深低温保存单采血小板凋亡相关miRNA的差异表达

Differential expression of apoptosis-associated miRNA in cryopreserved apheresis platelets

目的了解单采血小板在深低温保存过程中凋亡相关microRNA(miRNA)的表达改变。方法应用miRNA微孔板芯片技术分析-80℃保存不同时间单采血小板凋亡相关miRNA表达谱的差异,同时利用qRT-PCR技术验证该结果的准确性;采用生物信息学软件预测差异表达miRNA可能的靶基因。结果与新鲜单采血小板相比,凋亡相关miRNA表达谱在血小板-80℃保存5 d时无明显变化、保存7 d时仅有2种明显上调变化的miRNA:miR-216和miR-296。选择表达谱中miR-216 miR-296 miR-7和miR-16共4种miRNA做qRT-PCR验证:以新鲜血小板表达水平为基准值1,-80℃保存5、7 d后miR-216的相对表达量分别为1.81±0.21和2.88±0.23,miR-296的相对表达量分别为2.27±0.20和3.85±0.17,miR-7的相对表达量分别为0.89±0.06和0.76±0.03,miR-16的相对表达量分别为1.12±0.35和1.40±0.32,miR-216和miR-296在<7 d保存过程中表达上调(P<0.05),miR-7和miR-16的表达未发生明显变化(P>0.05)。结论短期(<7 d)深低温保存单采血小板凋亡相关miRNA的表达谱接近新鲜单采血小板。

Objective To explore the variant expression profile of apoptosis-related microRNA（ miRNA） of cryopreserved apheresis platelets. Methods The apoptosis-associated miRNA profile of cryopreserved apheresis platelets after different storage periods was analysed nith miRNA microarrays and the result was validated by quantitative real-time polymerase chain reaction（ qRT-PCR）. Various biological information software and databases were utilized to predict target genes of the selected miRNA. Results Microarray assays showed that,compared with fresh platelets,there was no significant change in platelet miRNA expression after cryopreserved for 5 days,and only the miR-216 and miR-296 levels were significantly increased in platelets cryopreserved for 7 days. Expression of miR-216,miR-296,miR-7 and miR-16 were further detected by qRT-PCR. As compared to the fresh platelets,the relative expression levels of miR-216 at different cryopreserved time（ 5 and 7 day） were 1. 81±0. 21 and 2. 88±0. 23,the levels of 296 were 2. 27±0. 20 and 3. 85±0. 17（ P〈0. 05）; the levels of miR-7 were 0. 89±0. 06 and 0. 76±0. 03,and the levels of miR-16 were 1. 12±0. 35 and 1. 40±0. 32（ P〉0. 05）. Conclusion The apoptosis-associated miRNA profile for short-term（〈7 d） cryopreserved platelets is closer to that of fresh platelets.