【作者】 高爽； 赵红； 张志民；

【Author】 Shuang Gao;Hong Zhao;Zhimin Zhang;Department of Endodontics, School of Dentistry, Jilin University;Department of Endodontics, Yantai Stomatological Hospital of Binzhou Medical University;

【机构】 吉林大学口腔医院牙体牙髓科； 滨州医科大学烟台口腔医院牙体牙髓科；

【摘要】 目的 :我课题组自行设计并筛选出靶向NF-κB p65 mRNA的反义寡核苷酸,在体内、外的早期牙髓炎模型中观察其对NF-κB表达的影响,评估其对早期牙髓炎的治疗效果,为临床抑制炎症、保存活髓提供了新的思路。方法:1.利用数据库分析,选定人和大鼠的NF-κB p65 mRNA同源序列的靶区域及其相应的互补候选反义寡核苷酸(Antisense oligonucleotides,ASOs)。评估所有候选ASOs与靶区域结合的可能性。最后,对评选出的ASOs(PMOs全称,PMOs)进行特异性分析。2.建立早期牙髓炎体外模型,分别采用qRT-PCR法和WB法检测相关基因表达情况。筛选出效果最佳的PMO进行下一步体内研究。3.建立早期牙髓炎动物模型,通过HE染色、IHC染色、qRT-PCR法和WB法检测相关基因表达。将大鼠的心、肝、脾、肺、肾等重要脏器进行HE染色观察:分析PMO3是否有药物毒性作用。结果:1.利用NCBI和BLAST数据可筛选出人和大鼠NF-κB p65 mRNA同源序列中的12段保守序列。在primer软件中,针对上述12段保守序列设计出了138个与靶区域互补的候选ASOs。接着,利用Sfold和RNAfold web server在线分析软件评估了所有候选ASOs与靶区域结合的可能性,筛选出第29、30、72号候选ASOs。最后,经Nucleotide BLAST网络数据库对比分析,所评选出的三个候选ASOs均具有特异性,分别将其命名为PMO1、PMO2和PMO3进行后续实验。2.免疫荧光实验:PMO1、PMO2、PMO3均能进入hDPCs和r DPCs内部。qRT-PCR和WB实验:与对照组相比,PMOs可以下调NF-κB p65、p-p65及其下游炎症因子IL-1β和TNF-α的表达水平。其中以PMO3的体外抗炎效果最佳,进一步用于体内牙髓炎模型的研究。3. HE染色:与对照组相比,PMO3可缩小炎症范围,减少炎症细胞浸润。体内IHC染色、qRT-PCR和WB实验:与对照组相比,PMO3可降低牙髓组织中的NF-κB p65、p-p65及其下游炎症因子IL-1β和TNF-α的表达。大鼠重要脏器的HE染色:与对照组比较,PMO3组大鼠各脏器的细胞形态无异常。结论:1.完成了靶向NF-κB p65 mRNA反义寡核苷酸的设计与合成。2.成功建立了牙髓炎细胞模型和动物模型。3.PMOs均能进入牙髓细胞内,并成功抑制牙髓炎模型中NF-κB p65及其下游炎症因子的表达。3.PMO3能治疗LPS引起的牙髓炎症,且具有生物安全性。更多还原

【Abstract】 Objectives:In this study, we designed the ASO targeting NF-κB p65 mRNA and observed its effect on the expression of NF-κB and its therapeutic effect on early pulpitis in vivo and in vitro, which provided a new idea for the protection of dental pulp tissue.Methods:1. Using database analysis, select the target regions of NF-κB p65 mRNA homologous sequences from humans and rats, as well as their corresponding complementary candidate antisense oligonucleotides(ASOs). Evaluate the potential of all candidate ASOs to bind to the target regions. Finally, a specificity analysis was conducted on the selected ASOs(PMOs)2. Establish an in vitro model of early pulpitis and detect the expression of related genes using qRT-PCR and WB methods, respectively. Select the PMO with the best performance for further in vivo research.3. Establish an animal model of early pulpitis and detect the expression of related genes through HE staining, IHC staining, qRT-PCR, and WB methods. Perform HE staining on important organs such as the heart, liver, spleen, lungs, and kidneys of rats to analyze whether PMO3 has drug toxicity effects.Results:1. Using NCBI and BLAST database, 12 conserved sequences of human and rat NF-κB p65 mRNA homologous sequences can be screened. In the Primer software,138 candidate ASOs complementary to the target region were designed for the 12conserved sequences mentioned above. Next, Sfold and RNAfold web server online analysis software were used to evaluate the potential binding of all candidate ASOs to the target region, and the 29th, 30th, and 72nd candidate ASOs were selected. Finally,after comparing and analyzing the Nucleotide BLAST network database, the three selected candidate ASOs were found to be specific and were named PMO1, PMO2,and PMO3 for subsequent experiments.2. Immunofluorescence experiment shows: PMO1, PMO2, and PMO3 can all enter the interior of hDPSCs and r DPSCs. QRT-PCR and WB experiments show:Compared with the control group, PMOs can downregulate the expression levels of NF-κB p65, p-p65, and their downstream inflammatory factors IL-1β and TNF-α.Among them, PMO3 has the best in vitro anti-inflammatory effect and is further used for the study of in vivo pulpitis models..3. HE staining shows: Compared with the control group, PMO3 can reduce the scope of inflammation and decrease the infiltration of inflammatory cells. In vivo IHC staining, qRT-PCR, and WB experiments show: Compared with the control group,PMO3 can reduce the expression of NF-κB p65, p-p65, and downstream inflammatory factors IL-1β and TNF-α in dental pulp tissue. HE staining of important organs in rats show: Compared with the control group, there were no abnormalities in the cell morphology of various organs in the PMO3 group rats.Conclusion:1. The design and synthesis of antisense oligonucleotides targeting NF-κB p65mRNA were completed2. All PMOs could enter into the cells and successfully inhibit the mRNA and protein expression of NF-κB p65 and downstream inflammatory factors in vitro.3. PMO3 has a good anti-inflammatory effect in vivo and has biological safety.更多还原