小鼠—大鼠异种心脏移植miRNA表达谱分析
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摘要
目的研究小鼠-大鼠异种异位心脏移植后供心miRNA表达谱的变化,为有效控制异种移植排斥反应奠定实验基础。
方法
1.采用改良Heron套袖法建立小鼠-小鼠／大鼠异位心脏移植模型,将小鼠心脏移植到受体右侧颈部。实验动物包括同系对照组、异种24h组和异种停跳组,每组含受体动物8只。
2.试剂盒法分离提取RNA, Cy5标记所提取总RNA中的miRNA,浓缩标记样品进行miRNA芯片杂交,使用Axon GenePix 4000B Microarray Scanner进行图像扫描,筛选出异种心脏移植排斥反应中差异表达显著的miRNA。
3. TargetScan软件预测差异表达显著的miRNA靶基因,寻找与异种移植排斥反应发生相关的基因。
4.选取杂交结果中差异表达显著的miR-146a、miR-155和miR-451进行相对定量研究,通过TaqMan miRNA Assays技术对三组样本进行检测,验证芯片结果。
结果
1.模型建立练习阶段、稳定阶段和成熟阶段手术成功率分别为53.33%、85.71%和92.31%,3个阶段手术成功率相比较具有统计学差异且后两阶段成功率高于练习阶段。
2.异种24h组和对照组芯片杂交结果显示24个miRNA差异表达显著,其中11个表达显著下调,13个表达显著上调。
3.异种停跳组和对照组芯片杂交结果显示25个miRNA差异表达显著,其中12个表达显著下调,13个表达显著上调。
4.异种停跳组和异种24h组芯片杂交结果显示6个miRNA差异表达显著,其中5个miRNA变化趋势一致,1个miRNA变化趋势相反。
5.芯片杂交结果中差异表达显著的miR-146a、miR-155和miR-451进行相对定量研究,与对照组相比较结果具有统计学差异,且3个miRNA的变化倍数符合芯片分析的结果。
结论
1．采用改良Heron套袖法建立小鼠-小鼠／大鼠异位心脏移植模型,此方法操作简便、易于掌握,是研究异种心脏移植排斥反应的良好动物模型。
2.在异种心脏移植排斥反应中出现多个miRNA显著的差异表达,涉及免疫、肿瘤的发生、胰岛素的合成与代谢、神经系统生理功能、细胞增殖、血管生成等多个方面,并广泛参与基因的转录调控。
3.在小鼠-大鼠异种心脏移植排斥反应中miR-146a和miR-155同时出现高表达,miR-451出现显著低表达,说明它们参与了免疫应答和炎症反应的调控,提示miRNA在异种心脏移植排斥反应中可能发挥着重要的调控作用。
4.微阵列芯片技术是有效的高通量分析心脏异种移植排斥反应miRNA差异表达的研究手段。
Objective To detect the miRNA expression profiles of the myocardial tissue after the heterotopic mouse to rat cardiac xenotransplantation and provide the experimental evidences for effective regulation of xenotransplantation rejection.
Methods
1. The heterotopic cardiac mouse-mouse/rat xenotransplantation models (in the right side of neck of recipients) were established by modified Heron sleeve method. The experimental animals were divided into normal control group, xenotransplantation group (24 hours) and xenotransplantation group (arresting). Each group included 8 recipients.
2. RNA was extracted with kit and miRNA was marked with Cy5. Then concentration and hybridization were finished by miRNA array. The slides were scanned with Axon GenePix 4000B Microarray Scanner and the miRNAs which significantly differential expressed in the cardiac xenotransplantation rejection were filtrated.
3. The target genes corresponded with the above miRNAs were predicted by software of Target Scan, which may be the causes of xenotransplantation rejection.
4. MiR-146a, miR-155 and miR-451 in the three groups, which were obviously changed during xenotransplantation, were analysis by TaqMan(?) miRNA Assays, verifying the results of miRNA array.
Results
1. In the three stages (practicing, stable, skilled) of establishing the models, the successful rates of surgery were 53.33%,85.71%, and 92.31%respectively, with significantly difference. The successful rates in stable and skilled stages were high.
2. There were 11 downregulated miRNAs and 13 upregulated miRNAs among the total 24 significantly differential expressed miRNAs found in xenotransplantation (24hours) group comparing with control group.
3. There were 12 downregulated miRNAs and 13 upregulated miRNAs among the total 25 significantly differential expressed miRNAa found in xenotransplantation (arresting) group comparing with control group.
4. Among the 6 significantly differential expressed miRNAs found in xenotransplantation (arresting) group comparing with xenotransplantation (24 hours) group,5 miRNAs had the consistent trend while the other one did not.
5. MiR-146a, miR-155 and miR-451 in the three groups were obviously changed following xenotransplantation comparing with control group and the results were accordant with the results of miRNA array.
Conclusions
1. Modified Heron sleeve method is a simple and easy-to-learn operation for setting up the mouse-mouse/rat heterotopic cardiac xenotransplantation model. It is a good animal model for xenotransplantation rejection.
2. It is demonstrated that there are significantly differential expressions of miRNAs in xenotransplantation involving in immune, tumor genesis, synthesis and metabolism of insulin, physiological functions of nervous system, cell proliferation, angiogenesis as well as transcriptional regulation.
3. Simultaneous high expression of miR-146a and miR-155 and low expression of miR-451 in mouse-rat cardiac xenotransplantation rejection reveal the relevance to the regulation of immune response and inflammation. The miRNA is proposed to play an important role in regulating the cardiac xenotransplantation.
