化进程及平滑肌细胞钙化过程中呈差异性表达的mi RNA谱进行比较分析,通过对mi RNA的变化趋势进行对比,筛选有共同变化趋势目标mi RNA,再应用q RT-PCR及原位杂交技术证实可能与动脉钙化进程相关的关键mi RNA。结果:1)关键mi RNA筛选:经对比分析,筛选出mi R-125b-5p、mi R-30a-5p、mi R-32-5p、mi R-133a-3p及mi R-29a-5p等16个可能与动脉钙化发生发展相关的mi RNA,并初步确认mi R-125b-5p、mi R-30a-5p、mi R-32-5p等8个关键mi RNA作为下一步实验研究的目标。2)关键mi RNA验证:(1)实时定量PCR验证:提取12周龄OPG-/-基因敲除及野生型小鼠主动脉组织中的RNA,对初步确认的8个关键mi RNA进行q RT-PCR的验证。结果显示:与野生型小鼠相比,存在明显钙化的OPG-/-基因敲除小鼠主动脉中mi R-125b-5p、mi R-30a-5p、mi R-32-5p表达上调,mi R-133a-3p、mi R-33-5p、mi R-29a-5p、mi R-210-3p、mi R-320-3p表达下调。除mi R-33-5p外,其他7个mi RNA趋势与12周龄组小鼠主动脉组织芯片结果吻合。趋势吻合的7个mi RNA中,mi R-30a-5p、mi R-32-5p、mi R-133a-3p、mi R-29a-5p、mi R-210-3p及mi R-320-3p均具有统计学差异(p<0.05)。(2)原位杂交:结果显示,mi R-32-5p在OPG-/-小鼠血管组织中的表达随钙化过程的进展而增高,且主要表达在动脉管壁的中膜及内膜。此实验中,mi R-32-5p变化的趋势与芯片结果一致。结论:mi R-125b-5p、mi R-32-5p、mi R-30a-5p、mi R-29a-5p、mi R-133a-3p、mi R-210-3p和mi R-320-3p等在OPG-/-小鼠动脉钙化过程中发挥重要作用,可能是调控血管钙化的关键mi RNA。更多还原

【Abstract】 Objectives : To screen the mi RNA profile during the process of vascular calcification in OPG-/- mouse, and to verify the potential mi RNAs witch could regulate vascular calcification.Methods : The mi RNA profile during the process of vascular calcification was screened by using mi RCURY? LNA Array(v.18.0)(Exiqon) with aortas of the WT mice and OPG-/- mice at 12 weeks of age and with the smooth muscle cell calcification model. QRT-PCR and in situ hybridization were utilized to verify the mi RNAs showing the common change trends in both in vivo and in vitro models.Results : Sixteen potential mi RNAs including mi R-125b-5p,mi R-30a-5p, mi R-32-5p, mi R-133a-3p and mi R-29a-5 were identified to be involved in vascular calcification progression from the in vivo and in vitro models.Verification of the key mi RNAs:1. Quantitative PCR analysis showed that seven of eight mi RNAs were validated to be consistent with the trends in the microarray analysis.The expression of mi R-125 b,mi R-30 a and mi R-32 were confirmed to be elevated, and mi R-133 a,mi R-29 a,mi R-210 and mi R-320 were proved to be decreased in the aortas of OPG-/- mice with vascular calcification(p<0.05).2. In situ hybridization confirmed that mi R-32 located mainly in the media of aorta and showed a slightly and markedly increased expression in aortas of OPG-/- mice at 4 and 12 weeks of age respectively, compared to those in WT mice.Conclusion : The 7 mi RNAs including mi R-125b-5p, mi R-32-5p,mi R-30a-5p, mi R-29a-5p, mi R-133a-3p, mi R-210-3P and mi R-320-3p were identified to be implicated in the progression of vascular calcification, and those mi RNAs might be served as candidates for biological marker and modulator of vascular calcification.更多还原