| Objective:This project used a mouse neck ectopic heart transplantation to establish a model to identify mi RNAs associated with transplantation immunity by studying the differential changes of estradiol on mi RNA expression profiles in mouse allogeneic heart transplantation rejection,to lay an experimental foundation for finding a more effective way to inhibit transplantation rejection,and to search for a possible novel biological marker for clinical diagnosis of non-invasive heart transplant rejection.Methods: 1.Using microsurgical techniques,a mouse neck heart transplantation model was established.The experimental animals were divided into homologous transplantation group,heterologous transplantation model group and heterologous transplantation estradiol group.2.Mental status and survival time of heart grafts were observed in each group.3.After assessing the success of the model,mouse transplanted heart tissues were obtained on postoperative day7 for HE staining and pathological grading.4.Total RNA was isolated and extracted from mouse spleen tissues using the kit method,quantified using Nano Drop ND-2000 and tested for RNA integrity by Agilent Bioanalyzer 2100.5.After passing the RNA quality check,the total RNA was first reverse transcribed into double-stranded c DNA,and then further synthesized into Cy3-labelled c RNA.Raw images were obtained.6.The differentially expressed mi RNAs were detected using Agilent mi RNA microarrays,and cluster analysis,volcano plot and scatter plot were constructed by R language software.7.Two databases,mi RWalk and mi RDB,were used to predict mi RNA target genes,which were displayed by venn to obtain intersection sets.8.The screened differentially expressed significant GO and KEGG enrichment analyses were performed on the screened sets of significantly differentially expressed target genes.Results:1.Mice were observed to be in good mental condition after neck heart transplantation,and the survival time of transplanted hearts was prolonged in the heterologous transplantation estradiol intervention group compared to the heterologous transplantation model group(p<0.01).2.The pathological grade of the allograft group was 0~1 by HE staining;the myocardial damage in the allograft model group was severe and the pathological grade was 2~3,which was significantly higher than that of the allograft group(p<0.01);the rejection reaction in the allograft estradiol group was significantly reduced and the pathological grade decreased compared with that of the allograft model group(p<0.01).3.A total of 24 mi RNAs were significantly(p<0.05)differentially expressed between the xenograft model group and the allograft estradiol group;10 of the 24 significantly differentially expressed mi RNAs were significantly up-regulated,namely mmu-mi R-144-3p,mmu-mi R-144-5p,mmu-mi R-486a-5p,mmu-mi R-466f-5p,mmu-mi R-1187,mmu-mi R-6990-5p,mmu-mi R-3470 a,mmu-mi R-144-5p,mmu-mi R-486a-3p,mmu-mi R-5128;14 of the 24 mi RNAs with significant expression differences were significantly down-regulated,namely mmu-mi R-146a-5p,mmu-mi R-150-5p,mmu-mi R-152-3p,mmu-mi R-155-5p,mmu-mi R-21a-5p,mmu-mi R-340-3p,mmu-mi R-342-3p,mmu-mi R-350-3p,mmu-mi R-28a-5p,mmu-mi R-139-5p,mmu-mi R-467a-3p,mmu-mi R-3058-3p,mmu-mi R-378 b,mmu-mi R-148 a.4.GO enrichment analysis showed that molecular functional ontology analysis was mainly in protein binding,metal ion binding,and RNA polymer transcriptional activator activity;cellular component ontology analysis was The KEGG pathway was enriched in several pathways,mainly involving: signaling pathways regulating stem cell pluripotency,T cell receptor signaling pathway,TGF-βsignaling pathway,retrograde endocrine signaling,Wnt signaling pathway,c AMP signaling pathway,etc.Conclusion:1.The animal model of mouse neck heart transplantation was successfully established,which demonstrated that estradiol could reduce the effect of immune rejection injury in allogeneic mouse heart transplantation.2.The mi RNAs that were significantly different in the allograft model group and the allograft estradiol group were screened by mi RNA microarray hybridization.24 mi RNAs with significant differences were screened,suggesting that the differential expression of mi RNAs may be closely related to the role of estradiol in attenuating immune rejection injury in allograft mice heart transplantation,providing a basis for the subsequent study of its related pathogenesis and intervention studies. |
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