4. Microarray chip is an effective method for the high-throughput analysis of miRNAs expression profile in cardiac xenotransplantation rejection.
方法
1.采用改良Heron套袖法建立小鼠-小鼠／大鼠异位心脏移植模型,将小鼠心脏移植到受体右侧颈部。实验动物包括同系对照组、异种24h组和异种停跳组,每组含受体动物8只。
2.试剂盒法分离提取RNA, Cy5标记所提取总RNA中的miRNA,浓缩标记样品进行miRNA芯片杂交,使用Axon GenePix 4000B Microarray Scanner进行图像扫描,筛选出异种心脏移植排斥反应中差异表达显著的miRNA。
3. TargetScan软件预测差异表达显著的miRNA靶基因,寻找与异种移植排斥反应发生相关的基因。
4.选取杂交结果中差异表达显著的miR-146a、miR-155和miR-451进行相对定量研究,通过TaqMan miRNA Assays技术对三组样本进行检测,验证芯片结果。
结果
1.模型建立练习阶段、稳定阶段和成熟阶段手术成功率分别为53.33%、85.71%和92.31%,3个阶段手术成功率相比较具有统计学差异且后两阶段成功率高于练习阶段。
2.异种24h组和对照组芯片杂交结果显示24个miRNA差异表达显著,其中11个表达显著下调,13个表达显著上调。
3.异种停跳组和对照组芯片杂交结果显示25个miRNA差异表达显著,其中12个表达显著下调,13个表达显著上调。
4.异种停跳组和异种24h组芯片杂交结果显示6个miRNA差异表达显著,其中5个miRNA变化趋势一致,1个miRNA变化趋势相反。
5.芯片杂交结果中差异表达显著的miR-146a、miR-155和miR-451进行相对定量研究,与对照组相比较结果具有统计学差异,且3个miRNA的变化倍数符合芯片分析的结果。
结论
1．采用改良Heron套袖法建立小鼠-小鼠／大鼠异位心脏移植模型,此方法操作简便、易于掌握,是研究异种心脏移植排斥反应的良好动物模型。
2.在异种心脏移植排斥反应中出现多个miRNA显著的差异表达,涉及免疫、肿瘤的发生、胰岛素的合成与代谢、神经系统生理功能、细胞增殖、血管生成等多个方面,并广泛参与基因的转录调控。
3.在小鼠-大鼠异种心脏移植排斥反应中miR-146a和miR-155同时出现高表达,miR-451出现显著低表达,说明它们参与了免疫应答和炎症反应的调控,提示miRNA在异种心脏移植排斥反应中可能发挥着重要的调控作用。
4.微阵列芯片技术是有效的高通量分析心脏异种移植排斥反应miRNA差异表达的研究手段。
Objective To detect the miRNA expression profiles of the myocardial tissue after the heterotopic mouse to rat cardiac xenotransplantation and provide the experimental evidences for effective regulation of xenotransplantation rejection.
Methods
1. The heterotopic cardiac mouse-mouse/rat xenotransplantation models (in the right side of neck of recipients) were established by modified Heron sleeve method. The experimental animals were divided into normal control group, xenotransplantation group (24 hours) and xenotransplantation group (arresting). Each group included 8 recipients.
2. RNA was extracted with kit and miRNA was marked with Cy5. Then concentration and hybridization were finished by miRNA array. The slides were scanned with Axon GenePix 4000B Microarray Scanner and the miRNAs which significantly differential expressed in the cardiac xenotransplantation rejection were filtrated.
3. The target genes corresponded with the above miRNAs were predicted by software of Target Scan, which may be the causes of xenotransplantation rejection.
4. MiR-146a, miR-155 and miR-451 in the three groups, which were obviously changed during xenotransplantation, were analysis by TaqMan(?) miRNA Assays, verifying the results of miRNA array.
Results
1. In the three stages (practicing, stable, skilled) of establishing the models, the successful rates of surgery were 53.33%,85.71%, and 92.31%respectively, with significantly difference. The successful rates in stable and skilled stages were high.
2. There were 11 downregulated miRNAs and 13 upregulated miRNAs among the total 24 significantly differential expressed miRNAs found in xenotransplantation (24hours) group comparing with control group.
3. There were 12 downregulated miRNAs and 13 upregulated miRNAs among the total 25 significantly differential expressed miRNAa found in xenotransplantation (arresting) group comparing with control group.
4. Among the 6 significantly differential expressed miRNAs found in xenotransplantation (arresting) group comparing with xenotransplantation (24 hours) group,5 miRNAs had the consistent trend while the other one did not.
5. MiR-146a, miR-155 and miR-451 in the three groups were obviously changed following xenotransplantation comparing with control group and the results were accordant with the results of miRNA array.
Conclusions
1. Modified Heron sleeve method is a simple and easy-to-learn operation for setting up the mouse-mouse/rat heterotopic cardiac xenotransplantation model. It is a good animal model for xenotransplantation rejection.
2. It is demonstrated that there are significantly differential expressions of miRNAs in xenotransplantation involving in immune, tumor genesis, synthesis and metabolism of insulin, physiological functions of nervous system, cell proliferation, angiogenesis as well as transcriptional regulation.
3. Simultaneous high expression of miR-146a and miR-155 and low expression of miR-451 in mouse-rat cardiac xenotransplantation rejection reveal the relevance to the regulation of immune response and inflammation. The miRNA is proposed to play an important role in regulating the cardiac xenotransplantation.
4. Microarray chip is an effective method for the high-throughput analysis of miRNAs expression profile in cardiac xenotransplantation rejection.
引文
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